CN101160322A - Molecules and chimeric molecules thereof - Google Patents

Abstract

The present invention relates generally to the fields of proteins, diagnostics, therapeutics and nutrition. More particularly, the present invention provides an isolated protein molecule in or related to the tumour necrosis factor (TNF) superfamily such as TNF-a, Lymphotoxin-a (LT-a), TNFRI, TNFRII, OX40, BAFF, NGFR, Fas Ligand or chimeric molecules thereof comprising at least a portion of the protein molecule, such as TNF-a-Fc, LT-a-Fc, TNFRI-Fc, TNFRII-Fc, OX40-Fc, BAFF-Fc, NGFR-Fc, Fas Ligand-Fc; wherein the protein or chimeric molecule thereof has a profile of measurable physiochemical parameters, wherein the profile is indicative of, associated with or forms the basis of one or more pharmacological traits. The present invention further contemplates the use of the isolated protein or chimeric molecule thereof in a range of diagnostic, prophylactic, therapeutic, nutritional and/or research applications.

Description

Molecule and its chimeric molecule

Technical field

The present invention broadly relates to proteinology, diagnostics, acology and nutrition.Especially, at least one of chimeric molecule the invention provides the protein molecule for belonging to TNF (TNF) superfamily or associated separation and comprising the protein molecule, the protein of described separation, such as TNF-a, lymphotoxin-a (LT-a), TNFRI, TNFRII, OX40, BAFF, NGFR, FasL；At least one of chimeric molecule for including the protein molecule, such as TNF-a-Fc, LT-a-Fc, TNFRI-Fc, TNFRII-Fc, OX40-Fc, BAFF-Fc, NGFR-Fc, FasL-Fc.Wherein the protein or its chimeric molecule have measurable parameter attribute, and wherein this feature represents, is associated with one or more pharmacological characteristics or formed the basis of one or more pharmacological characteristics.It is still further contemplated that the protein of separation or its chimeric molecule are used to diagnosing, prevented, is treated, in nutrition and/or research application.

Background technology

Any prior art being related in this specification also not shall not be construed as forming prior art a kind of accreditation or any type of prompting of a part for universal general knowledge.

TNF superfamilies are related to cell growth, survival, apoptosis, necrosis and immune response.TNF molecules possess significant selecting cell toxic action to tumour cell, also induce non-cancerous cells apoptosis.Acceptor in TNF superfamilies includes rich cysteine in extracellular domain and repeated.The member of TNF superfamilies includes TNF-a, LT-a, TNFRI, TNFRII, OX40, BAFF, NGFR, FasL.

TNF-a (TNF-alpha, Tumor necrosis factor ligand superfamily member 2, TNFSF2) is a transmembrane protein containing 233 amino acid, forms a homotrimer with bioactivity.The molecule related to its structure, lymphocytotoxin-α (LT- α, TNF-β, TNFSF2) it is a peptide containing 205 amino acid, including one section the signal sequence containing 34 amino acid, it is different from other TNF superfamilies parts, instructs the secretion of its mature peptide.

TNF-α and the alpha mediated cells of the LT- especially necrosis of mutant or apoptosis, while inducing inflammatory reaction, cell propagation, the release of cell factor and the activation of T and bone-marrow-derived lymphocyte.In addition, locally low-level expression participates in tissue remodeling and heat shock response by TNF-α and LT- α, including destroy the cell of virus infection and strengthen the antibacterial action of granulocyte.Also, it has proven that, in embryo development procedure, TNF-α and LT- α are an important components, to promote the organ of periphery lymphatic system to occur, such as lymph node, spleen and Peyer spots.TNF-α and LT- α expression are out of control to be played an important role in autoimmune disease such as rheumatoid arthritis, and the generation of inflammatory bowel disease such as Crohn diseases and multiple sclerosis (MS).

TNF-α and LT- α are played a role by TNF acceptors, the mediation of tumor necrosis factor receptor I (TNFRI) and tumor necrosis factor receptor I I (TNFRII).Two kinds of TNF acceptors and TNF-α and LT- α combination all have high-affinity, and two kinds of acceptors are present in all cell types in addition to erythrocyte.The deletion analysis of TNFRI C-terminal intracellular region has shown that it has a death domain, and participating in signal transduction process causes apoptosis.TNFRI death domain can be acted on other multi-signal adaptor molecules, including TRADD and RIP.TNFRII is more present in the cell of endothelial cell and hematopoietic cell system.TNFRII soluble form is identified in human urine, is 30kDa and 45kDa albumen.These proteolytic cleavages of soluble TNF acceptor albumen from membrane-bound receptor, are demonstrated by TNF inhibition.It is worth noting that, the expression of the stimulants also inducing soluble TNF acceptors of many induction TNF-αs expression, this shows that soluble recepter may play effect in regulation TNF activity.Especially, it may be played a role during the disease such as psoriasis for the patient that TNFRI and TNFRII crosses measure feature in treatment with TNF-α.At present, psoriasis is 2-3% or so (Nickoloff et al.J Clin Invest.113 in global infection rate：1664-1675,2004).Psoriatic does not only exist impaired etc the cutaneous lesions of itching and face, and 10-30% patient also has psoriatic arthritis with onychodystrophy.Therefore, psoriasis is not only a kind of skin disease, while it has an effect on many daily lifes, the use of such as hand, walk, sleep and sexual life.It is reported that in fact, at least 30% psoriatic intends to commit suiside (Nickoloff et al., supra, 2004).Other scytitises with the too high feature of TNF-α level include behcet's disease, bullous dermatitis, eczema, mycotic infection, leprosy, neutrophilic dermatitis, pityriasis (or pityriasis rosea), tinea nigra (or tinea nigra), pityriasis rubra pilaris, systemic loupus erythematosus, systemic many blood vessels and toxic epidermal necrolysis (Evereklioglu Expert Opin Pharmacother5 (2)：317-28,2004；Lipozecic et al.Acta Dermatovenerol Croat12(1)：35-41,2004；Mche et al..Ann Dermatol Venereol 129(12)：1374-9,2002；Teo et al..Microbes Infect4(11)：1193-202,2002).May be by being caused using other treatments in addition, TNF-α level is too high.For example, using the patient of Aldara emulsifiable pastes (imiquimod) be likely to form skin below react, including erythema, burn into ulcer, peeling, fish scale, drying, formed pimple, crust, skin diffusate.

People OX40 (A member of the TNF receptor family 4, TNFSF4) is 50kDa transmembrane protein, is mainly expressed in the CD4+T cell surfaces of activation.OX40 is a costimulator, participates in the immune response that T cell is relied on, the i.e. activation of T cell and propagation, by the generation of effector induced t cell cell factor, the generation of memory T cell, and hinders the tolerance of periphery T cell in vivo.OX40 induced expressions in a few hours or a couple of days after CD28 signals are started.It is reported that either in the height of immune response, or on memory T cell is produced, OX40 has played effect with the interaction of its part in amplification T cell number.When lacking CD28, the OX40-OX40L interactions also propagation of mediate T cell and IL-2 generation.But the OX40 T cell deficients of activation are extremely sensitive to apoptosis, even if IL-2 generation, cell division and expansion are all relatively normal.It is reported that, OX40 level or OX40-OX40L interaction are handled during immune response to being conducive in the disease for the treatment of T cell mediation, especially anaphylaxis, inflammatory and autoimmune disease.Recently, several research groups are by blocking OX40-OX40L interaction to reduce the clinical sign of autoimmune disease animal model.

BAFF (being also known Tumor necrosis factor ligand superfamily member 13B, TNFSF13B) is an II type membrane glycoprotein containing 285 amino acid.BAFF is expressed by B cell, T cell, dendritic cells, macrophage and neutrophil leucocyte.BAFF is a B cell survival factors, mainly promotes the propagation of activating B cell, immunoglobulin and IgD⁺The connection of B cell, and immunoglobulin secretion type cell survival, and participate in the maturation of B cell.This shows that BAFF is an important regulatory factor in humoral immune reaction.Research shows with BAFF processing B cell original-survival oncogene can be caused to include Bc1-xL, Bc1-2 and Mc1-1 expression.Because BAFF is a B cell survival factors, therefore its degraded can promote the survival of autoreaction B cell and the generation of autoimmune disease.In addition, also detecting that BAFF levels are raised in the patient of autoimmune disease, in the joint for being included in rheumatoid arthritis (RA) and inflammatory arthritis patient, the BAFF levels of its synovia are higher than the level in serum.BAFF is useful in the bioprocess of regulation B cell, T cell, dendritic cells, macrophage and Neutrophil-mediated, especially on activation BAFFR, such as increases the propagation of bone-marrow-derived lymphocyte, activates and survive.Especially, BAFF can be used for treating immune deficiency, such as B lymphocyte proliferation, activate and insufficient patient of surviving, or typically variation immune deficiency (CVID), or IgA defects patient.BAFF can be used for increasing the generation of antibody during vaccine inoculation.In addition, BAFF is connected into radionuclide available for targeted therapy and B cell malignant tumor is killed.

Trk C (NGFR) is also an i.e. TNFRSF16 of A member of the TNF receptor family 16.NGFR is an I type memebrane protein, is the signal peptide containing 28 amino acid by the glycoprotein of 427 Amino acid synthesis.NGFR is combined with all neurotrophic factors all has identical affinity, but can reach higher binding affinity with combining for NGFR by TrkA, B, C.Ligand binding can promote survival or the apoptosis of neuron in NGFR.Effect of the neurotrophic factor to cell needs a compound to act between NGFR acceptors and Trk A, B, C acceptors, and the compound is not completely understood also.But, in the neuron of NGFR defects, apoptosis caused by NGF processing neurons is not observed, and Trk A mainly suppress NGFR apoptosis activity.It is more complicated, promote apoptosis and anti-apoptotic by way of all being produced by NGFR signal inductions, and dependent on cell type and functional status.There are a variety of possible clinical practices on neurological disorder in NGFR, including Alzheimer diseases, Parkinson's, neuromuscular and motoneuron disorders, multiple sclerosis, cerebral paralysis, diabetic neuropathy and treatment pain, because the interaction of Trk A and NGFR in sensory neuron participates in the development of chronic ache.Soluble NGFR can be used for suppressing the growth of breast cancer and other tumours, because NGF and other NGFR parts are mitogens.

FasL (FasL or tnf ligand superfamily member 6, TNFSF6) is an II type memebrane protein containing 281 amino acid.FasL also serves as a soluble protein presence, and the proteolysis cutting or alternative splicing by ECD are produced.FasL activity form is a homotrimer.FasL participates in regulation apoptosis (apoptosis), immunologic balance and immune privilege and tumor cell survival.Initial experiment shows that the CD4+T cells of activation induce dissolved cell activity in the cell that Fas is expressed.Hereafter, FasL has been cloned, and has shown that FasL is apoptosis-induced by the interaction with Fas.FasL is incorporated into its acceptor Fas, causes the assembling of dead inducement signal compound (DISC), so as to originate apoptotic signal cascade.DISC includes related death domain (FADD) albumen of Fas, raises and activates caspase8 and 10, so as to originate caspase cascades and Apoptosis.FasL played an important role in normal immunologic balance, and systemic autoimmune disease occurs in the animal model of FasL defects.Have shown that FasL participates in the apoptosis of 3 types：The T cell of activation is removed at the end of immune response；The cell or tumour cell of virus infection are killed by cytolytic T lymphocyte or NK；And at eyes and testis, inflammatory cell is killed by non-lymphocyte.In addition, FasL expression can also promote the inflammatory reaction of Neutrophil-mediated by neutrophilia chemotactic activity.In addition, FasL also participates in erythroid differentiation, the balance and the stress reaction of cell of the generation such as eyes and skin of blood vessel.

The biological effect factor acts through protein and their own protein-bonded interaction and played, it means that TNF superfamilies and GAP-associated protein GAP and their own part or acceptor have the significant potentiality as the treatment factor for adjusting physiological processes.However, the small change of molecule such as one-level, two grades, three or four structure and co- or rear-translation modification pattern may be to the activity of albumen, secretion, antigenicity and remove and produce significant impact.Therefore, with specific one-level, two grades, structure or the issuable albumen of assembling after three or four structure, or common translation or translation, it assigns unique or significant useful properties.Therefore, it is necessary to assess the physiology characteristic of the albumen under different Production conditions to determine whether that it has useful physiologic character or other pharmacological characteristics.

So far, problem be to be carried out in the cell originated with the species of the remote edge of people's evolution available commercially as the production of protein, cell such as bacterium, yeast, fungi, and insect.These cell marking proteins lack glycosylation or presentation different from the glycosylation repertoire of people's cell and it substantially have impact on their applications clinically.For example, the protein expressed in yeast or fungal systems such as aspergillus has high density mannose, albumen (Herscovics et al.FASEB J 7 invalid in the treatment are caused：540-550,1993).

Even in non-human mammal expression system such as Chinese hamster ovary (CHO) cell, it has proved that there is marked difference compared with people's cell on glycosylation pattern.For example, most of mammals, including rodent, enzyme (α 1,3) galactosyl transferase is expressed, it produces Gal (α 1,3)-Gal (β Isosorbide-5-Nitraes)-GlcNAc oligosaccharides on glycoprotein.But in people, ape and Old World Monkeys, the expression of the enzyme is inactivated by the frameshift mutation of gene.(Larsen et al.J Biol Chem265：7055-7061, although 1990) most of Chinese hamster ovary celI systems synthesize for recombinant protein, such as Dux-B11, (the α 1 of inactivation gene expression, 3) galactosyl transferase, they still lack feature (α 2,6) sialyltransferase for the synthesis of present in human cell (α 2,6)-connection terminal sialic acid.Further, there is sialic acid motif in the Chinese hamster ovary celI of expression glycoprotein to tend to decompose (Gramer et al.Biotechnology13 (7) by the endogenous sialidase of Chinese hamster ovary celI：692-8,1995).

As a result, the albumen produced by these inhuman expression systems shows the physics and chemistry different from people's cell derived protein and pharmacological characteristics such as half-life period, antigenicity, stability and feature effect.

The progress of nearest stem cells technology substantially enhances the potentiality that such as transplantation treatment, drug screening, toxicologic study and functional genomics are applied to using stem cell.However, stem cell routinely maintains in the culture medium including non-human protein's matter, due to the possibility that inhuman infectious substance pollutes, therefore clinical practice is not suitable for.Further, the stem cell culture in inhuman derivative medium may cause to introduce inhuman carbohydrate portions therefore endanger graft application.(Martin et al Nature Medicine 11(2)：228-232,2005).Therefore, use of the special people's derived protein in the maintenance and/or differentiation of stem cell will improve the introducing of allogene protein and increase the clinical practice of stem cell.

Correspondingly, it is necessary development protein and their acceptor, it has special desired physics and chemistry and pharmacological characteristic for diagnosis, prevention, treatment, nutrition and/or research application, and is used for clinical, business and research application the invention provides TNF superfamilies and associated protein is belonged to.

Summary of the invention

Throughout the specification, unless stated otherwise, term " containing " or its variant for example " have " or "comprising", it will accordingly be understood that to imply the group of the group or entirety that include the key element or whole or key element, but be not excluded for any other key element or whole.

Nucleotides and amino acid sequence are expressed as sequence identification number (SEQ ID NO：).These SEQ IDNO：Sequence identification number is corresponded in number<400>1(SEQ ID NO：1),<400>2(SEQ IDNO：2), the rest may be inferred.The summary info of sequence identification number is shown in table 1.Sequence list is provided after claim.

The present invention broadly relates to belong to TNF superfamilies or the protein or its chimeric molecule of the separation related to the family with physical and chemical parameter feature, wherein described character representation, the basis for being associated with the pharmacological characteristics of one or more characteristics or forming the pharmacological characteristics of one or more characteristics.The protein or its chimeric molecule of the particularly separation that the present invention is provided are selected from TNF-α, TNF-α-Fc, LT- α, LT- α-Fc, TNFRI, TNFRI-Fc, TNFRII, TNFRII-Fc, OX40, OX40-Fc, BAFF, BAFF-Fc, NGFR, NGFR-Fc, FasL, FasL-Fc, and it, which has, includes the physicochemical characteristicses { [P of multiple measurable physical and chemical parameters_x]₁、[P_x]₂、...[P_x]_n, wherein P_xRepresent measurable physical and chemical parameter and " n " be >=1 integer, wherein between and be included in [P_x]₁To [P_x]_nBetween parameter be respectively different measurable physical and chemical parameters, the value of any of which or more than one measurable physicochemical property represents, is associated with the pharmacological characteristics T of a characteristic_yBasis or series of features pharmacological characteristics { [T_y]₁、[T_y]₂、....[T_y]_mOr formed a characteristic pharmacological characteristics T_yBasis or series of features pharmacological characteristics { [T_y]₁、[T_y]₂、....[T_y]_mBasis, wherein T_yThe pharmacological characteristics and m for representing a characteristic are >=1 integers, wherein [T_y]₁To [T_y]_mFor different pharmacological characteristics.

Here term " characteristic " is related to protein or the pharmacological characteristics of its chimeric molecule, refer in the present invention protein or its chimeric molecule one or more be different from further feature physicochemical property pharmacological characteristics.Specifically, the characteristic of same protein of one or more pharmacological characteristics of protein isolate or its chimeric molecule from being produced in prokaryotic or low eukaryotic or even inhuman higher eucaryotic cells is different or has relevant difference.In other specific embodiment, help to realize a certain function as the albumen of the separation of target or the pharmacological characteristic of its chimeric molecule.Here term " measurable physical and chemical parameter " or Px represents protein isolate or one or more measurable characteristics of its chimeric molecule.In one embodiment of the invention, as the albumen of the separation of target or measurable physicochemical property of its chimeric molecule or contribute to or generate pharmacological characteristics Ty.

The protein or chimeric molecule of the separation of the present invention have physical and chemical parameter (P_x), the parameter is used to intactly define protein molecule protein molecule or chimeric molecule.The physical and chemical parameter can be selected from：Apparent molecular weight (P₁), isoelectric point (pI) (P₂), isoform number (P₃), the relative intensity (P of different isoform number₄), carbohydrate percetage by weight (P₅), the actual measurement molecular weight (P after N- connection oligosaccharides deglycosylations₆), N- connections and O- connections oligosaccharides deglycosylation after actual measurement molecular weight (P₇), the percentage (P of acid contents of monosaccharides₈), contents of monosaccharides (P₉), sialic acid content (P₁₀), sulfate and phosphate content (P₁₁)、Ser/Thr：GalNAc ratios (P₁₂), the neutral percentage (P of N- connection oligosaccharides₁₃), the acid percentage (P of N- connection oligosaccharides₁₄), the neutral percentage (P of O- connection oligosaccharides₁₅), the acid percentage (P of O- connection oligosaccharides₁₆), the ratio (P of N- connection oligosaccharides₁₇), the ratio (P of O- connection oligosaccharides₁₈), the structure (P of N- connection oligosaccharide ingredients₁₉), the structure (P of O- connection oligosaccharide ingredients₂₀), the position of N- connection oligosaccharides and composition (P₂₁), the position of O- connection oligosaccharides and composition (P₂₂), common translation modification (P₂₃), posttranslational modification (P₂₄), acylated (P₂₅), acetylation (P₂₆), amidatioon (P₂₇), deamidation (P₂₈), biotinylation (P₂₉), carbamylation (P₃₀), carboxylation (P₃₁), decarboxylation (P₃₂), disulfide formation (P₃₃), fatty-acylation (P₃₄), myristoylation (P₃₅), palmitoylation (P₃₆), octadecane be acylated (P₃₇), formylated (P₃₈), saccharification (P₃₉), glycosylation (P₄₀), glycophosphatidyl inositol grappling (P₄₁), hydroxylating (P₄₂), the combination (P of selenocysteine₄₃), lipid (P₄₄), the addition (P of lipoic acid₄₅), methylate (P₄₆), N or C-terminal closing (P₄₇), N or C-terminal remove (P₄₈), nitrification (P₄₉), methionine oxidized (P₅₀), phosphorylation (P₅₁), protease digestion (P₅₂), prenylation (P₅₃), farnesylation (P₅₄), Mang ox base (P₅₅), phosphopyridoxal pyridoxal phosphate addition (P₅₆), sialylated (P₅₇), asialoglycoprotein (P₅₈), sulfation (P₅₉), ubiquitination (P_6o), the addition (P of ubiquitin sample molecule₆₁), primary structure (P₆₂), secondary structure (P₆₃), tertiary structure (P₆₄), quaternary structure (P₆₅), chemical stability (P₆₆), heat endurance (P₆₇).The feature of these parameters is provided in table 2.

In a particular embodiment, to next or multiple physicochemical property (P_x) and pharmacological characteristics (T_y) characterize the present invention TNF-α feature, it includes：

- apparent molecular weight (P₁) it is about 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, in a specific embodiment, for 10-30kDa；

-pI(P₂) scope is about 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 4-8.5 in a specific embodiment；

- it there are about 2 to 50 isoform (P₃), such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, in a specific embodiment, it is 10-40 isoform；

- carbohydrate percetage by weight (P₅) it is about 1 to 99%, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, in a specific embodiment, for 0-10%；

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) it is about 8 to 30kDa；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 8 to 25kDa, it is 10 to 20kDa in a specific embodiment；

- immune response feature (T₁₃) it is different from the humanTNF-α expressed in nonhuman cells' system, in a specific embodiment, when the protein concentration of the EL I SA kit measurements TNF-α of the present invention with the humanTNF-α expressed in containing nonhuman cells' system, the protein concentration of TNF-α of the present invention is underestimated.

In a particular embodiment, to next or multiple physicochemical property (P_x) and pharmacological characteristics (T_y) characterize the present invention LT- α feature：

- apparent molecular weight (P₁) it is about 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, in a specific embodiment, 32kDa is arrived for 15；

-pI(P₂) scope is about 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 5 to 11 in a specific embodiment；

- it there are about 2 to 100 isoform (P₃), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 isoforms, in a specific embodiment, for 7-33 isoform；

- carbohydrate percetage by weight (P₅) it is about 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, in a specific embodiment, for 0 to 42%；

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) it is about 10 to 30kDa, it is 12 to 25kDa in a particular embodiment；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 10 to 25kDa, it is 12 to 23kDa in a specific embodiment；

- immune response feature (T₁₃) it is different from the people LT- α expressed in nonhuman cells' system, in a specific embodiment, with the LT- α of the kit measurement present invention of the ELI SA comprising the people LT- α expressed in nonhuman cells' system protein concentration, LT- α of the invention protein concentration is underestimated.

In a particular embodiment, to next or multiple physicochemical property (P_x) and pharmacological characteristics (T_y) characterize the present invention TNFRI-Fc feature, it includes：

- apparent molecular weight (P₁) it is about 5 to 120, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, in a specific embodiment, 75kDa is arrived for 45；

-pI(P₂) scope is about 2 to 12, such as 2,3,4,5,6,7,8,9,10,11,12, it is 5.5-9.5 in a specific embodiment；

- it there are about 2 to 20 isoform (P₃), such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 isoforms are 8-16 isoform in a specific embodiment；

- carbohydrate percetage by weight (P₅) it is about 10 to 90%, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90%, in a specific embodiment, for 0-36%；

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) it is about 35 to 65kDa, it is 36 to 60kDa in a specific embodiment；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 35 to 65kDa, it is 36 to 60kDa in a specific embodiment；

Percentage (the P of-acidity contents of monosaccharides₈) it is about 0-50%, such as 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50, in a specific embodiment, it is 0-10%；

- contents of monosaccharides (P₉) and sialic acid content (P₁₀), during with GalNAc as standard, for 1: 0.1-8 trehalose, 1: 7-27 GlcNAc, 1: 1-14 galactolipin, 1: 2-17 mannose and 1: 0-3 NeuNAc, in a specific embodiment, the trehalose for being 1: 1-4.5,1: 10-18 GlcNAc, 1: 3-9 galactolipin, 1: 4-11 mannose and 1: 0.1-2 NeuNAc；During with 3 times of mannoses as standard, for 3: 0.01-3 trehalose, 3: 0.01-3 GalNAc, 3: 1-17 GlcNAc, 3: 0.1-5 galactolipin and 3: 0-3 NeuNAc, in a specific embodiment, for 3: 0.1-1.5 trehalose, 3: 0.1-1 GalNAc, 3: 3-11 GlcNAc, 3: 1-2.5 galactolipin and 3: 0-2 NeuNAc；

- sulphates content (P₁₁), during with GalNac as standard, the sulfate for being 1: 0.1-21, in a specific embodiment, the sulfate for being 1: 1.5-14；During with 3 times of mannoses as standard, the sulfate for being 3: 0.1-6, in a specific embodiment, the sulfate for being 3: 0.5-4；

- sulfation (P₅₉) it is expressed as the percentage of contents of monosaccharides in molecule, for 0-50%, such as 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50, in a specific embodiment, it is 10-16%；

Neutral percentage (the P of-N- connection oligosaccharides₁₃) it is about 30 to 100%, such as 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, in a specific embodiment, for 80 to 100%, in further specific embodiment, for 94 to 97%；

Acid percentage (the P of-N- connection oligosaccharides₁₄) it is about 0 to 50%, such as 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50%, in a specific embodiment, for 0 to 20%, it is 3 to 6% in further specific embodiment；

Neutral percentage (the P of-O- connection oligosaccharides₁₅) it is about 24 to 67%, such as 24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67%, in a specific embodiment, for 29 to 62%, it is 34 to 57% in further specific embodiment；

Acid percentage (the P of-O- connection oligosaccharides₁₆) it is about 10 to 80%, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80%, in a specific embodiment, for 38 to 71%, in further specific embodiment, for 43 to 66%；

Position (the P of-N- connection oligosaccharides₂₁) include N-299 (being numbered from the section start of signal sequence), give after PNGase, identified by PMF.

In a particular embodiment, to next or multiple physicochemical property (P_x) and pharmacological characteristics (T_y) characterize the present invention TNFRII-Fc feature, it includes：

- apparent molecular weight (P₁) it is about 10 to 150, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, in a specific embodiment, 118kDa is arrived for 46；

-pI(P₂) scope is about 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 4 to 10 in a specific embodiment；

- it there are about 2 to 52 isoform (P₃), such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52, in a specific embodiment, it is 10 to 40 isoforms；

- carbohydrate percetage by weight (P₅) it is about 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, in a specific embodiment, for 0 to 56%；

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) it is about 40 to 100kDa, it is 46 to 87kDa in a specific embodiment；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 40 to 95kDa, it is 42 to 80kDa in a specific embodiment；

Percentage (the P of-acidity contents of monosaccharides₈) it is about 0-50%, such as 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50, in a specific embodiment, it is 1 to 10%；

- contents of monosaccharides (P₉) and sialic acid content (P₁₀), during with GalNAc as standard, the trehalose for being 1: 0.01-3,1: 0.1-5 GlcNAc, 1: 0.1-3 galactolipin, 1: 0.1-3 mannose and 1: 0.01-3 NeuNAc；In a specific embodiment, the trehalose for being 1: 0.01-2,1: 0.1-3 GlcNAc, 1: 0.1-2 galactolipin, 1: 0.1-2 mannose and 1: 0.01-2 NeuNAc；During with 3 times of mannoses as standard, for 3: 0.01-3 trehalose, 3: 1-17 GalNAc, 3: 2-32 GlcNAc, 3: 1-9 galactolipin and 3: 0.1-3 NeuNAc, in a specific embodiment, for 3: 0.1-2 trehalose, 3: 3-11 GalNAc, 3: 5-21 GlcNAc, 3: 3-6 galactolipin and 3: 0.1-2 NeuNAc；

- sulphates content (P₁₁), during with GalNac as standard, the sulfate for being 1: 0.1-6, in a specific embodiment, the sulfate for being 1: 1-4；During with 3 times of mannoses as standard, the sulfate for being 3: 4-29, in a specific embodiment, the sulfate for being 3: 9-19；

- sulfation (P₅₉) it is expressed as the percentage of contents of monosaccharides in molecule, for 10-90%, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90%, in a specific embodiment, for 27 to 41%；

Neutral percentage (the P of-N- connection oligosaccharides₁₃) it is about 10 to 100%, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, in a specific embodiment, for 69 to 89%, in further specific embodiment, for 74 to 84%；

Acid percentage (the P of-N- connection oligosaccharides₁₄) it is about 0 to 80%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80%, in a specific embodiment, for 11 to 31%, in further specific embodiment, for 16 to 26%；

Neutral percentage (the P of-O- connection oligosaccharides₁₅) it is about 5 to 90%, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, in a specific embodiment, for 17 to 54%, in further specific embodiment, for 22 to 49%；

Acid percentage (the P of-O- connection oligosaccharides₁₆) it is about 5 to 99%, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, in a specific embodiment, for 46 to 83%, in further specific embodiment, for 51 to 78%；

The structure of-one or more N- glycan is listed in (P in the ratio of the N- of table 37 (a) connection oligosaccharides₁₉)；

The structure of-one or more O- glycan is listed in (P in the ratio of the O- of table 37 (b) connection oligosaccharides₂₀)；

- bioactivity is distinguished in the people TNFRII-Fc expressed in nonhuman cells' system, and in a specific embodiment, TNFRII-Fc of the present invention neutralizes the cytotoxicity (T of TNF-α induction in L-929 cells₃₀) people TNFRII-Fc of the ability than being expressed in E.coli cell ability it is high 8-18 times.

In a particular embodiment, to next or multiple physicochemical property (P_x) and pharmacological characteristics (T_y) characterize the present invention OX40-Fc feature, it includes：

- apparent molecular weight (P₁) it is about 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, in a specific embodiment, 75kDa is arrived for 46；

-pI(P₂) scope is about 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 4 to 9 in a specific embodiment；

- it there are about 2 to 50 isoform (P₃), such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, in a specific embodiment, it is 8-16 isoform；

- carbohydrate percetage by weight (P₅) it is about 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, in a specific embodiment, for 0 to 36%；

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) it is about 40 to 75kDa, it is 44 to 72kDa in a specific embodiment；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 38 to 75kDa, it is 41 to 70kDa in a specific embodiment；

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) it is about 46 to 65kDa；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 46 to 65kDa；

- contents of monosaccharides (P₉) and sialic acid content (P₁₀), during with GalNAc as standard, the trehalose for being 1: 0.01-3,1: 1-4 GlcNAc, 1: 0.1-3 galactolipin, 1: 0.1-3 mannose and 1: 0-3 NeuNAc；In a specific embodiment, the trehalose for being 1: 0.1-1,1: 2-3 GlcNAc, 1: 0.5-2 galactolipin, 1: 0.5-1 mannose and 1: 0-2 NeuNAc；During with 3 times of mannoses as standard, for 3: 0.1-3 trehalose, 3: 1-7 GalNAc, 3: 3-15 GlcNAc, 3: 2-9 galactolipin and 3: 0-3 NeuNAc, in a specific embodiment, for 3: 0.5-2 trehalose, 3: 3-5 GalNAc, 3: 6-10 G l cNAc, 3: 4-5 galactolipin and 3: 0-2 NeuNAc；

- sialic acid content (P₁₀) it is expressed as the percentage of contents of monosaccharides in molecule, about 0 to 50%, such as 0,1st, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50%, in a specific embodiment, it is 0-10%；

- sulphates content (P₁₁), during with GalNac as standard, the sulfate for being 1: 0-3, in a specific embodiment, the sulfate for being 1: 0.30-2；During with 3 times of mannoses as standard, the sulfate for being 3: 0.1-7, in further specific embodiment, the sulfate for being 3: 1-5；

- sulfation (P₅₉) it is expressed as the percentage of contents of monosaccharides in molecule, for 0-50%, such as 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50, in a specific embodiment, it is 9 to 15%；

Neutral percentage (the P of-N- connection oligosaccharides₁₃) it is about 69 to 100%, it is 74 to 100% in a specific embodiment, is 79 to 95% in further specific embodiment；

Acid percentage (the P of-N- connection oligosaccharides₁₄) it is about 0 to 31%, it is 0 to 26% in a specific embodiment, is 5 to 21% in further specific embodiment；

Neutral percentage (the P of-O- connection oligosaccharides₁₅) it is about 20 to 100%, it is 40 to 90% in a specific embodiment, is 45 to 80% in further specific embodiment；

Acid percentage (the P of-O- connection oligosaccharides₁₆) it is about 0 to 80%, it is 10 to 60% in a specific embodiment, is 20 to 55% in further specific embodiment；

Position (the P of-N- connection oligosaccharides₂₁) include N-160 to N-298 (being numbered from the section start of signal sequence), give after PNGase, identified by PMF.

In a particular embodiment, to next or multiple physicochemical property (P_x) and pharmacological characteristics (T_y) characterize the present invention BAFF feature, it includes：

- apparent molecular weight (P₁) it is about 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, in a specific embodiment, 22kDa is arrived for 10；

-p I(P₂) scope is about 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 4 to 8 in a specific embodiment；

- it there are about 2 to 50 isoform (P₃), such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, in a specific embodiment, it is 5 to 10 isoforms.

- carbohydrate percetage by weight (P₅) it is about 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, in a specific embodiment, for 0 to 25%.

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) it is about 8 to 22kDa, it is 10 to 22kDa in a specific embodiment；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 8 to 22kDa, it is 10 to 22kDa in a specific embodiment；

- bioactivity is distinguished in the people BAFF expressed in nonhuman cells' system, in a specific embodiment, BAFF induction RPMI8226 cell propagation T of the present invention₃₂Ability be 1.1-2.4 times of the people BAFF expressed in E.coli cells.

In a particular embodiment, to next or multiple physicochemical property (P_x) and pharmacological characteristics (T_y) characterize the present invention NGFR-Fc feature, it includes：

- apparent molecular weight (P₁) it is about 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, in a specific embodiment, 105kDa is arrived for 55；

-pI(P₂) scope is about 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 3: 6 in a specific embodiment；

- it there are about 2 to 50 isoform (P₃), such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, in a specific embodiment, it is 8 to 16 isoforms；

- carbohydrate percetage by weight (P₅) it is about 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, in a specific embodiment, for 11 to 53%.

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) for 45 between 100kDa, be 48 to arrive 90Da in a specific embodiment；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 45 to 95kDa, it is 48 to 85kDa in a specific embodiment；

In a particular embodiment, to next or multiple physicochemical property (P_x) and pharmacological characteristics (T_y) characterize the present invention Fa s parts characteristic, it includes：

- apparent molecular weight (P₁) it is about 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, in a specific embodiment, 35kDa is arrived for 15；

-pI(P₂) scope is about 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14；

- it there are about 2 to 50 isoform (P₃), such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms；

- carbohydrate percetage by weight (P₅) it is about 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, in a specific embodiment, for 0 to 51%.

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) for 10 between 28kDa, be 12 to arrive 21kDa in a specific embodiment；

Position (the P of-N- connection oligosaccharides₂₁) include N-184 (being numbered from the section start of signal sequence), give after PNGase, identified by PMF.

In a detailed embodiment, present invention contemplates TNF superfamilies or the protein or its chimeric molecule of relative separation, it is selected from TNF-α, TNF-α-Fc, LT- α, LT- α-Fc, TNFRI, TNFRI-Fc, TNFRII, TNFRII-Fc, OX40, OX40-Fc, BAFF, BAFF-Fc, NGFR, NGFR-Fc, Fa s parts, FasL-Fc.The protein or its chimeric molecule of the separation of the present invention include a series of measurable pharmacological parameters, selected from containing or comprising therapeutic effect (T₁), dose therapeutically effective (TCID₅₀)(T₂), bioavilability (T₃), from the time (T for being administered into maintaining treatment level₄), absorption rate (T₅), discharge rate (T₆), special activity (T₇), heat endurance (T₈), lyophilized stability (T₉), serum/plasma stability (T₁₀), serum half-life (T₁₁), the solubility (T in blood flow₁₂), immune response feature (T₁₃), immunogenicity (T₁₄), neutralizing antibody suppress (T_14A), side effect (T₁₅), receptor/ligand affinity (T₁₆), receptor/ligand activation (T₁₇), tissue or cell category specificity (T₁₈), penetration capacity (such as intestines, lung, blood-brain barrier, skin etc.) (T of biomembrane or barrier₁₉), generation blood vessel ability (T_19A), tissue resorption (T₂₀), degraded stability (T₂₁), freeze-thaw stability (T₂₂), protease stability (T₂₃), ubiquitin stability (T₂₄), administration reduce (T₂₅), mode of administration (T₂₆), the compatibility (T with other pharmaceutical excipients or carrier₂₇), the residual (T in organism or environment₂₈), preserve during stability (T₂₉), (T such as toxicity in organism or environment₃₀)。

In addition, the protein or chimeric molecule of the present invention can have different biological effect (T in different cell categories₃₁), including but not limited to people's primary cell, such as lymphocyte, red blood cell, retina cell, liver cell, neuron, horn cell, endothelial cell, endoderm cell, ectoderm cell, mesoblastema, epithelial cell, nephrocyte, liver cell, osteocyte, bone marrow cell, lymph node cells, dermal cell, fibroblast, T cell, B cell, thick liquid cell, NK, macrophage, bite neutrophil leucocyte, grain cell of Langerhan, BMDC, bite sour granulocyte, bite alkali granulocyte, mammary cell, small leaf cell, prostatic cell, pneumonocyte, esophageal cells, pancreatic cell, Beta cells (insulin secretory cell), angioblast, myocyte, elliptocyte (liver cell), mesenchymal cell, brain microvessel endothelial cells in vitro, astroglia, spongiocyte, a variety of stem cells include adult and embryonic stem cell, a variety of progenitor cells；With other people permanent, conversion or cancer cell systems.

Biological effect in cell includes multiplication effect (T₃₂), differentiation (T₃₃), apoptosis (T₃₄), the growth (T of cell size₃₅), cell factor adhesion (T₃₆), cell adhesion (T₃₇), cell amplification (T₃₈), cell mobility (T₃₉), migration and intrusion (T₄₀), chemotaxis (T₄₁), cell phagocytosis (T₄₂), signal transduction (T₄₃), rich protein to receptor/ligand (T₄₄), the activation (T of JAK/STAT approach₄₅), the activation (T of Ras-erk approach₄₆), the activation (T of AKT approach₄₇), the activation (T of PKC approach₄₈), the activation (T of PKA approach₄₉), src activation (T₅₀), fas activation (T₅₁), TNFR activation (T₅₂), NFkB activation (T₅₃), p 38MAPK activation (T₅₄), c-fos activation (T₅₅), secretion (T₅₆), acceptor caves in (T₅₇), acceptor reciprocation (T₅₈), the up-regulation of surface markers or downward (T₅₉), before FACS/change (T of other scattering signatures₆₀), the change (T of subgroup ratio₆₁), differential gene expression (T₆₂), meronecrosis (T₆₃), cell agglutination (T₆₄), cellular rejection (T₆₅) and heparin sulfate combination (T₆₆) and glycosylation structure combination (T₆₇) and chondroitin sulfate combination (T₆₈) and extracellular matrix combination (such as collagen, fibronectin) (T₆₉) and artificial material combination (such as support) (T₇₀) and carrier combination (T₇₁) and confactor combination (T₇₂), individually or the effect (T in the mixture containing other protein to stem cells hyperplasia, differentiation and/or self-renewing₇₃) etc..The feature of these characteristics is provided in table 3.

Invention further provides the chimeric molecule for including the albumen of separation or its segment, such as constant region (Fc) or framework region that the ectodomain of one membrane bound protein passes through one or more protein connexon combination human immunoglobulin(HIg)s.Such chimeric molecule also illustrates that into albumen-Fc herein.Include present invention contemplates the example of such albumen-Fc chimeras for example including TNF-α-Fc, LT- α-Fc, TNFRI-Fc, TNFRII-Fc, OX40-Fc, BAFF-Fc, NGFR-Fc, FasL-Fc.Such protein-Fc has the characteristic of measurable physical and chemical parameter, these personality presentations, the albumen-Fc of association separation one or more specific pharmacological characteristics.It is contemplated by the invention that other chimeric molecules include albumen or albumen-Fc or its segment, be connected with an aliphatic radical such as polyunsaturated fatty acid molecule.Here aliphatic radical can be with an amino acid residue in binding molecule skeleton a amino acid residue or side chain.

Invention further provides the chimeric molecule for including protein isolate or its segment, such as Fc or framework region that the ectodomain of one embrane-associated protein passes through one or more protein connexon combination human immunoglobulin(HIg)s.In other respects, the constant region (Fc) or framework region of mammalian immunoglobulin derive from and include primate, including the mankind, suede, chimpanzee and gorilla, domestic animal (such as ox, sheep, pig, horse, donkey), experimental animal (such as mouse, rat, cavy, hamster, rabbit), wild animal (such as rodent of pet (such as cat, dog) and capture, fox, deer, kangaroo).In a further embodiment, Fc or framework region are human immunoglobulin(HIg)s.Mammal is the mankind in a particular embodiment.Such chimeric molecule also illustrates that into albumen-Fc herein.Include albumen or albumen-Fc or its segment present invention contemplates other chimeric molecules, be connected with the aliphatic radical of a such as polyunsaturated fatty acid molecule.Here an amino acid residue in the possible binding molecule skeleton of aliphatic radical or an amino acid residue in side chain.Chimeric molecule in the present invention, including TNF-α-Fc, LT- α-Fc, TNFRI-Fc, TNFRII-Fc, OX40-Fc, BAFF-Fc, NGFR-Fc, FasL-Fc, it has the characteristic of measurable physical and chemical parameter, these personality presentations, the albumen-Fc of association separation one or more specific pharmacological characteristics.

Especially, the term " TNFRI-Fc " used here and " TNFRII-Fc " refer to, by the segment of the extracellular domain TNFR polypeptides (such as TNFRI or TNFRII) containing one or more TNFRI or TNFRII, directly or being connected in the constant region of human immunoglobulin(HIg) (Fc) or framework region or its segment through one or more protein connexons known in the art and merging to form chimeric protein with this.The segment of TNFR (TNFRI or TNFRII) polypeptide can be selected from SEQ ID NO：64th, 66,68,92,94,96, one or more of 98.Fc areas can be selected from IgG1 (examples：Basic such as SEQ ID NO：2, SEQ ID NO：4), IgG2 (examples：Basic such as SEQ ID NO：6), IgG3 (examples：Basic such as SEQ ID NO：8), IgG4 (examples：Basic such as SEQ ID NO：10), IgA1 (examples：Basic such as SEQ ID NO：12), IgA2 (examples：Basic such as SEQ ID NO：14), IgM (examples：Basic such as SEQ ID NO：16), IgE (examples：Basic such as SEQ ID NO：Or IgD (examples 18)：Basic such as SEQ ID NO：20) the Fc areas of people's isoform (isotype).In a particular embodiment, the Fc receptor binding domains or complement activation area in Fc areas can be modified by recombinating, compared with the amino acid sequence in Fc areas, include insertion, missing or the displacement of one or more amino acid.In addition, the receptor binding domain or complement activation area in Fc areas can be chemically modified, and in amino acid backbone or after any related common translation or translation on product, change its glycoforms, add or remove carbohydrate fraction, increase polyunsaturated fatty acid part or other parts based on fat.Fc areas can also exist in the form of truncation, and this is to be hydrolyzed what is obtained by the protease including papain, pepsin or other any site-specifics.Fc areas can promote chimeric protein to spontaneously form the polymer of dimer, tripolymer or higher level, the single phase ratio with equivalent, and they combine TNF-α molecule and to prevent it from being incorporated into the ability of cell bound receptor higher.Therefore, it is contemplated by the invention that " TNFRI-Fc polypeptides " and " TNFRI I-Fc polypeptides " be TNF-α activity antagonist.

" TNF " used here includes being related to TNF-α.

Therefore, the invention provides the polypeptide by nucleotide sequence coded separation, the sequence is selected from SEQ ID NO：27, 29, 31, 33, 35, 37, 39, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 127, 129, 131, 133, 135, 137, 139, 141, 143, 147, 149, 151, 153, 155, 157, 159, 163, 165, 167, 169, 171, 173, 175, 177, 179, 183, 185, 187, 189, or have the nucleotide sequence of at least 65% uniformity with above-mentioned sequence any of which one, or the sequence that can hybridize with any one in above-mentioned sequence or its complementary type under low stringency condition.

Another aspect provides the polypeptide of separation, the polypeptide is as coded by respectively correspond toing sequences of the respective mRNA of following sequences by cell processing montage, and the sequence is by SEQ IDNO：191,192,193 are constituted.

Another aspect provides the polypeptide of separation, its amino acid sequence included is selected from SEQ ID NO：28, 30, 32, 34, 36, 38, 40, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 128, 130, 132, 134, 136, 138, 140, 142, 144, 148, 150, 152, 154, 156, 158, 160, 164, 166, 168, 170, 172, 174, 176, 178, 180, 184, 186, 188, 190, or there is the amino acid sequence of at least 65% similitude with above-mentioned sequence any of which one.

Present invention further contemplates the pharmaceutical composition combined including at least a portion protein or its chimera, pharmacologically acceptable carrier, confactor and/or diluent.

In terms of primary structure, the invention provides the protein of separation or its chimera or its segment, its nucleotide coding sequence is selected from SEQ ID NO：27, 29, 31, 33, 35, 37, 39, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 127, 129, 131, 133, 135, 137, 139, 141, 143, 147, 149, 151, 153, 155, 157, 159, 163, 165, 167, 169, 171, 173, 175, 177, 179, 183, 185, 187, 189, or have the nucleotide sequence of at least 60% uniformity with above-mentioned sequence any of which one, or the sequence that can hybridize with any one in above-mentioned sequence or its complementary type in low stringency condition.

In addition, other aspects of the present invention provide the isolated nucleotide molecule of encoding proteins matter or its chimeric molecule or its functional area, comprising with SEQ ID NO：27, 29, 31, 33, 35, 37, 39, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 127, 129, 131, 133, 135, 137, 139, 141, 143, 147, 149, 151, 153, 155, 157, 159, 163, 165, 167, 169, 171, 173, 175, 177, 179, 183, 185, 187, 189 have at least 60% similitude, or after optimal comparison and/or can be with SEQ I D NO：27, 29, 31, 33, 35, 37, 39, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 127, 129, 131, 133, 135, 137, 139, 141, 143, 147, 149, 151, 153, 155, 157, 159, 163, 165, 167, 169, 171, 173, 175, 177, 179, 183, 185, 187, 189 or the nucleotide sequence that hybridizes under low stringency condition of their complementary type.

In a specific embodiment, present invention is generally directed to the nucleic acid molecule of separation, it has the nucleotide sequence for encoding following molecules, and the molecule is TNF superfamilies albumen or associated albumen or its chimeric molecule, and it is selected from：TNF-α, TNF-α-Fc, LT- α, LT- α-Fc, TNFRI, TNFRI-Fc, TNFRII, TNFRII-Fc, OX40, OX40-Fc, BAFF, BAFF-Fc, NGFR, NGFR-Fc, Fa s parts, Fa s part-Fc or its segment, substantially with SEQ IDNO：28, 30, 32, 34, 36, 38, 40, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 128, 130, 132, 134, 136, 138, 140, 142, 144, 148, 150, 152, 154, 156, 158, 160, 164, 166, 168, 170, 172, 174, 176, 178, 180, 184, 186, 188, one or more amino acid sequences shown in 190, or with SEQ ID NO：28, 30, 32, 34, 36, 38, 40, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 128, 130, 132, 134, 136, 138, 140, 142, 144, 148, 150, 152, 154, 156, 158, 160, 164, 166, 168, 170, 172, 174, 176, 178, 180, 184, 186, 188, one or more sequences after 190 optimal comparisons have the amino acid sequence of at least 60% similitude.

In other respects, the invention provides the nucleic acid molecule of separation, coding, which is selected from, belongs to TNF superfamilies or associated albumen or its chimeric molecule, aforementioned proteins are selected from TNF-α-Fc, LT- α-Fc, TNFRI-Fc, TNFRII-Fc, OX40-Fc, BAFF-Fc, NGFR-Fc, FasL-Fc or its segment, comprising selected from SEQ ID NO：31st, 33,35,45,47,49,51,63,65,67,91,93,95,97,129,131,151,153,155,165,167,185,187 nucleotide sequence, it directs or through the nucleotide sequence of well known encoding proteins matter connexon on one or more this areas, it is connected with constant (Fc) or framework region of encoding human immunoglobulin nucleotide sequence, constant (Fc) or framework region of described encoding human immunoglobulin nucleotides sequence are substantially such as SEQ ID NO：1st, 3,5,7,9,11,13,15,17 or 19 it is one or more shown in.In a specific embodiment, the nucleotide sequence of encoding proteins matter connexon is selected from coding IP, GSSNT, TRA or VDGIQWIP nucleotide sequence.

In other respects, the invention provides the protein of separation, the protein is the protein for belonging to TNF superfamilies or associated separation, it is selected from TNF-α-Fc, LT- α-Fc, TNFRI-Fc, TNFRII-Fc, OX40-Fc, BAFF-Fc, NGFR-Fc, FasL-Fc or its segment, and it, which includes to be selected from, has SEQ ID NO：32nd, 34,36,46,48,50,52,64,66,68,92,94,96,98,130,132,152,154,156,166,168,186,188 amino acid sequence, it directs or through well known protein connexon on one or more this areas, it is connected with constant (Fc) or framework region of human immunoglobulin(HIg) nucleotide sequence, constant (Fc) or framework region of described encoding human immunoglobulin nucleotide sequence are substantially such as SEQ ID NO：2nd, 4,6,8,10,12,14,16,18 or 20 it is one or more shown in.

Invention further provides the albumen of separation or its chimeric molecule or encode its nucleic acid molecule diagnosis, prevent, treatment, nutrition and/or research application in application.Especially it is noted that the invention provides treating or preventing disease on subject animal or alleviating the method for disease symptomses, described method includes giving the protein or its chimeric molecule of the separation of effective dose to the subject animal.In a specific embodiment, the present invention provides treatment method for the patient with following illnesss, the feature of the illness is that TNF-α level is too high, state of an illness aggravation or other too high related to TNF-α level, the pharmaceutical composition of chimeric molecule containing TNFRI and/or TNFRII and/or TNFRI or TNFRII of this method including giving the effective therapeutic dose of patient.In a specific embodiment, illness is selected from：Psoriasis, behcet's disease, bullous dermatitis, eczema, mycotic infection, leprosy, neutrophilic dermatitis, pityriasis (or pityriasis rosea), tinea nigra (or tinea nigra), pityriasis rubra pilaris, systemic loupus erythematosus, systemic many blood vessels and toxic epidermal necrolysis.In addition, disease disease can be as caused by using other medicines, for example, using Aldara emulsifiable pastes, including but not limited to：The erythema of skin, burn into ulcer, peeling, fish scale, drying, formed pimple, crust, diffusate.

Moreover, present invention contemplates be screened to have different diagnosis with the albumen or its chimeric molecule of the present invention, preventing, treatment, the small molecule of nutrition and/or research application effect.

Present invention further contemplates can be using protein isolate or its chimeric molecule as immunogene, to produce the antibody for treating and diagnosing.

Present invention further contemplates the culture medium for cultivating stem cell or related methods for the treatment of can be added using protein isolate or its chimeric molecule.

Present invention provides the purposes that people's source protein or its chimeric molecule are used as immunoassays or the standard protein of its kit.Present invention provides the method for determining people's cell expression people's albumen or its chimeric molecule level in biological agent.

Be used to diagnosing with certain composition present invention provides a protein or its chimeric molecule, prevent, treating, nutrition and/or research.Especially, the invention provides the preparation suitable for topical application, comprising TNFRI and/or TNFRII and/or chimeric TNFRI or TNFRII molecules, chimeric TNFRI or TNFRII molecules are included TNFRI or TNFRII molecules by being fused to directly or through one or more protein connexons on the Fc areas of antibody or its function homologue.In a specific embodiment, one or more TNFRI-Fc or TNFRII-Fc described here are contained in topical application preparation.

Table 1：Sequence identifier

Sequence identifier	Sequence
		SEQ ID N0：1	Human IgG1's Fc nucleotide sequences
SEQ ID NO：2	Human IgG1's Fc amino acid sequences
		SEQ ID NO：3	Human IgG1 Fc nucleotide sequences (variant)
SEQ ID NO：4	Human IgG1 Fc amino acid sequences (variant)
		SEQ ID NO：5	Human IgG2's Fc nucleotide sequences
SEQ ID NO：6	Human IgG2's Fc amino acid sequences
		SEQ ID NO：7	The Fc nucleotide sequences of human IgG 3
SEQ ID NO：8	The Fc amino acid sequences of human IgG 3
		SEQ ID NO：9	The Fc nucleotide sequences of human IgG 4
SEQ ID NO：10	The Fc amino acid sequences of human IgG 4
		SEQ ID NO：11	People's IgA1 Fc nucleotide sequences
SEQ ID NO：12	People's IgA1 Fc amino acid sequences
		SEQ ID NO：13	People's IgA2 Fc nucleotide sequences
SEQ ID NO：14	People's IgA2 Fc amino acid sequences
		SEQ ID NO：15	People's IgM Fc nucleotide sequences
SEQ ID NO：16	People's IgM Fc amino acid sequences
		SEQ ID NO：17	People's IgE Fc nucleotide sequences
SEQ ID NO：18	People's IgE Fc amino acid sequences
		SEQ ID NO：19	People's IgD Fc nucleotide sequences
SEQ ID NO：20	People's IgD Fc amino acid sequences
		SEQ ID NO：21	Human IgG1 Fc forward primers (being used for pIRESbleo XIP clones) (nucleotide sequence)
SEQ ID NO：22	Human IgG1 Fc reverse primers (being used for pIRESbleo XIP clones) (nucleotide sequence)
		SEQ ID NO：23	Human IgG1 Fc forward primers (being used for pIRESbleo GSSNT clones) (nucleotides sequence Row)
SEQ ID NO：24	Human IgG1 Fc reverse primers (being used for pIRESbleo GSSNT clones) (nucleotides sequence Row)
		SEQ ID NO：25	TNF-α forward primer (nucleotide sequence)
SEQ ID NO：26	TNF-α reverse primer (nucleotide sequence)
		SEQ ID NO：27	TNF-α nucleotide sequence (precursor peptide)
SEQ ID NO：28	TNF-α amino acid sequence (precursor peptide)
		SEQ ID NO：29	TNF-α nucleotide sequence (precursor peptide (variant))
SEQ ID NO：30	TNF-α amino acid sequence (precursor peptide (variant))
		SEQ ID NO：31	TNF-α nucleotide sequence (mature peptide)

Sequence identifier	Sequence
		SEQ ID NO：32	TNF-α amino acid sequence (mature peptide)
SEQ ID NO：33	TNF-α nucleotide sequence (precursor peptide+mature peptide)
		SEQ ID NO：34	TNF-α amino acid sequence (precursor peptide+mature peptide)
SEQ ID NO：35	TNF-α nucleotide sequence (precursor peptide (variant)+mature peptide)
		SEQ ID NO：36	TNF-α amino acid sequence (precursor peptide (variant)+mature peptide)
SEQ ID NO：37	Nucleotide sequence (the precursor peptide+mature peptide+GSSNT of TNF-α-Fc entire constructs Connexon IgG1 Fc)
		SEQ ID NO：38	Amino acid sequence (the precursor peptide+mature peptide+GSSNT of TNF-α-Fc entire constructs Connexon IgG1 Fc)
SEQ ID NO：39	TNF-α-Fc entire constructs nucleotide sequence (precursor peptide (variant)+mature peptide+ GSSNT connexon IgG1 Fc)
		SEQ ID NO：40	TNF-α-Fc entire constructs amino acid sequence (precursor peptide (variant)+mature peptide+ GSSNT connexon IgG1 Fc)
SEQ ID NO：41	LT- α forward primers (nucleotide sequence)
		SEQ ID NO：42	LT- α reverse primers (nucleotide sequence)
SEQ ID NO：43	LT- α nucleotide sequences (signal peptide)
		SEQ ID NO：44	LT- alpha amino acids sequence (signal peptide)
SEQ ID NO：45	LT- α nucleotide sequences (mature peptide)
		SEQ ID NO：46	LT- alpha amino acids sequence (mature peptide)
SEQ ID NO：47	LT- α nucleotide sequences (mature peptide (variant))
		SEQ ID NO：48	LT- alpha amino acids sequence (mature peptide (variant))
SEQ ID NO：49	LT- α nucleotide sequences (signal peptide+mature peptide)
		SEQ ID NO：50	LT- alpha amino acids sequence (signal peptide+mature peptide)
SEQ ID NO：51	LT- α nucleotide sequences (signal peptide+mature peptide (variant))
		SEQ ID NO：52	LT- alpha amino acids sequence (signal peptide+mature peptide (variant))
SEQ ID NO：53	(signal peptide+mature peptide+GSSNT connects the nucleotide sequence of LT- α-Fc entire constructs Meet sub- IgG1 Fc)
		SEQ ID NO：54	(signal peptide+mature peptide+GSSNT connects the amino acid sequence of LT- α-Fc entire constructs Meet sub- IgG1 Fc)
SEQ ID NO：55	LT- α-Fc entire constructs nucleotide sequence (signal peptide+mature peptide (variant)+ GSSNT connexon IgG1 Fc)
		SEQ ID NO：56	LT- α-Fc entire constructs amino acid sequence (signal peptide+mature peptide (variant)+ GSSNT connexon IgG1 Fc)
SEQ ID NO：57	TNFRI forward primers (nucleotide sequence)
		SEQ ID NO：58	TNFRI reverse primers (nucleotide sequence)
SEQ ID NO：59	TNFRI nucleotide sequences (signal peptide)
		SEQ ID NO：60	TNFRI amino acid sequences (signal peptide)
SEQ ID NO：61	TNFRI nucleotide sequences (signal peptide (variant))

Sequence identifier	Sequence
		SEQ ID NO：62	TNFRI amino acid sequences (signal peptide (variant))
SEQ ID NO：63	TNFRI nucleotide sequences (mature peptide)
		SEQ ID NO：64	TNFRI amino acid sequences (mature peptide)
SEQ ID NO：65	TNFRI nucleotide sequences (mature peptide) (variant)
		SEQ ID NO：66	TNFRI amino acid sequences (mature peptide) (variant)
SEQ ID NO：67	TNFRI nucleotide sequences (signal peptide+mature peptide)
		SEQ ID NO：68	TNFRI amino acid sequences (signal peptide+mature peptide)
SEQ ID NO：69	TNFRI-Fc nucleotide sequence (mature peptide+IP connexons+IgG1Fc)
		SEQ ID NO：70	TNFRI-Fc amino acid sequence (mature peptide+IP connexons+IgG1Fc)
SEQ ID NO：71	TNFRI-Fc nucleotide sequence (mature peptide (variant)+IP connexons+IgG1Fc)
		SEQ ID NO：72	TNFRI-Fc amino acid sequence (mature peptide (variant)+IP connexons+IgG1Fc)
SEQ ID NO：73	TNFRI-Fc nucleotide sequence (mature peptide+IP connexons+IgG1Fc (variant))
		SEQ ID NO：74	TNFRI-Fc amino acid sequence (mature peptide+IP connexons+IgG1Fc (variant))
SEQ ID NO：75	TNFRI-Fc nucleotide sequence (mature peptide (variant)+IP connexons+IgG1Fc (variant))
		SEQ ID NO：76	TNFRI-Fc amino acid sequence (mature peptide (variant)+IP connexons+IgG1Fc (variant))
SEQ ID NO：77	TNFRI-Fc nucleotide sequence (mature peptide+GSSNT connexons+IgG1Fc)
		SEQ ID NO：78	TNFRI-Fc amino acid sequence (mature peptide+GSSNT connexons+IgG1Fc)
SEQ ID NO：79	TNFRI-Fc nucleotide sequence (mature peptide (variant)+GSSNT connexons+IgG1   Fc)
		SEQ ID NO：80	TNFRI-Fc amino acid sequence (mature peptide (variant)+GSSNT connexons+IgG1   Fc)
SEQ ID NO：81	Nucleotide sequence (the signal peptide+mature peptide+IP connexons of TNFRI-Fc entire constructs   +IgG1Fc)
		SEQ ID NO：82	Amino acid sequence (the signal peptide+mature peptide+IP connexons of TNFRI-Fc entire constructs   +IgG1Fc)
SEQ ID NO：83	Nucleotide sequence (the signal peptide+mature peptide+IP connexons of TNFRI-Fc entire constructs + IgG1Fc (variant))
		SEQ ID NO：84	Amino acid sequence (the signal peptide+mature peptide+IP connexons of TNFRI-Fc entire constructs + IgG1Fc (variant))
SEQ ID NO：85	(signal peptide+mature peptide+GSSNT connects the nucleotide sequence of TNFRI-Fc entire constructs Meet son+IgG1Fc)
		SEQ ID NO：86	(signal peptide+mature peptide+GSSNT connects the amino acid sequence of TNFRI-Fc entire constructs Meet son+IgG1Fc)
SEQ ID NO：87	TNFRII forward primers (nucleotide sequence)
		SEQ ID NO：88	TNFRII reverse primers (nucleotide sequence)
SEQ ID NO：89	TNFRII nucleotide sequences (signal peptide)
		SEQ ID NO：90	TNFRII amino acid sequences (signal peptide)

Sequence identifier	Sequence
		SEQ ID NO：91	TNFRII nucleotide sequences (mature peptide)
SEQ ID NO：92	TNFRII amino acid sequences (mature peptide)
		SEQ ID NO：93	TNFRII nucleotide sequences (mature peptide) (variant)
SEQ ID NO：94	TNFRII amino acid sequences (mature peptide) (variant)
		SEQ ID NO：95	TNFRII nucleotide sequences (signal peptide+mature peptide)
SEQ ID NO：96	TNFRII amino acid sequences (signal peptide+mature peptide)
		SEQ ID NO：97	TNFRII nucleotide sequences (signal peptide+mature peptide (variant))
SEQ ID NO：98	TNFRII amino acid sequences (signal peptide+mature peptide (variant))
		SEQ ID NO：99	TNFRII-Fc nucleotide sequence (mature peptide+IP connexons+IgG1Fc)
SEQ ID NO：100	TNFRII-Fc amino acid sequence (mature peptide+IP connexons+IgG1Fc)
		SEQ ID NO：101	TNFRII-Fc nucleotide sequence (mature peptide (variant)+IP connexons+IgG1Fc)
SEQ ID NO：102	TNFRII-Fc amino acid sequence (mature peptide (variant)+IP connexons+IgG1Fc)
		SEQ ID NO：103	TNFRII-Fc nucleotide sequence (mature peptide+IP connexons+IgG1Fc (variant))
SEQ ID NO：104	TNFRII-Fc amino acid sequence (mature peptide+IP connexons+IgG1Fc (variant))
		SEQ ID NO：105	TNFRII-Fc nucleotide sequence (mature peptide (variant)+IP connexons+IgG1Fc (variant))
SEQ ID NO：106	TNFRII-Fc amino acid sequence (mature peptide (variant)+IP connexons+IgG1Fc (variant))
		SEQ ID NO：107	TNFRII-Fc nucleotide sequence (mature peptide+GSSNT connexons+IgG1Fc)
SEQ ID NO：108	TNFRII-Fc amino acid sequence (mature peptide+GSSNT connexons+IgG1Fc)
		SEQ ID NO：109	TNFRII-Fc nucleotide sequence (mature peptide (variant)+GSSNT connexons+IgG1   Fc)
SEQ ID NO：110	TNFRII-Fc amino acid sequence (mature peptide (variant)+GSSNT connexons+IgG1   Fc)
		SEQ ID NO：111	Nucleotide sequence (the signal peptide+mature peptide+IP connections of TNFRII-Fc entire constructs Son+IgG1Fc)
SEQ ID NO：112	Amino acid sequence (the signal peptide+mature peptide+IP connections of TNFRII-Fc entire constructs Son+IgG1Fc)
		SEQ ID NO：113	TNFRII-Fc entire constructs nucleotide sequence (signal peptide+mature peptide (variant)+ IP connexons+IgG1Fc)
SEQ ID NO：114	TNFRII-Fc entire constructs amino acid sequence (signal peptide+mature peptide (variant)+ IP connexons+IgG1Fc)
		SEQ ID NO：115	Nucleotide sequence (the signal peptide+mature peptide+IP connections of TNFRII-Fc entire constructs Son+IgG1Fc (variant))
SEQ ID NO：116	Amino acid sequence (the signal peptide+mature peptide+IP connections of TNFRII-Fc entire constructs Son+IgG1Fc (variant))
		SEQ ID NO：117	TNFRII-Fc entire constructs nucleotide sequence (signal peptide+mature peptide (variant)+ IP connexons+IgG1Fc (variant))
SEQ ID NO：118	TNFRII-Fc entire constructs amino acid sequence (signal peptide+mature peptide (variant)+

Sequence identifier	Sequence
			IP connexons+IgG1Fc (variant))
SEQ ID NO：119	Nucleotide sequence (the signal peptide+mature peptide+GSSNT of TNFRII-Fc entire constructs Connexon+IgG1Fc)
		SEQ ID NO：120	Amino acid sequence (the signal peptide+mature peptide+GSSNT of TNFRII-Fc entire constructs Connexon+IgG1Fc)
SEQ ID NO：121	TNFRII-Fc entire constructs nucleotide sequence (signal peptide+mature peptide (variant)+ GSSNT connexons+IgG1Fc)
		SEQ ID NO：122	TNFRII-Fc entire constructs amino acid sequence (signal peptide+mature peptide (variant)+ GSSNT connexons+IgG1Fc)
SEQ ID NO：123	OX40-Fc forward primers 1 (nucleotide sequence)
		SEQ ID NO：124	OX40 reverse primers 1 (nucleotide sequence)
SEQ ID NO：125	OX40 forward primers 2 (nucleotide sequence)
		SEQ ID NO：126	OX40 reverse primers 2 (nucleotide sequence)
SEQ ID NO：127	OX40 nucleotide sequences (signal peptide)
		SEQ ID NO：128	OX40 amino sequences (signal peptide)
SEQ ID NO：129	OX40 nucleotide acid sequences (mature peptide)
		SEQ ID NO：130	OX40 amino acid sequences (mature peptide)
SEQ ID NO：131	OX40 nucleotide sequences (signal peptide+mature peptide)
		SEQ ID NO：132	OX40 amino sequences (signal peptide+mature peptide)
SEQ ID NO：133	OX40-Fc nucleotide sequences (mature peptide+IP connexons+IgG1Fc)
		SEQ ID NO：134	OX40-Fc amino acid sequences (mature peptide+IP connexons+IgG1Fc)
SEQ ID NO：135	OX40-Fc nucleotide sequences (mature peptide+IP connexons+IgG1Fc (variant))
		SEQ ID NO：136	OX40-Fc amino acid sequences (mature peptide+IP connexons+IgG1Fc (variant))
SEQ ID NO：137	OX40-Fc nucleotide sequences (mature peptide+GSSNT connexons+IgG1Fc)
		SEQ ID NO：138	OX40-Fc amino acid sequences (mature peptide+GSSNT connexons+IgG1Fc)
SEQ ID NO：139	Nucleotide sequence (the signal peptide+mature peptide+IP connexons of OX40-Fc entire constructs   +IgG1Fc)
		SEQ ID NO：140	Amino acid sequence (the signal peptide+mature peptide+IP connexons of OX40-Fc entire constructs   +IgG1Fc)
SEQ ID NO：141	Nucleotide sequence (the signal peptide+mature peptide+IP connexons of OX40-Fc entire constructs + IgG1Fc (variant))
		SEQ ID NO：142	Amino acid sequence (the signal peptide+mature peptide+IP connexons of OX40-Fc entire constructs + IgG1Fc (variant))
SEQ ID NO：143	(signal peptide+mature peptide+GSSNT connects the nucleotide sequence of OX40-Fc entire constructs Meet son+IgG1Fc)
		SEQ ID NO：144	(signal peptide+mature peptide+GSSNT connects the amino acid sequence of OX40-Fc entire constructs Meet son+IgG1Fc)
SEQ ID NO：145	BAFF forward primers (nucleotide sequence)
		SEQ ID NO：146	BAFF reverse primers (nucleotide sequence)

Sequence identifier	Sequence
		SEQ ID NO：147	BAFF nucleotide sequences (precursor peptide)
SEQ ID NO：148	BAFF amino acid sequences (precursor peptide)
		SEQ ID NO：149	BAFF nucleotide sequences (precursor peptide (variant))
SEQ ID NO：150	BAFF amino acid sequences (precursor peptide (variant))
		SEQ ID NO：151	BAFF nucleotide sequences (mature peptide)
SEQ ID NO：152	BAFF amino acid sequences (mature peptide)
		SEQ ID NO：153	BAFF nucleotide sequences (precursor peptide+mature peptide)
SEQ ID NO：154	BAFF amino acid sequences (precursor peptide+mature peptide)
		SEQ ID NO：155	BAFF nucleotide sequences (precursor peptide (variant)+mature peptide)
SEQ ID NO：156	BAFF amino acid sequences (precursor peptide (variant)+mature peptide)
		SEQ ID NO：157	(precursor peptide+mature peptide+GSSNT connects the nucleotide sequence of BAFF-Fc entire constructs Meet son+IgG1Fc)
SEQ ID NO：158	(precursor peptide+mature peptide+GSSNT connects the amino acid sequence of BAFF-Fc entire constructs Meet sub- IgG1Fc)
		SEQ ID NO：159	BAFF-Fc entire constructs nucleotide sequence (precursor peptide (variant)+mature peptide+ GSSNT connexon IgG1Fc)
SEQ ID NO：160	BAFF-Fc entire constructs amino acid sequence (precursor peptide (variant)+mature peptide+ GSSNT connexon IgG1Fc)
		SEQ ID NO：161	NGFR forward primers (nucleotide sequence)
SEQ ID NO：162	NGFR reverse primers (nucleotide sequence)
		SEQ ID NO：163	NGFR nucleotide sequences (signal peptide)
SEQ ID NO：164	NGFR amino acid sequences (signal peptide)
		SEQ ID NO：165	NGFR nucleotide sequences (mature peptide)
SEQ ID NO：166	NGFR amino acid sequences (mature peptide)
		SEQ ID NO：167	NGFR nucleotide sequences (signal peptide+mature peptide)
SEQ ID NO：168	NGFR amino acid sequences (signal peptide+mature peptide)
		SEQ ID NO：169	NGFR-Fc nucleotide sequences (mature peptide+IP connexons+IgG1Fc)
SEQ ID NO：170	NGFR-Fc amino acid sequences (mature peptide+IP connexons+IgG1Fc)
		SEQ ID NO：171	NGFR-Fc nucleotide sequences (mature peptide+IP connexons+IgG1Fc (variant))
SEQ ID NO：172	NGFR-Fc amino acid sequences (mature peptide+IP connexons+IgG1Fc (variant))
		SEQ ID NO：173	NGFR-Fc nucleotide sequences (mature peptide+GSSNT connexons+IgG1Fc)
SEQ ID NO：174	NGFR-Fc amino acid sequences (mature peptide+GSSNT connexons+IgG1Fc)
		SEQ ID NO：175	Nucleotide sequence (the signal peptide+mature peptide+IP connexons of NGFR-Fc entire constructs   +IgG1Fc)
SEQ ID NO：176	Amino acid sequence (the signal peptide+mature peptide+IP connexons of NGFR-Fc entire constructs   +IgG1Fc)
		SEQ ID NO：177	Nucleotide sequence (the signal peptide+mature peptide+IP connexons of NGFR-Fc entire constructs + IgG1Fc (variant))

Sequence identifier	Sequence
		SEQ ID NO：178	Amino acid sequence (the signal peptide+mature peptide+IP connexons of NGFR-Fc entire constructs + IgG1Fc (variant))
SEQ ID NO：179	(signal peptide+mature peptide+GSSNT connects the nucleotide sequence of NGFR-Fc entire constructs Meet son+IgG1Fc)
		SEQ ID NO：180	(signal peptide+mature peptide+GSSNT connects the amino acid sequence of NGFR-Fc entire constructs Meet son+IgG1Fc)
SEQ ID NO：181	FasL forward primer (nucleotide sequence)
		SEQ ID NO：182	FasL reverse primer (nucleotide sequence)
SEQ ID NO：183	FasL nucleotide sequence (precursor peptide)
		SEQ ID NO：184	FasL amino acid sequence (precursor peptide)
SEQ ID NO：185	FasL nucleotide sequence (mature peptide)
		SEQ ID NO：186	FasL amino acid sequence (mature peptide)
SEQ ID NO：187	FasL nucleotide sequence (precursor peptide+mature peptide)
		SEQ ID NO：188	FasL amino acid sequence (precursor peptide+mature peptide)
SEQ ID NO：189	Nucleotide sequence (the precursor peptide+mature peptide+GSSNT of FasL-Fc entire constructs Connexon IgG1Fc)
		SEQ ID NO：190	Amino acid sequence (the precursor peptide+mature peptide+GSSNT of FasL-Fc entire constructs Connexon IgG1Fc)
SEQ ID NO：191	TNF-α genome nucleotide sequence
		SEQ ID NO：192	LT- α genome nucleotide sequences
SEQ ID NO：193	FasL genome nucleotide sequence
		SEQ ID NO：194	The sialyltransferase forward primers of α 2,6 (are used to be cloned into pIRESbleo3-a2,6ST)
SEQ ID NO：195	The sialyltransferase reverse primers of α 2,6 (are used to be cloned into pIRESbleo3-a2,6ST)
		SEQ ID NO：196	The sialyltransferase forward primers of α 2,6 (are used to be cloned into pIRESpuro3-a2,6ST)
SEQ ID NO：197	The sialyltransferase reverse primers of α 2,6 (are used to be cloned into pIRESpuro3-a2,6ST)
		SEQ ID NO：198	TNFRII-Fc forward primers (nucleotide sequence), for being cloned into pCEP-4
SEQ ID NO：199	TNFRII-Fc reverse primers (nucleotide sequence), for being cloned into pCEP-4

The list of conventional abbreviation is provided in table 4 and table 5 herein.

Table 4：Abbreviation and alias

Referred to as	Description
		AAA	Amino acid analysis
AFC	Affinity chromatography
		APC	Antigen presenting cell
BAFF	B cell activity factor；TNF to APO L it is related leucocyte expression ligand 1；TNF The leucocyte expression part -1 (TALL-1, TALL1) related to ApoL；B drenches Bar LSF (BlyS)；B cell activity factor；BMDC derives TNF sample molecules；UNQ401/PRO738；TNF alloactivation apoptosis；Nuclear factor The kinases (THANK) at-к B and c-Jun NH2 ends；ZTNF4；Tumor necrosis factor Sub- part superfamily member 13B (TNFSF13B).
		bFGF	Basic Fibroblast Growth Factor, FGF2
BSA	Bovine serum albumin(BSA)
		cDLC	Composite fuel part is chromatographed
CRD	Carbohydrate recognition domain
		CSF	Colony stimulating factor
DCS	Donor calf serum
		DeoxGlc
	1,5-anhydroglucitol
		DLC	The false affinity chromatography of fuel part
DSC	Differential scanning calorimetry
		ECD	Extracellular domain
EGF	EGF
		ELISA	Enzyme Linked Immunoadsorbent Assay
EPO	Hematopoietin；
		EST	The sequence label of expression
Fc	Crystalline fragments or constant region for immunoglobulin
		FCS	Hyclone
FGF2	Basic Fibroblast Growth Factor, bFGF
		FTIS	Fourier transform infrared spectroscopy
Fuc	Trehalose
		G-CSF	Granulocyte colony stimulating factor
Gal	Galactolipin
		GalNAc,   galactosamine	2- deoxidations, 2 amine-galactoses
GFC	Gel permeation chromatography
		GlcA	Glucuronic acid
GlcNAc,   glucosamine	2- deoxidations, 2 Glucosamines
		Glc	Glucose
GM-CSF	Granulocyte-macrophage colony stimutaing factor
		HBS	Hepes buffer salts
hES	Human embryo stem cell
		HIC	Hydrophobic interaction chromatograph
HPAEC-PAD	Use the high pH anion-exchange chromatographies of pulsed amperometric current detecting
		HPLC	High pressure liquid chromatography or high performance liquid chromatography
HSA	Human serum albumins
		HTS	High flux screening

Referred to as	Description
		IdoA	Iduronic acid
IEC	Ion-exchange chromatography
		IEF	Isoelectric focusing
IFN	Interferon
		Ig	Immunoglobulin
IL	Interleukin
		lacNAc	N-acetyl lactosamine
lacdiNAc	N, N '-diacetyllactosediamine
		LC	Fluid is chromatographed
LT-α	Lymphocytotoxin α；Lymphocytotoxin a；LTA；TNF surpasses house Race I (TNFSFI)；TNF (derived from lymphocyte)；TNFB；TNFβ； Coley's toxin；CTX (cytotoxin)；DIF (differentiation induction factor)；F-1 (because Son -1)；Homorrhagic factor；Necrosin；NKCF (NK virulence factor)； NK-CIA (NKT bacterium colony inhibitory activity)
		Man	Mannose
MALDI-TOF	Matrix-assisted laser desorption ionization
		Man	Mannose
MCC	Metal chelate chromatography
		MS	Mass spectrum
NacSial, NeuAc or   NeuNAc	N- acetyl nerve ammonia (N-acetyl neuraminic acid)
		NGlySial, NeuGc or   NeuGly	NeuGc ALPHA2-3Gal (N-glycolyl neuraminic acdi)
NGFR	Trk C (NGFR)；p75NGFR；Gp80-LNGFR；p75ICD； Low-affinity neurotrophic factor acceptor；P75 neurotrophic factor acceptors (p75   NTR)；A member of the TNF receptor family 16 (TNFRSF16).
		OX40	ACT-35；CD134；A member of the TNF receptor family 4 (TNFRSF4)； The acceptor (TXGP1L) of tax- transcriptional activations glycoprotein 1.
PBS	Phosphate buffer solution
		PCS	Photon correlation spectroscopy
PDGF-AA	Platelet-derived growth factor A homodimers
		PNGase	Tire-N4- (N- acetyl group-β-D-glucosaminyl) asparagine acid amides enzyme
PUVA	Psoralen-UVA
		RMLP	Receptor-mediated part chromatography
RPC	Reversed phase chromatography
		SDS PAGE	Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SEC	Exclusion chromatography
		Sia	Aluminosilicate
TCA	Trichloroacetic acid
		TFF	Tangential flow filtration
TGF	TGF
		TNF	TNF
TNF-α	TNF (TNF)；Tumor necrosis factor ligand superfamily member α (TNFRSF2), TNF-alpha；TNFα；TNFA；TNF (unicellular derivative), TNF (derived from macrophage)；DIF；cachectin
		TNFR	Tumor Necrosis Factor Receptors
TNFRI	Tumour necrosis factor receptor-1 (TNFRI)；TNF-RI；TNFR1；TNF-R1；TNFAR；   CD120a；p55；p60；TNF receptor superfamily member 1A (TNFRSF1A).
		TNFRII	Tumor necrosis factor receptor I I (TNFRII, TNF-RII)；TNFR2；TNF-R2；

Referred to as	Description
			CD120b；p75；p80；TNF-α acceptor；TNFBR；TNF receptor superfamilies into Member 1B (TNFRSF1B).
TNFRI-Fc	TNFRI (ECD)-Fc fusions
		TNFRII-Fc	TNFRII (ECD)-Fc fusions
UVA	Ultraviolet light,long wave
		UVB	UV-B
Xyl	Xylose

Table 5：Amino acid is referred to as

Amino acid	Three-letter codes	Single letter code
			Alanine	Ala	A
Arginine	Arg	R
			Asparagine	Asn	N
Aspartic acid	Asp	D
			Cysteine	Cys	C
Glutamic acid	Glu	E
			Glutamine	Gln	Q
Glycine	Gly	G
			Histidine	His	H
Isoleucine	Ile	I
			Leucine	Leu	L
Lysine	Lys	K
			Methionine	Met	M
Phenylalanine	Phe	F
			Proline	Pro	P
Serine	Ser	S
			Threonine	Thr	T
Tryptophan	Trp	W
			Tyrosine	Tyr	Y
Valine	Val	V

The code of table 5 (a)-unconventional amino acid

Table 5 (b) monoamino-acid polarity and electric charge packet

Table 6：Stem cell list

Cell type
	Common cell types
Embryonic stem cell
	Thick liquid cell stem cell
Dry cell of microorganism
	Human embryo stem cell
Human epidermal stem cell
	The stem cell of adipose-derived
Brain
	Adult neural stem cell
Human neure
	People's astroglia
Epithelium
	Human keratinocyte stem cell
Human keratinocyte's transient amplification cell
	Human melanocytes stem cell
Human melanocytes
	Skin
Human foreskin fibroblasts
	Pancreas
People's urine output solencyte
	Human pancreas' island
Human pancreas' beta cell
	Kidney
The ripe kidney stem cell of people
	Human embryo kidney (HEK) epithelial stem cell
People's kidney epithelia cell
	Liver
People's Hepatic oval cells
	People's liver cell
People's list ductal epithelial cell
	Human embryo endodermal stem cells
People's adult human liver stem cell (has dispute) to it
	Breast
Human breast epithelial stem cell
	Lung
The stem cell of bone marrow derived
	Human lung cancer cell A549
Human bronchial epithelial cell
	The non-race II type pneumonocytes of people
Muscle
	Human Skeletal Muscle stem cell (satellite cell)
Heart
	Human Cardiomyocytes
Bone marrow interstital stem cell
	Simple squamous cell

Cell type
	Descending main artery epithelial cell
Main artery arch epithelial cell
	Aortic smooth muscle cells
Eyes
	Limbal stem cells
Corneal epithelial cell
	CD34+ candidate stem cells
Mescenchymal stem cell
	Osteoblasts (precursor is mescenchymal stem cell)
Peripheral blood mononuclear progenitor cells (candidate stem cell)
	Interstitial (precursor is above-mentioned cell type)
Interstitial cell
	Spleen
People's spleen precursor stem cell
	Human spleen cell
Immunocyte
	People's CD4+T- cells
People's CD8+T- cells
	NK cells of human beings
Person monocytic cell
	Human macrophage
Human dendritic cell
	People's B- cells
Nose
	Goblet cells (the mucilage secretion cell of nose)
The pseudostrimatic stanchion cell (being located at below nose regio olfactoria) for having a cilium
	The pseudostratified epithelial cell for having a cilium (cell arrangement is in ductus nasopharyngeus)
Tracheae
	Lamination epidermal cell (cell arrangement and constitute tracheae)
There is columnar cell's (cell arrangement and constitute tracheae) of cilium
	Goblet cells (cell arrangement and constitute tracheae)
Basal cell (cell arrangement and constitute tracheae)
	Oesophagus
Cricopharyngeus cell
	Reproduction
Female originally vesica
	Male spermatogonium

Brief description of the drawings

Fig. 1 is the diagram of the cloning procedure of the cDNA insertion pIRESbleo3 or pIRESbleo3-Fc carriers of the protein of the coding present invention.

Fig. 2 (a) represents to be released from one group of LC-MS chromatogram of the TNFRII-Fc of present invention N- glycan.Upper figure：Total ion chromatogram；Figure below：Base peak chromatograms.

Fig. 2 (b) represents that N- glycan is present in one group of MS/MS spectrum when on the TNFRII-Fc of the present invention.(1) [M-H] -1461, Rt 22.0min；(2)[M-2H]²- 811, Rt 23.9min；(3)[M-2H]²- 892, Rt 24.6min；(4)[M-2H]²- 1037, Rt 27.2min.

Fig. 2 (c) represents one group of LC-MS from the TNFRII-Fc that Chinese hamster ovary cell (Enbrel) the is expressed N- glycan discharged.Upper figure：Total ion chromatogram；Figure below：Base peak chromatograms.

Fig. 2 (d) represents that N- glycan is present in one group of MS/MS spectrum when on the TNFRII-Fc of Chinese hamster ovary cell (Enbrel) expression.(1) [M-H] -1462, Rt 22.5min；(2)[M-2H]²- 893, Rt 23.6min；(3)[M-2H]²- 1038, Rt 26.1min；(4)[M-2H]²- 1184, Rt 30.1min；(5)[M-H]^-1598, Rt 39.1min；(6)[M-H]^-1906, Rt 39.2min.

Fig. 2 (e) represents one group of LC-MS from the TNFRII-Fc of the present invention O- glycan discharged.Upper figure：Total ion chromatogram；Figure below：Base peak chromatograms.

Fig. 2 (f) represents that O- glycan is present in one group of MS/MS spectrum when on the TNFRII-Fc of the present invention.(1-A and 1-B) [M-H]^-676, Rt 21.3min；(2-A and 2-B) [M-H]^-967, Rt 23.2min；(3)[M-H]^-749, Rt 24.3min；(4-A and 4-B) [M-H]^-1041, Rt 28.9min；(5-A and 5-B) [M-H]^-1332, Rt 33.4min.

Fig. 2 (g) represents one group of LC-MS from the TNFRII-Fc that Chinese hamster ovary cell (Enbrel) the is expressed O- glycan discharged.Upper figure：Total ion chromatogram；Figure below：Base peak chromatograms.

Fig. 2 (h) represents that O- glycan is present in one group of MS/MS spectrum when on the TNFRII-Fc of Chinese hamster ovary cell (Enbrel) expression.(1-A and 1-B) [M-H]^-676, Rt22.8min；(2-A and 2-B) [M-H]^-967, Rt 23.2min.

Fig. 3 (a) is the picture of the hand of a pityriasis rubra pilaris (pityriasis rubria pilaris) patient before the treatment.Note the skin and open injury (openlesion) of red.

Fig. 3 (b) is that the same hand shown in Fig. 3 (a) gives 2ml TNFRII-Fc topical preparations (the 250 μ g/ml TNFRII-Fc of the present invention；20mg/ml Distavals) continue the picture after 2 weeks.Note red reduce and damage disappearance.

Fig. 4 diagram represents the cell death of the WEHI164 cells of the TNF-α processing of the invention gradually risen through concentration.

Fig. 5 diagram represents the cell death of the WEHI164 cells of the LT- α processing of the invention gradually risen through concentration.

Fig. 6 diagram represents the neutralising capacity for the cytotoxicity that the TNFRI-Fc of the present invention is mediated to TNF-α in WEHI-164 cells.

Fig. 7 figure represents the neutralising capacity for the cytotoxicity that the TNFRII-Fc of the present invention is mediated to TNF-α in WEHI-164 cells.

Fig. 8 figure represents the depression effect for the cytotoxicity that the TNFRII-Fc (rhombus) that TNFRII-Fc (fork-shaped) more of the invention and non-human cell express is mediated to TNF-α in mouse L-929 cells.

The multiplication effect of the cells of RPMI 8226 of Fig. 9 BAFF (open circles) inductions for scheming expression BAFF (filled circles) more of the invention and non-human cell's expression.

Figure 10 figure represents the neutralising capacity of the NGFR-Fc of the present invention TF-1 cell propagation beta induced to NGF-.

Figure 11 represents humanTNF-α (horizontal line, R＆D systems that TNF-α of the present invention (square) is expressed with Bacillus coli cells；Triangle WHO) the external of immunoreactivity feature compare.ELISA kit standard curve (circle).

Figure 12 represents that the external of the immunoreactivity feature for the people LT- α (rhombus) that the LT- α (square) of the present invention are expressed with Bacillus coli cells is compared.

Figure 13 diagram represent in mouse for percutaneous administration of the present invention TNFRII-Fc topical preparations after, TNFRII-Fc bio distribution.

Embodiment

Be interpreted as except as otherwise noted, the invention is not restricted to special composition, preparation method, diagnostic method, analysis experimental design, nutrition experimental record or research experiment record or and so on possible change.It is further appreciated that only to be description specific embodiment for term purpose as used herein and does not limit specially.

It should be noted that what this specification was used, the indefinite article and definite article of singulative include plural number, unless context is otherwise indicated.Thus, for example, on " a kind of protein ", " a kind of cell factor " or " a kind of chimeric molecule " or " a kind of acceptor ", including single parameter and also including two or more parameters.

Term " compound ", " active factors ", " chemokines ", " pharmacologically active agents ", " medicament ", " active matter " and " medicine " exchanges use herein, is related to a kind of compound and the particularly a kind of desired physics and chemistry of induction and/or the protein of pharmacological effect or its chimeric molecule.The term also includes the pharmaceutically acceptable and pharmacological active component of these active factorses, and salt, esters, amide-type, pro-drug, active metabolite, analog etc. are more particularly to included but is not limited to herein.During using term " compound ", " active factors ", " chemokines ", " pharmacologically active agents ", " medicament ", " activity " and " medicine ", it is interpreted as its include active factors and pharmaceutically acceptable, pharmacological activity salt, esters, amide-type, pro-drug, active metabolite, analog etc. in itself.

Include the composition of two or more active materials on " compound ", " active factors ", " chemokines ", " pharmacologically active agents ", " medicament ", " active matter " and " medicine ", such as two or more cell factors." composition " also includes many part such as two parts compositions, wherein before formula, the factor is separately provided and given or prepare respectively or mixes.

For example, many part packs (multi-part pharmaceutical pack) can belong to TNF superfamilies or relative protein or chimeric molecule with two or more, and it is selected from TNF-a, TNF-a-Fc, LT- α, LT- α-Fc, TNFRI, TNFRI-Fc, TNFRII, TNFRII-Fc, OX40, OX40-Fc, BAFF, BAFF-Fc, NGFR, NGFR-Fc, FasL, the FasL-Fc for being individually separated preserving.

The term " effective dose " of reagent as used herein and " therapeutically effective amount " represent protein or its chimeric molecule individually or in the composition for have other reagents to provide the sufficient amount of desired treatment or physiological effect or result.Undesirable effect, such as side effect, are proved with desired therapeutic effect sometimes；Therefore, doctor balances possible benefit with possible risk to determine that what is appropriate " effective dose ".According to the species of subject, age and comprehensive condition, mode of administration etc., required definite amount can change between subject and subject.Therefore, it is not possible to specify one accurate " effective dose ".However, appropriate " effective dose " to any individual case can be determined by those skilled in the art using unique routine test.

" pharmaceutically acceptable " carrier is used, excipient or diluent represent that pharmaceutical carriers include abiotic or non-undesirable substance, i.e. material and selected active factors are administered to subject without causing any side effect or substantial side effect together.Carrier can include auxiliary material and other additives, such as diluent, detergent, colouring agent, wetting agent or emulsifying agent, pH buffer, preservative.

Similarly, " pharmaceutically acceptable " salt, esters, amide-type, pro-drug or the derivative of composition refer to abiotic or non-bad salt, esters, amide-type, pro-drug or derivative provided herein.

Term " treatment " as used herein and " therapy " are related to severity and/or the mitigation of frequency of the symptom of disease being treated, prevention and the improvement of the infringement of adjoint disease or remedy or take a turn for the better that the symptom of the elimination of symptom and/or potential cause, disease and/or their potential cause occurs.

" treatment " subject can be included in the disease in susceptible individual or the prevention and the treatment individual to clinical symptoms by improving the symptom of disease of other bad physiologic results.

" subject " as used herein is related to animal, in specific specific embodiment, mammal, and in further specific embodiment, the people that can be benefited from the pharmaceutical preparation and method of the present invention.The species of animal at this to that can be benefited from presently described pharmaceutical preparation and method is not limited.Whether people or non-human animal can be referred to as individual, patient, animal, host or acceptor to subject.The Compounds and methods for of the present invention is applied to physianthropy, veterinary science and general, animal breeding raise and train or wild.

Indicated above, in specific specific embodiment, animal is people or other primates such as orangutan, gorilla, ape, livestock animals, laboratory test animal, pairing animal or the wild animal and birds being captured.

Laboratory test animal citing includes mouse, mouse, rabbit, cavy and hamster.There is provided convenient pilot system or animal model for rabbit and rodent, such as mouse and mouse.Livestock animals include sheep, ox, pig, goat, horse and donkey.Nonmammalian such as birds, fish and amphibian include Xenopus, procaryon and non-lactation eucaryote.

Term " cytokine " " is used for its most universal meaning and including any various protein secreted by cell, and it adjusts the functional activity of immune system, regulation individual cells and/or tissue, and/or induces a series of physiological reactions.Term " cytokine " as used herein " is construed as being related to the cell factor of " complete " and includes the increase of one or more amino acid; missing is substituted; and it is kept substantially its fragment of the biological activity of the intact cell factor, derivative or homologue or chimeric molecule.

" cytokine receptor " is that cell membrane is associated or solvable albuminous cell factor acceptor, relevant with cytokine signaling system or regulation.Term " cytokine " acceptor as used herein " is construed as being related to the cytokine receptor of " complete " and includes the increase of one or more amino acid; missing is substituted, and is kept substantially its fragment, derivative or the homologue or chimeric molecule of the biological activity of intact cell factor acceptor.

Term " protein " is used for its most universal meaning and including cell factor and cytokine receptor.It is as used herein, term " protein " should be understood to be related to the protein of " complete " and include the increase of one or more amino acid, missing is substituted, and is kept substantially its fragment, derivative or the homologue or chimeric molecule of the biological activity of whole protein.

Term " polypeptide " is related to the condensate and its equivalent of amino acid, but does not limit the specific length of product, and therefore, peptide, oligopeptides, peptide and protein are included in the definition of " polypeptide ".The term also includes the modification of all common translations or the posttranslational modification product of polypeptide.Having in this definition is also included within simultaneously, for example, the amino acid for the non-natural generation that the analog containing certain one or more amino acid, such as table 5 (a) are provided or the polypeptide with substitution chain.

Present invention contemplates the protein of separation or its chimeric molecule, it has measurable physical and chemical parameter (P_x) feature, wherein this feature represents, is associated with one or more special pharmacological characteristics (T_y) or the one or more special pharmacological characteristics (T of formation_y) basis.The protein or chimeric molecule of separation are the protein or associated protein for belonging to TNF superfamilies, selected from comprising TNF-a, TNF-a-Fc, LT- α, LT- α-Fc, TNFRI, TNFRI-Fc, TNFRII, TNFRII-Fc, OX40, OX40-Fc, BAFF, BAFF-Fc, NGFR, NGFR-Fc, FasL, FasL-Fc group.TNF-a, TNF-a-Fc, LT- α, LT- α-Fc as used herein, TNFRI, TNFRI-Fc, TNFRII, TNFRII-Fc, OX40, OX40-Fc, BAFF, BAFF-Fc, NGFR, NGFR-Fc, FasL, FasL-Fc include whole polypeptide and its segment being related to.

More particularly, the invention provides a kind of protein of separation or its chimeric molecule, it, which has, includes a series of physicochemical characteristicses of measurable physical and chemical parameters, { [P_x]₁, [P_x]₂、··[P_x]_n, wherein P_xRepresent measurable physical and chemical parameter and " n " be >=1 integer, wherein [P_x]₁To [P_x]_nIndividually one different measurable physical and chemical parameter, the numerical value of the one or more measurable physicochemical characteristicses of any of which represents, is associated with the pharmacological characteristics T of a characteristic_yOr the pharmacological characteristics ([T of series of features_y]₁、[T_y]₂、....[T_y]_mOr formed a characteristic pharmacological characteristics T_yOr the pharmacological characteristics ([T of series of features_y]₁、[T_y]₂、....[T_y]_mBasis, wherein T_yThe pharmacological characteristic and " m " for representing a characteristic are >=1 integer, and [T_y]₁To [T_y]_mIndividually one different pharmacological characteristics.

Term " measurable physical and chemical parameter " (P as used herein_x) it is related to the protein of one or more measurable separation or the feature of its chimeric molecule.Representational " special measurable physical and chemical parameter " includes, but are not limited to：Apparent molecular weight (P₁), isoelectric point (pI) (P₂), isoform number (P₃), the relative intensity (P of different isoform number₄), carbohydrate percetage by weight (P₅), the actual measurement molecular weight (P after N- connection oligosaccharides deglycosylations₆), N- connections and O- connections oligosaccharides deglycosylation after actual measurement molecular weight (P₇), the percentage (P of acid contents of monosaccharides₈), contents of monosaccharides (P₉), sialic acid content (P₁₀), sulfate and phosphate content (P₁₁)、Ser/Thr：GalNAc ratios (P₁₂), the neutral percentage (P of N- connection oligosaccharides₁₃), the acid percentage (P of N- connection oligosaccharides₁₄), the neutral percentage (P of O- connection oligosaccharides₁₅), the acid percentage (P of O- connection oligosaccharides₁₆), the ratio (P of N- connection oligosaccharides₁₇), the ratio (P of O- connection oligosaccharides₁₈), the structure (P of N- connection oligosaccharide ingredients₁₉), the structure (P of O- connection oligosaccharide ingredients₂₀), the position of N- connection oligosaccharides and composition (P₂₁), the position of O- connection oligosaccharides and composition (P₂₂), common translation modification (P₂₃), posttranslational modification (P₂₄), acylated (P₂₅), acetylation (P₂₆), amidatioon (P₂₇), deamidation (P₂₈), biotinylation (P₂₉), carbamylation (P₃₀), carboxylation (P₃₁), decarboxylation (P₃₂), disulfide formation (P₃₃), fatty-acylation (P₃₄), myristoylation (P₃₅), palmitoylation (P₃₆), octadecane be acylated (P₃₇), formylated (P₃₈), saccharification (P₃₉), glycosylation (P₄₀), glycophosphatidyl inositol grappling (P₄₁), hydroxylating (P₄₂), the combination (P of selenocysteine₄₃), lipid (P₄₄), the addition (P of lipoic acid₄₅), methylate (P₄₆), N or C-terminal closing (P₄₇), N or C-terminal remove (P₄₈), nitrification (P₄₉), methionine oxidized (P₅₀), phosphorylation (P₅₁), protease digestion (P₅₂), prenylation (P₅₃), farnesylation (P₅₄), Mang ox base (P₅₅), phosphopyridoxal pyridoxal phosphate addition (P₅₆), sialylated (P₅₇), asialoglycoprotein (P₅₈), sulfation (P₅₉), ubiquitination (P₆₀), the addition (P of ubiquitin sample molecule₆₁), primary structure (P₆₂), secondary structure (P₆₃), tertiary structure (P₆₄), quaternary structure (P₆₅), chemical stability (P₆₆), heat endurance (P₆₇).The summary of these parameters is provided in table 2.

It is to include any pharmacological or clinically relevant characteristic of the protein or chimeric molecule of the present invention that term " (distinctive) pharmacological characteristics of characteristic ", which has been readily appreciated by one skilled in the art,.Representational " pharmacological characteristics " not only shall be limited only to the extent what invention included：Therapeutic effect (T₁), dose therapeutically effective (TCID₅₀)(T₂), bioavilability (T₃), from the time (T for being administered into maintaining treatment level₄), absorption rate (T₅), discharge rate (T₆), special activity (T₇), heat endurance (T₈), lyophilized stability (T₉), serum/plasma stability (T₁₀), serum half-life (T₁₁), the solubility (T in blood flow₁₂), immune response feature (T₁₃), immunogenicity (T₁₄), neutralizing antibody suppress (T_14A), side effect (T₁₅), receptor/ligand affinity (T₁₆), receptor/ligand activation (T₁₇), tissue or cell category specificity (T₁₈), penetration capacity (such as intestines, lung, blood-brain barrier, skin etc.) (T of biomembrane or barrier₁₉), generation blood vessel ability (T_19A), tissue resorption (T₂₀), degraded stability (T₂₁), freeze-thaw stability (T₂₂), protease stability (T₂₃), ubiquitin stability (T₂₄), administration reduce (T₂₅), mode of administration (T₂₆), the compatibility (T with other pharmaceutical excipients or carrier₂₇), the residual (T in organism or environment₂₈), preserve during stability (T₂₉), (T such as toxicity in organism or environment₃₀)。

In addition, the protein or chimeric molecule of the present invention can have different biological effect (T in different cell categories₃₁), including but not limited to people's primary cell, such as lymphocyte, red blood cell, retina cell, liver cell, neuron, horn cell, endothelial cell, endoderm cell, ectoderm cell, mesoblastema, epithelial cell, nephrocyte, liver cell, osteocyte, bone marrow cell, lymph node cells, dermal cell, fibroblast, T cell, B cell, thick liquid cell, NK, macrophage, bite neutrophil leucocyte, grain cell of Langerhan, BMDC, bite sour granulocyte, bite alkali granulocyte, mammary cell, small leaf cell, prostatic cell, pneumonocyte, esophageal cells, pancreatic cell, Beta cells (insulin secretory cell), angioblast, myocyte, elliptocyte (liver cell), mesenchymal cell, brain microvessel endothelial cells in vitro, astroglia, spongiocyte, a variety of stem cells include adult and embryonic stem cell, a variety of progenitor cells；With other people permanent, conversion or cancer cell systems.Biological effect in cell includes multiplication effect (T₃₂), differentiation (T₃₃), apoptosis (T₃₄), the growth (T of cell size₃₅), cell factor adhesion (T₃₆), cell adhesion (T₃₇), cellular invasion (T₃₈), cell mobility (T₃₉), migration and intrusion (T₄₀), chemotaxis (T₄₁), cell phagocytosis (T₄₂), signal transduction (T₄₃), raise albumen to receptor/ligand (T₄₄), the activation (T of JAK/STAT approach₄₅), the activation (T of Ras-erk approach₄₆), the activation (T of AKT approach₄₇), the activation (T of PKC approach₄₈), the activation (T of PKA approach₄₉), src activation (T₅₀), fas activation (T₅₁), TNFR activation (T₅₂), NFkB activation (T₅₃), p38MAPK activation (T₅₄), c-fos activation (T₅₅), secretion (T₅₆), acceptor caves in (T₅₇), acceptor reciprocation (T₅₈), the up-regulation of surface markers or downward (T₅₉), before FACS/change (T of other scattering signatures₆₀), the change (T of subgroup ratio₆₁), differential gene expression (T₆₂), meronecrosis (T₆₃), cell agglutination (T₆₄), cellular rejection (T₆₅) and heparin sulfate combination (T₆₆) and glycosylation structure combination (T₆₇) and chondroitin sulfate combination (T₆₈) and extracellular matrix combination (such as collagen, fibronectin) (T₆₉) and artificial material combination (such as support) (T₇₀) and carrier combination (T₇₁) and confactor combination (T₇₂), individually or the effect (T in the mixture containing other protein to stem cells hyperplasia, differentiation and/or self-renewing₇₃) etc..The summary of these characteristics is provided in table 3.

Term " characteristic " as used herein is relevant with the protein of the present invention or the pharmacological characteristics of chimeric molecule, is related to one or more protein or the pharmacological characteristics of its chimeric molecule, it is characteristic for special physicochemical characteristicses.In specific specific embodiment, one or more pharmacological characteristics of the protein of separation or its chimeric molecule are different from, or particularity relative to the same protein or the form of chimeric molecule produced in protokaryon or low eukaryotic or even high inhuman eukaryotic.In specific embodiment, the protein isolate matter or the pharmacological characteristics of its chimeric molecule tested are substantially similar to or function equivalence is in the protein of generation naturally.

Term " protokaryon " as used herein is related to any prokaryotic, and it includes any bacterial cell (including actinomycetes cells) or archeabacterial cell.Term " non-human eucaryote " as used herein means self evident.However, for clarity, the term especially includes any non-human eucaryote, it includes：Yeast such as saccharomyces or pichia；Other fungies；Insect, including Drosophila and insect cell culture；Fish, including chub mackerel category；Amphibian, including Xenopus；Plant and plant cell cultures.

It is related to " stem cell " including embryo or adult stem cell and is included in the stem cell listed in table 6.The protein or chimeric molecule of the present invention can be used alone or be used with the protein in cocktail, to induce one or more stem cells hyperplasias, differentiation or self-renewing.

The primary structure of protein or its chimeric molecule can be measured as amino acid sequence.Secondary structure can be measured as the quantity and/or relative position of one or more secondary protein structures, such as alpha-helix, parallel beta sheet, anti-parallel ss-sheet or corner.Tertiary structure describes the folding of polypeptide chain, and different Secondary structural elements are assembled into special arrangement.Spiral and folding are secondary building units, and domain is tertiary structural elements.In multi-domain proteins, tertiary structure includes arrangement domain each other.Accordingly, the presence that tertiary structure can be to one or more protein domains, missing, quantity and/or relative position are measured.Representational domain be not only the present invention limit include：Single-screw, helix turn helix domain, four-helix bundle, DNA binding domain, three helical bundles, Greece's key helical bundle, coiled-coil packaging structure domain, β-sandwich, β-tubbiness, anti-parallel ss-sheet up and down, Greece's key topological structure domain, jam volume topological structure domain, β-propeller, β-clover, β-spiral, Rossman is folded, α/β horseshoe, α/β bucket, alpha+beta topology, rich disulfide bond is folded, serine stretch protein enzyme level domain, actinocongestin domain, EGF spline structures domain, complement C- modular domains, wheat plant toxin domain, cobra (Cobra) neurotoxin domain, greenery cobra anticholinesterase domain, Kringle domains, mucoprotein sample area, spherical region, spacer region.The arrangement of different polypeptide chains of the quaternary structure description with protein structure, each chain has unique one-level, two grades and tertiary structure elements.Citing include with-or miscellaneous-oligomer multimerization (for example dimer formation or tripolymer formed).

For the primary structure being related to, the invention provides the protein of separation or its chimeric molecule, or its fragment, by including SEQ ID NO selected from sequence table：27, 29, 31, 33, 35, 37, 39, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 127, 129, 131, 133, 135, 137, 139, 141, 143, 147, 149, 151, 153, 155, 157, 159, 163, 165, 167, 169, 171, 173, 175, 177, 179, 183, 185, 187, 189, or there is the nucleotide sequence of at least about 60% homogeneity with above-named any bar sequence, or coded by the nucleotide sequence that can hybridize with any of the above-described sequence or their complementary type under low stringent conditions.

Another aspect of the present invention provides a kind of polypeptide of separation, and it is by the their own mRNA processed by cell spliced nucleotide sequence SEQ ID NO：191st, 192,193 coding.

The present invention is in yet a further aspect there is provided a kind of separation, encoding proteins matter or its chimeric molecule or its Functional portions nucleotide sequence molecule, and the nucleic acid molecule is included with including SEQ ID NO selected from sequence table：27, 29, 31, 33, 35, 37, 39, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 127, 129, 131, 133, 135, 137, 139, 141, 143, 147, 149, 151, 153, 155, 157, 159, 163, 165, 167, 169, 171, 173, 175, 177, 179, 183, 185, 187, the sequence similarity of the nucleotides selected in 189 at least 60%, or after optimal comparison and/or can be with SEQI D No：27, 29, 31, 33, 35, 37, 39, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 127, 129, 131, 133, 135, 137, 139, 141, 143, 147, 149, 151, 153, 155, 157, 159, 163, 165, 167, 169, 171, 173, 175, 177, 179, 183, 185, 187, 189 or one or more nucleotide sequences hybridized under low stringency condition of their complementary type.

In a specific embodiment, present invention is generally directed to a kind of nucleic acid molecule of separation, the molecule includes encoding a kind of protein or its chimeric molecule, or its fragment nucleotide sequence, it has substantially such as SEQ ID NO：28, 30, 32, 34, 36, 38, 40, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 128, 130, 132, 134, 136, 138, 140, 142, 144, 148, 150, 152, 154, 156, 158, 160, 164, 166, 168, 170, 172, 174, 176, 178, 180, 184, 186, 188, a 190 shown amino acid sequence or multiple, or with SEQ ID NO：28, 30, 32, 34, 36, 38, 40, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 128, 130, 132, 134, 136, 138, 140, 142, 144, 148, 150, 152, 154, 156, 158, 160, 164, 166, 168, 170, 172, 174, 176, 178, 180, 184, 186, 188, 190 one or more amino acid sequences with least about 60% similitude after optimal comparison.

On the other hand, the invention provides a kind of nucleic acid molecule of separation, encoding proteins matter molecule, or its fragment, including selected from SEQ ID NO：31st, the nucleotide sequence in 33,35,45,47,49,51,63,65,67,91,93,95,97,129,131,151,153,155,165,167,185,187, its directly or the nucleotide sequences through one or more coding protein connexons known in the art with basic such as SEQ ID NO：1st, the nucleotide sequence connection of the constant region (Fc) of 3,5,7,9,11,13,15,17 or 19 one or more shown encoding human immunoglobulins or framework region.In a specific embodiment, the nucleotide sequence of encoding proteins connexon is selected from coding IP, GSSNT, TRA or VDGIQWIP nucleotide sequence.

On the other hand, the invention provides a kind of protein molecule of separation or its fragment, including selected from SEQ ID NO：32nd, 34,36,46,48,50,52,64,66,68,92,94,96,98,130,132,152,154,156,166,168,186,188 amino acid sequence, it is directly or through one or more protein connexons known in the art and basic such as SEQ ID NO：2nd, the constant region (Fc) of 4,6,8,10,12,14,16,18 or 20 one or more shown human immunoglobulin(HIg)s or framework region connection.

Another aspect of the present invention provides the protein or its chimeric molecule or its fragment of a kind of separation, including the SEQ ID NO selected from sequence table：28, 30, 32, 34, 36, 38, 40, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 128, 130, 132, 134, 136, 138, 140, 142, 144, 148, 150, 152, 154, 156, 158, 160, 164, 166, 168, 170, 172, 174, 176, 178, 180, 184, 186, 188, 190 amino acid sequence, or one or more amino acid sequences with least about 65% similitude with above-mentioned sequence.

In a particular embodiment, protein similarities percentage or nucleotide identity level include at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% similitude or homogeneity.

Section or part of " derivative " of the polypeptide of the present invention also including total length parental polypeptide, it retains the part transcriptional activity of parental polypeptide and including variant.Such " biological active fragment " includes depletion mutant and small peptide, for example, having at least 10, in a particular embodiment, the at least 30 continuous amino acid with least 20 and in further specific embodiment, necessary to described continuous amino acid is displaying activity.This peptide can be obtained by the application of standard recombinant nucleotide technology or synthesized with conventional liquid phase or solid phase synthesis technique.For example, object of reference can be prepared with described solution synthesis or synthesis in solid state, e.g., including by Nicholson edit by Blackwell Scientific Publications publish entitled " the 9th chapter in Synthetic Vaccines " publication is named as " Peptide Synthesis " by Atherton and Shephard.Optionally, peptide can be produced by using protease such as endoLys-C, endoArg-C, endoGlu-C and the staphylococcus V8 protease digestions present invention amino acid sequence.Digestion fragment can be purified, for example, high performance liquid chromatography (HPLC) technology.Any such fragment, the method with generation is unrelated, is construed as being included in terminology used herein " derivative ".

Therefore, term " variant " is related to, and is shown as the substantially identical nucleotide sequence of sequence and reference nucleotide sequence or the polynucleotides hybridized under strict conditions with reference sequences being defined below.Term " nucleotide sequence ", " polynucleotides " and " nucleic acid molecule " can be exchanged herein to be used and including being added or lacking with one or more nucleotides, or the polynucleotides replaced with different nucleotides.In this respect, it is known in the art that some change including that can be mutated to reference nucleotide sequence, add, missing and replacement, the polynucleotides thus changed keep the biological function or activity of the polypeptide of reference polynucleotides or coding.Term " variant " also includes the variant of spontaneous allele.

The nucleic acid molecule of the present invention can be carrier or other constructs forms.

In a specific embodiment, carrier is DNA and comprising arbitrary selected marker.

The example of selected marker includes assigning the gene to compound such as antibiotic resistance, assigns the gene of the ability grown in Basic selective material, and coding produces the gene for the protein that can survey signal such as fluorescence.A variety of such genes are known and are available, including, such as antibiotics resistance gene such as neomycin resistance gene (neo) and hygromycin gene (hyg).Selected marker also includes assigning gene such as the tk genes (thymidine kinase) or hprt genes (hypoxanthine phosphoribosyltransferase) of the growth ability in some culture matrixes ability (hypoxanthine, ammonia petrin and thymidine) that its imparting grows in HAT culture mediums；With bacterium gp t genes (guanine/xanthine phosphoribosyl transferase), it allows to grow (mycophenolic acid, Ade and Xan) in MAX culture mediums.Other are used for selected markers of mammalian cell and carry the plasmid of multiple choices mark in Sambrook equimolecular Cloning-A Laboratory Manuals, Cold SpringHarbor, New York, USA, are described in 1990.

Selected marker can be obtained (the protokaryon marker gene in being used for example in purpose mammalian cell) by the expression of its own promoter and marker gene from the organism very different with purpose organism.However, replacing original promoter to be useful with the transcription structure of known function in recipient cell.Substantial amounts of transcription initiation region is useful to such purpose, for example, metallothionein promoter, thymidine kinase promoter, beta-actin promoter, immunoglobulin promoter, SV40 promoters and human cytomegalovirus promoter.Widely used example is pSV2-neo plasmids, its ability (a kind of related antibiotic of neomycin) for having the bacterial neomycin phosphoric acid transferase gene under the control of SV40 early promoters and being endowed the anti-G418 of mammalian cell.Other substantial amounts of mutation can be used for strengthening expression of the selected marker in zooblast, the addition of the translation initiation sequence of the addition and synthesis of such as poly (A) sequence.Composing type and inducible promoter can be used.

The genetic constructs of the present invention can also include 3 ' non-translated sequences.3 ' non-translated sequences are related to the part of gene, comprising containing polyadenylation signal and any other can influence mRNA process or gene expression Regulate signal DNA fragmentation.Polyadenylation signal has the feature for influenceing polyadenylic acid chain to be added to the end of mRNA precursor 3 '.Polyadenosine acid signal is generally identified by the presence with the homology of 5 ' AATAAA-3 ' normal forms, although variation is much.

Correspondingly, the genetic constructs of the nucleic acid molecule comprising the present invention, are effectively connected with promoter, can be cloned into suitable carrier to be delivered in regulation mistake, dysfunction or the cell or tissue of missing, to repair and/or provide appropriate regulation.Carrier containing suitable genetic constructs can be delivered in purpose eukaryotic by many distinct methods known to the technical staff of biology field.

Term " similitude " as used herein is included in accurate homogeneity between the sequence that nucleotides or amino acid levels compare.There is nonidentity in nucleotide level, " similitude " includes the difference between sequence, it causes the difference of amino acid, the difference of amino acid still with mutual structure, function, physics and chemistry and/or conformational levels are relevant.There is nonidentity in amino acid levels, " similitude " includes and mutual structure, function, physics and chemistry and/or conformational levels still relevant amino acid.This is included in polarity and/or electric charge aspect " conservative " amino acid residue of equal value.Table 5 (b) lists in polarity and/or electric charge the amino acid of " equivalence ".In specific specific embodiment, the comparison of nucleotides and sequence is carried out rather than similitude in level of sequence identity.

The term of sequence relation for describing two or more polynucleotides or polypeptide include " canonical sequence ", " comparison block ", " sequence similarity ", " sequence identity ", " sequence similarity percentage ", " Percentage of sequence identity ", " substantially similar " and " substantially same " " canonical sequence " be at least with 12, but often 15 to 18 and usually at least 25 or more, such as 30 monomeric units including nucleotides and amino acid residue, in length.Because two polynucleotides can all include (1) sequence similar between two polynucleotides (such as the part for there was only complete polynucleotide sequence), (2) sequence different between two polynucleotides, the similitude that progress is typically relatively compared by the sequence of two polynucleotides, identification and comparative sequences regional area are removed by " comparison block " of sequence between two (or a plurality of) polynucleotides." comparison block " is related to notional fragment of general 12 consecutive residues, and it is contrasted with canonical sequence.For the optimal comparison of two sequences, comparison block can include about 20% or less addition or missing (such as gaps) compared with canonical sequence (wherein containing addition or missing).In order to arrange comparison block, the optimal comparison of sequence can be by the computerization of algorithm or by checking and being realized by the optimal comparison (such as most high percentage homology is finally given between whole comparison block) of a variety of any generations of selected method.Control can also be obtained by the BLAST races of program, such as (the Nucl Acids Res 25 as disclosed in Altschul：389th, 1997) being discussed in detail for sequence analysis can find (In in Ausubel etc. Unit 19.3：Current Protocolsin Molecular Biology、John Wiley & Sons Inc.1994-1998).

Term " sequence similarity " as used herein and " sequence identity " are related to sequence in comparison block, on the basis of nucleotides is than nucleotides or amino acid than amino acid on the basis of same or function or the similar scope of structure.Therefore, for example, the calculating of " percentage of sequence identity " is compared by the sequence of two optimal comparisons in comparison block, measure, which is present in two sequences, has identical nucleotide base (such as A, T, C, G, ) or identical amino acid residue (such as Ala I, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) site numerical value, to produce the numerical value with loci, with the sum in site in numerical value divided by comparison block with loci (for example, the size of frame), and result is multiplied by 100 to produce Percentage of sequence identity.For the purposes of the present invention, " sequence identity " will be understood to refer to by DNASIS computer programs (for windows of Version 2.5；Available from Hitachi SoftwareEngineering Co., Ltd., South San Francisco, California, USA) " the pairing percentage " that is calculated with the standard error that is used in the comparison handbook appended by software.Similar explanation application and sequence similarity.

Low stringency as used herein is included and comprising being used to from least 0 at least about 15%v/v formamides and from least 1M at least about 2M salt hybridize, and at least about 1M is used for wash conditions at least about 2M salt.It is general, low stringency from about 25-30 DEG C to about 42 DEG C, such as 25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41 and 42 DEG C.Temperature can change and higher temperature is used to replace formamide and/or provides optional stringent conditions.Optional stringent conditions can be used in the place of needs, such as moderate stringency, it is included and comprising from least 16%v/v at least about 30%v/v formamides, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30% and from least about 0.5M at least about 0.9M salt, such as 0.5, 0.6, 0.7, 0.8 or 0.9M is used for wash conditions, or high stringency, it is included and comprising from least about 31%v/v at least about 50%v/v formamides, such as 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50% and from least about 0.01M at least about 0.15M salt, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 and 0.15M is used for wash conditions.General, wash in T_m=69.3+0.41 (G+C) % carries out (Marmur and Doty, J Mol Biol 5：109、1962).However, the T of double-stranded DNA_mThe quantity of 1 DEG C of base mismatch pair of often successively decreasing increases by 1% (Bonner andLaskey, Eur J Biochem 46：83、1974.Formamide is optional under these hybridization conditions.Accordingly, severity is defined below in specific embodiment：Low stringency is 6 × SSC buffer solutions, and 0.1%w/v SDS are at 25-42 DEG C；Middle stringency is 2 × SSC buffer solutions, and 0.1%w/v SDS are at 20 DEG C to 65 DEG C of temperature range；High stringency is 0.1 × SSC buffer solutions, and 0.1%w/v SDS are at a temperature of at least 65 DEG C.

As used herein, term " common translation is modified or posttranslational modification " is related to when peptide chain is translated or occurred after translation covalent bond modification.Common translation is modified or posttranslational modification includes but is not limited to acylated (including acetylation), amidatioon or deamidation, biotinylation, carbamylation (or carbamylation), carboxylation or decarboxylation, two sulphur alkali are formed, fatty-acylation (including myristoylation, palmitoylation and octadecane are acylated), formylated, saccharification, glycosylation, hydroxylation, selenocysteine is combined, lipid, class resin acid addition, methylate, N- or C- endcappeds, N- or C- ends are removed, nitrification, it is methionine oxidized, phosphorylation, proteolytic cleavage, prenylation (including farnesylation, Mang ox base), phosphopyridoxal pyridoxal phosphate addition, sialylated or asialoglycoprotein, sulfation, ubiquitination (or ubiquitination) or Ubiquitin Like Proteins addition.

Acylation includes the hydrolysis of N- ends initial methionine and acetyl group is attached to new N-terminal amino acid.Acetyl group Co-A is the acetyl donor of acylation.

Amidatioon is the c-terminus of peptide and the covalent bond of amide groups and the stability for bioactivity and albumen is generally necessary.Go the hydrolysis removal that amidatioon is amide groups.Acid amides comprising amino acid residue goes amidatioon to be rare deformation, and it is by organism completion to recombinate 3D structures and change electric charge ratio/pI.

Biotinylation effect is that thus biotinyl is attached on molecule a technology; it is catalyzed or is carried out in vitro by holocarboxylase synthetase in the biosynthetic process of enzyme; it is intended to the visible special substrate of probe and the avidin hatching by using biotin labeling, or is intended to be connected to any streptavidin of many kinds of substance by physics and chemistry analytical control.

Carbamyl is transferred to acceptor portion such as amino by carbamylation (or carbamylation) from the molecule (such as carbamyl phosphate) containing carbamyl.

The carboxylation of glutaminic acid residue is the formation (Gla residues) that vitamin k-dependent reacts that it causes gamma-carboxyl glutamate.Gla residues are present in some protein of coagulation cascade, and it is required for the biological function of protein.Carboxylation can also betide asparatate residue.

Disulfide bond is the covalent bond of the disulphide formed when the sulfydryl of two cysteines is oxidized.Many mammalian proteins include disulfide bond, and it is for the generation and maintenance of tertiary protein structure, and such biological activity is conclusive.

Protein synthesis in bacterium includes the formylated of N- tenninal methionines and goes formylated.This formylated/go not occur in the formylated cytoplasm for circulating in eukaryotic and be the exclusive feature of bacterial cell.In addition to occurring a part of the hydroxylating in glycine residue as amidation process, under proline and lysyl hydroxylase catalysis (Kivirikko et al.FASEB Journal 3 can also occur on proline and lysine for hydroxylating：1609-1617、1989).

Saccharification is that glucose or other carbohydrates are uncontrolled, the amino acid backbone for being attached to protein of non-enzymatic.

Glycosylation is that sugar unit is attached to polypeptide backbone and will described further below.

Hydroxylation is the reaction of the vitamin C dependence as confactor.Hydroxylation is due to binding site of the hydroxyl lysine as glycosylation as the increase of the importance of posttranslational modification.

Selenoprotein is the protein of the selenium containing rare element, by adding unique amino acid, selenocysteine in translation process.TRNA for selenocysteine substitute serine and then enzyme seleno to produce selenocysteine-tRNA.Selenocystine-tRNA antisense codon and the terminator codon in mRNA (UGA) influence each other replacement serine codon.An element in selenoprotein mRNAs 3 ' non-translational regions (UTR) determines that UGA pronounces termination codon and given or selenocysteine codon.

Lipidization is a covalently bound general name for including lipid on protein, and it includes fatty-acylation effect and prenylation.

Fatty-acylation effect includes covalent attachment thing such as 14 myristic acids (myristoylation) of aliphatic acid, 16 carbon palmitic acids (palmitoylation) and 18 carbon stearic acid (octadecane acylation).Aliphatic acid is connected to protein in preceding-Gorky separates and can be with targeting (the Blenis and Resh Curr Opin Cell Biol 5 (6) of regulatory protein confrontation film：984-9,1993).Therefore fatty-acylation effect is important (BernsteinMethods Mol Biol 237 in the functional activation of protein：195-204,2004).

Prenylation includes the combination of prenyl, i.e. 15 carbon farnesyls or 20 carbon Mang oxen-Mang ox base and receptor protein.Isoprenoid compounds, including farnesyl chloroquine or Mang ox benzylacetone chloroquine, are obtained in Biosynthesis of cholesterol approach.On the cysteine residues that isoprenoid base is attached in appropriate consensus sequence CAAX by thioether bond (wherein A is any aliphatic amino acid in addition to alanine), the c-terminus of protein is positioned at.Prenylation change protein and the united ability of lipid membrane and it is all known to GTP- combinations aminosal (G-protein) modify in this way so that prenylation is conclusive to signal transduction.(RandoBiochim Biophys Acta 1300(1)：5-16、1996；Gelbet al.Curr OpinChem Biol 2(1)j：40-8、1998).

Class resin acid is vitamin-like antioxidant, is used as the free radical of scavenger.Lipoic acid lysine is formed by lipoic acid protein ligase and class resin acid is attached on the lysine with reference to acid amides.

It is that a kind of common modification can be with the activity of regulatory protein matter or the new amino acid classes of generation that albumen, which methylates,.Protein methyltransferase by methyl from SAMe be transferred to albumen in nucleophilic oxygen, nitrogen or sulphur atom.The effect that methylates is divided into two kinds of general classification.First, the relative level of transmethylase and methyl esterase can control methylation on special carboxyl, the activity of its regulatory protein matter in turn.This methylate is reversible.Second group of protein methylation reaction includes the irreversible modification of sulphur or nitrogen-atoms in protein.This reaction produces the new amino acid with the physicochemical characteristicses changed, and it changes activity (the Clarke Curr Opin Cell Biol5 of protein：977 983、1993).

Protein nitration is important posttranslational modification, and it is carried out in nitrous oxide signal transduction.The nitrification regulation catalytic activity of protein, cell signal and cytoskeleton organization.

Phosphorylation is related to the phosphate addition to protein kinase.Serine, threonine and tyrosine residue are the amino acid being phosphorylated.Phosphorylation is a kind of important mechanism, the bioactivity of its regulatory protein matter.

Most of protein is also modified by proteolytic cleavage.It can only include the removal of initial methionine.Other protein are synthesized in inactive precursor form, are activated by restricted or specific proteolysis.In order to secrete or signal sequence of the synthesis with the main hydrophobic amino acids of 12-36 with the albumen (preceding albumen) of film combination, it is after by being removed during ER films.

Phosphopyridoxal pyridoxal phosphate is the coenzyme derivative of vitamin B6 and participates in the transamination of amino acid side chain, decarboxylation, racemization, and many modifications.All phosphopyridoxal pyridoxal phosphates-desirability enzyme is worked by the formation of schiff bases between amino acid and coenzyme.The enzyme that most of dependence phosphopyridoxal pyridoxal phosphate bases are combined with lysine residue is self activation.

Sialylation is related to the terminal position that sialic acid is attached to glycoprotein by various sialyltransferases；And asialoglycoprotein is related to the excision of sialic acid.Sialic acid includes but is not limited to, N-acetyl-neuraminate (NeuAc) and NeuGc ALPHA2-3Gal (NeuGc).Sialic acid structure is caused by glycoprotein is sialylated, including sialic acid Lewis structures, for example, sialic acid Lewisa and sialic acid Lewis x, and sialic acid T structures, for example, sialic acid-TF and sialic acid Tn.

Sulfation occurs in tyrosine residue and is catalyzed by the enzyme tyrosine protein sulfurtransferase for being present in wire side on the outside of Gorky.Have determined that by the albumen within 1 to 20 that HepG2 cells are secreted and by least one tyrosine sulphate residue of the albumen within the 1 to 3 of fibroblasts to secrete.Sulfation is found to have an impact the bioactivity of albumen.It is particularly interesting that CCR5, main HIV co-receptors, optimal attachment and optimal HIV co- function of receptors of the discovery by the sulfation of one or more tyrosine residues in tyrosine sulfation and CCR5 N- terminal extracellular domains for MIP-1alpha/CCL3, MIP-1 beta/CCL4 and RANTES/CCL5 are necessary (Moore J Biol Chem 278 (27)：24243-24246,2003).Sulfation can also occur on carbohydrate.In addition, the sulfation of the carbohydrate fraction of glycoprotein can occur by the activity of the sulfurtransferases of sugared sulfurtransferase such as GalNAc (β 1-4) GlcNAc (β 1-2) Man α 4.

Posttranslational modification can include protein-protein bonding.Ubiquitin is a kind of 76 aminoacid protein, and it both can also be covalently attached to other protein with itself combination in mammalian cell.Adhered to by the peptide bond between the amino of the lysine residue in the C-terminal of ubiquitin and other protein.The attachment of the chain and target protein of ubiquitin molecule, which is targeted, to be tended to by proteasome proteolytic and for the steady state levels of regulation regulatory protein matter, such as relevant with cell cycle protein, is a kind of important mechanism (Wilkinson Annu Rev Nutr 15：161-89、1995).On the contrary, single ubiquitination can be played an important role in the direct regulation of protein function.Ubiquitin Like Proteins can also be covalently attached on protein to influence their functional metabolism, including NEDD-8, SUMO-1 and Apg12.

Glycosylation is attachment of the saccharide residue on polypeptide backbone.Saccharide residue, such as monose, disaccharides and oligosaccharides include but is not limited to：Trehalose (Fuc), galactolipin (Gal), glucose (Glc), amine-galactose (GalNAc), gucosamine (GlcNAc), mannose (Man), N-acetyllactosamine (lacNAc), N, N '-diacetylamino lactose (lacdiNAc).These sugar units can be attached on polypeptide backbone at least seven kinds modes, i.e.

(1) consensus Asn-X-Ser is attached to by N- glycosidic bonds；Asn-X-Thr；Or the R- bases (N- glycosylations) of the asparagicacid residue in Asn-X-Cys.

(2) serine is attached to by O glycosidic bonds, threonine, hydroxy-proline, the R- bases (O- glycosylations) of tyrosine or oxylysine.

(3) the R- bases C- connection mannoses of tyrosine are passed through；

(4) glycophosphatidyl inositol grappling is used to some protein being fixed to cell membrane；

(5) GlcNAc of R- bases of serine or threonine is connected to as signal monose.The connection is usually reversible to adhere to (Yin-o-Yang) with inorganic phosphate；

(6) linear polysaccharide is to serine, the attachment (proteoglycans) of threonine or aspartic acid；

(7) the R- bases of cysteine are connected to by S-glycosides key.

Glycosylation structure can include one or more sugar antigens determinants following in table 7.

Table 7

Sugar antigens determinant list

Antigen title	O antigen polysaccharide o structure
		Blood group H (O), 1 type	Fuc(α1-2)Gal(β1-3)GlcNAc-R
Blood group H (O), 2 types	Fuc(α1-2)Gal(β1-4)GlcNAc-R
		Blood group A, 1 type	GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-3)GlcNAc-R
Blood group A, 2 types	GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)GlcNAc-R
		Blood group B, 1 type	Gal(α1-3)[Fuc(α1-2)]Gal(β1-3)GlcNAc-R
Blood group B, 2 types	Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)GlcNAc-R
		Blood group i	[Gal(β1-4)GlcNAc(β1-3)]nGal(β1-R
Blood group I	Gal(β1-4)GlcNAc(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(   β1-4)GlcNAc(β1-3)Gal(β1-R
		Lewisa(Lea)	Gal(β1-3)[Fuc(α1-4)]GlcNAc-R
Sialic acid l Lewis a (sLea)	NeuAc(α2-3)Gal(β1-3)[Fuc(α1-4)]GlcNAc-R
		Lewis b(Leb)	Fuc(α1-2)Gal(β1-3)[Fuc(α1-4)]GlcNAc-R
Lewis x(Lex)	Gal(β1-4)[Fuc(α1-3)]GlcNAc-R
		Sialic acid l Lewis x (sLex)	NeuAc(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc-R
Lewis y(Ley)	Fuc(α1-2)Gal(β1-4)[Fuc(α1-3)]GlcNAc-R
		Forssman	GalNAc(α1-3)GalNAc(β1-3)Gal-R

Antigen title	O antigen polysaccharide o structure
		Thomsen-Friedenreich(TF   or T)	Gal(β1-3)GalNAc(α1-O)-Ser/Thr
Sialic acid l-TF (sTF) or Sialic acid l-T (sT)	Gal(β1-3)[NeuAc(α2-6)]GalNAc(α1-O)-Ser/Thr
		Tn	GalNAc(α1-O)-Ser/Thr
Sialic acid l Tn (sTn)	NeuAc(α2-6)GalNAc(α1-O)-Ser/Thr

Carbohydrate can also include some feeler structures, including list, double, three and four outboard structures.

Glycosylation can be by N linked glycosylations, O linked glycosylations, C connection mannose structures, and glycophosphatidyl inositol grappling presence, missing or pattern；Carbohydrate mass percent；Ser/Thr-GalNAc ratios；It is single, two, three and tetrose structure ratio or pass through agglutinin or antibody binding is determined.

The sialylation of protein can be determined by the immunoreactivity of protein and a kind of anti-antibody of specific sialic acid structure.For example, Lewis x distinct antibodies and the CEACAM1 reactions expressed by granulocyte but recombined human CEACAM1 reactions (the Luckaet al Glycobiology 15 (1) not expressed with 293 cells：87-100、2005).Optionally, presence of the sialic acid structure in protein can be by the mixture of glucosides ferment treatment through appropriate measurement process such as mass spectrum (MS), high performance liquid chromatography (HPLC) or sugared mass fingerprint (GMF) detection.

The apparent molecular weight of protein includes all constituents (confactor and non-covalent bond domain) and the modification of all common translations or the posttranslational modification of albumen composition (covalent groups cut off covalent groups to the attachment of peptide or on peptide).Apparent molecular weight is generally influenceed by common translation modification or posttranslational modification.The apparent molecular weight of protein can determine that it is also two dimension in its two-way analog, 2D-PAGE (two dimensional polyacrylamide gel electrophoresis) by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).However, the apparent molecular weight of protein can be by mass spectrum (MS)-by laser desorption Ionization-Time of Flight (MALDI-TOF) MS of the matrix-auxiliary for producing the electronic and ionic changed or can produce more sensitive electro-spray ionization (ESI) MS at multiple electrically charged peaks and more accurately determine.The apparent molecular weight of protein or its chimeric molecule can be in the range of 1 to 1000kDa.Accordingly,The protein or chimeric molecule of the separation of the present invention have 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,429,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444,445,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,510,511,512,513,514,515,516,517,518,519,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542,543,544,545,546,547,548,549,550,551,552,553,554,555,556,557,558,559,560,561,562,563,564,565,566,567,568,569,570,571,572,573,574,575,576,577,578,579,580,581,582,583,584,585,586,587,588,589,590,591,592,593,594,595,596,597,598,599,600,601,602,603,604,605,606,607,608,609,610,611,612,613,614,615,616,617,618,619,620,621,622,623,624,625,626,627,628,629,630,631,632,633,634,635,636,637,638,639,640,641,642,643,644,645,646,647,648,649,650,651,652,653,654,655,656,657,658,659,660,661,662,663,664,665,666,667,668,669,670,671,672,673,674,675,676,677,678,679,680,681,682,683,684,685,686,687,688,689,690,691,692,693,694,695,696,697,698,699,700,701,702,703,704,705,706,707,708,709,710,711,712,713,714,715,716,717,718,719,720,721,722,723,724,725,726,727,728,729,730,731,732,733,734,735,736,737,738,739,740,741,742,743,744,745,746,747,748,749,750,751,752,753,754,755,756,757,758,759,760,761,762,763,764,765,766,767,768,769,770,771,772,773,774,775,776,777,778,779,780,781,782,783,784,785,786,787,788,789,790,791,792,793,794,795,796,797,798,799,800,801,802,803,804,805,806,807,808,809,810,811,812,813,814,815,816,817,818,819,820,821,822,823,824,825,826,827,828,829,830,831,832,833,834,835,836,837,838,839,840,841,842,843,844,845,846,847,848,849,850,851,852,853,854,855,856,857,858,859,860,861,862,863,864,865,866,867,868,869,870,871,872,873,874,875,876,877,878,879,880,881,882,883,884,885,886,887,888,889,890,891,892,893,894,895,896,897,898,899,900,901,902,903,904,905,906,907,908,909,910,911,912,913,914,915,916,917,918,919,920,921,922,923,924,925,926,927,928,929,930,931,932,933,934,935,936,937,938,939,940,941,942,943,944,945,946,947,948,949,950,951,952,953,954,955,956,957,958,959,960,961,962,963,964,965,966,967,968,969,970,971,972,973,974,975,976,977,978,979,980,981,982,983,984,985,986,987,988,989,990,991,992,993,994,995,996,997,998,999,1000kDa apparent molecular weight.The molecular weight or molecular mass of protein can pass through any convenient method determination, such as electrophoresis, mass spectrum, gradient centrifugation.

The isoelectric point (or pI) of protein is pH when albumen does not carry net charge.The attribute can be determined by isoelectric focusing (IEF), and it is also the one-dimensional of 2D-PAGE.Experimental determination pI values can be up to 5 units by the difference between the pI and the pI of theory that the scope of common translation modification or posttranslational modification is influenceed and is therefore tested.Accordingly,The protein or chimeric molecule of the separation of the present invention can have 0,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2.0,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4.0,4.1,4.2,4.3,4.4,4.5,4.6,4.7,4.8,4.9,5.0,5.1,5.2,5.3,5.4,5.5,5.6,5.7,5.8,5.9,6.0,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7.0,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8.0,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9,9.0,9.1,9.2,9.3,9.4,9.5,9.6,9.7,9.8,9.9,10.0,10.1,10.2,10.3,10.4,10.5,10.6,10.7,10.8,10.9,11.0,11.1,11.2,11.3,11.4,11.5,11.6,11.7,11.8,11.9,12.0,12.1,12.2,12.3,12.4,12.5,12.6,12.7,12.8,12.9,13.0,13.1,13.2,13.3,13.4,13.5,13.6,13.7,13.8,13.9,Or 14.0 p I.

Term " isoform " as used herein represents a kind of different kinds of molecules form of given albumen, and is included in protein (1) primary structures (such as due to displacement RNA montages, or polymorphism) different in following level；(2) secondary structure (such as due to different common translation modifications or posttranslational modification)；And/or (3) three or four structure (such as because different subunits interacts, with-or iso- oligomer multimerization).Special, term " isoform " includes sugar-type, and it includes with continuous primary structure but modified or posttranslational modification in two grades or tertiary structure, or common translation, such as different glycosylation form, different protein or its chimeric molecule in level.

The chemical stability of protein can be measured in " half-life period " form of protein in special solvent or environment.Representational, the protein having less than 50kDa molecular weight has the half-life period of about 5 to 20 minutes.The protein or chimeric molecule of the present invention is contemplated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, the half-life period of 99 or 100 hours.Another chemical stability is especially easily measured as the resistance that protein or its resistance molecule are acted on protease digestion, such as trypsase or pancreas milk reducing protease digesting effect.

Protein or its chimeric molecule can be measured its part or the affinity of acceptor in the form of equilibrium segregation coefficient (Kd) or function equivalence measurement.

The dissolubility of protein can be by being dissolved in the Tot Prot of given solvent and/or the wherein measurement of the ratio of proteolytic.Further, protein or its chimeric molecule are in heterogeneity such as polarity, pH, and the ratio and/or level dissolved in the solvent of temperature etc. can also provide protein or measurable physicochemical property of its chimeric molecule.

Any " measurable physical and chemical parameter " can determine that measurement quantifies or limited with known any method for a person skilled in the art.It is described below and is determined for, measures, quantify or limit the scope of the methodology of the protein of one or more separation or measurable physical and chemical parameter of its chimeric molecule.However, it should be understood to that the present invention is never simply defined as described special method, or it is measurable physical and chemical parameter to be defined to using these methods.

Glycoprotein can be described as having two interactions to produce one as overall molecule element-amino acid sequence and carbohydrate or sugared side chain.The carbohydrate part of molecule exists with the monose or oligosaccharide side chains form that are attached to by N- or O- keys on the hydroxyl side chains of Asn amino side chains or Ser/Thr residues respectively.Monose is the term on carbohydrate least unit, and it is considered as a sugar, with (CH₂O)_nBasic chemical formula and most of be usually formed 5 or six-membered cyclic structure (being respectively pentose and hexose).Oligosaccharides is the compound of the monose molding structure with Various Complex, and it can be linear or branch, but does not have the long-chain of tandem repeat unit generally (it is a kind of form of polysaccharide).The branch levels and the branch of end that oligosaccharides contains, which are replaced, significantly influences the feature of the glycoprotein as an entirety, and is played an important role on the biological function of molecule.Oligosaccharides is prepared and is attached in amino acid backbone in the endoplasmic reticulum (ER) and golgiosome of cell.Different organisms and the species of cell have the ratio of different glycosyl transferase and endoglycosidase and exoglycosidase and therefore produce different oligosaccharide structures.One of main defense mechanism of body is to find and destroy abnormal isoform, and same have correct glycosylated biopharmaceuticals to be neutralized being the discovery that for antibody important not only for heightening the effect of a treatment also for reducing.

Polysaccharide chains are generally expressed with branched form, even and if when it is linear, such chain is generally by a variety of modifications.Therefore, the complete sequence of oligosaccharides is difficult to be completed by single method and therefore need the combination repeatedly of physics and chemical method and finally obtain the details of studied structure.

Determining for the glycosylation pattern of protein can be carried out by using many different methods, for example, use SDS-PAGE.The fact that this technology is generally divided a word with a hyphen at the end of a line dependent on glycosylated protein in SDS-PAGE with different diffusion zones.The progress of differentiation between different isoforms is by using a series of agent treatment protein.For example, considering the glycosylated discrimination of N connections with the significant reduction of bandwidth after the digestion of peptide-N4- (N- acetyl-β-GLUCOSAMINE base) asparagine acid amides enzyme (PNGase) and the change for position of dividing a word with a hyphen at the end of a line.

In order to determine the glycosylated component of N connections, N- sugar chains from flavobacterium meningosepticum with cloning and cut off from protein in the PNGase of expression in escherichia coli.The N- sugar chains of excision can be from such as Packer et al Glycoconj J 5 (8)：737-47, the Alltech Carbograph SPE carbon posts (Deerfield, Illinois, USA) described in 1998 are reclaimed.Then, sample can carry out Monosaccharide analysis, saliva acid analysis or sulfate analysis with the Dionex systems with GP50 pumps ED50 pulsed amperometrics meter or electric conductivity detector and a variety of pH anion-exchange columns.

The glycosylated degree of O- connections can be determined by cutting off O- sugar chains from target protein first through β-elimination.The O- sugar chains of excision can be reclaimed as described in Packer et al. (1998, as above) from Alltech Carbograph SPE carbon posts (Deerfield, Illinois, USA).Then, sample can carry out Monosaccharide analysis, saliva acid analysis or sulfate analysis with the Dionex systems with GP50 pumps ED50 pulsed amperometrics meter or electric conductivity detector and a variety of pH anion-exchange columns.

The monose subunit of oligosaccharides has variable sensitivity to acid and therefore can discharged under the conditions of slight trifluoroacetic acid (TFA) condition, moderate TFA conditions, and strength hydrochloric acid (HCl) from destination protein.Then, mixture of monosaccharides is separated by using the high pH anion-exchange chromatographies (HPAEC) of a variety of post filled medias, and is detected with pulsed amperometric detection method (PAD).

High pH anion-exchange chromatographies have been widely used in measure monosaccharide component with Pulse amperometric detection method (HPAEC-PAD).Fluorescence-labeling method has been incorporated into and much used in a kit form.The obvious advantage of fluorescent method is sensitiveness enhancing (about 50 times).When one potential deficiency is coupling reaction, in hydrolysate and in external standard mixture, different monose can show different selectivity to different fluorophors.However, the enhancing of sensitiveness and differentiate the ability which monose is present from the sub-fraction of the total amount of actual glycoprotein, and the stronger sensitiveness with laser induced fluorescence potentiality so that this method is very attractive.Other electric conductivity detector can be used for determining sulfate and phosphate component.According to code is used, peak area can calculate the total amount of every kind of monose of presence.These data can represent the glycosylated level of N- and O- connections, sialylated degree, and amino acid composition in compound, glycosylate percentage by weight, acidoglycoprotein percentage by weight.

A small amount of monosaccharide composition analysis of protein is carried out preferably after the electric marking with PVDF (PSQ) film, or, less amount is analyzed with dot blot.PVDF is the preferable matrix of carbohydrate analysis, because being once all not joined to through peracid or enzyme hydrolysis, monose and oligosaccharides on film.

The measure of the oligosaccharide content of molecules of interest is carried out by many technologies.Sugar is cut off first from amino acid backbone by (such as being eliminated with hydroxide β) method of (such as being digested with PNGase) of enzyme or chemistry.Sugar can be by reducing stable or being made to be easy to detection with fluorescence labeling.Then, the free oligosaccharides of generation is separated, high pH anion-exchange chromatographies and pulsed amperometric detection method (HPAEC-PAD) can be passed through, it can be used in known standard to determine the ratio and sialylated level of various structures, or pass through fluorescence assisted carbohydrate electrophoresis (FACE), a kind of method separated similar to protein s DS-PAGE.In this process, oligosaccharides is marked with fluorophor, and it has impact on electrophoretic mobility.The banding pattern that they are separated and obtained in the polyacrylamide gel of high percentage provides the feature of the oligosaccharide content of molecules of interest.By using standard sample, obtaining some information or band of the practical structures existed can be cut and be analyzed with mass spectrum, can determine the structure of each of which.

Fluorescence assisted carbohydrate electrophoresis (FACE) is a kind of polyacrylamide gel electrophoresis system, designed for separating the single oligosaccharides discharged from glycoconjugate.Oligosaccharides by chemistry or enzyme method from sample protein matter be removed, reducing end is remained in this way.Then, oligosaccharides is digested as monose or keeps complete, and is fluorescently labeled (electrically charged or uncharged).High percentage polyacrylamide gel and a variety of buffer systems are used to migrate oligosaccharides/monose, and it is migrated relative to their size/component in the mode almost identical with protein.Carbohydrate is visualized by optical densitometric method and the relative amount of sugar can be measured by fluoroscopic examination.This process is consistent with MALDI-TOF MS, therefore this method can be used for illustrating practical structures.

Quartz crystal microbalance and surface plasma resonance (being respectively QCM and SPR) are the two methods of the physicochemical property acquisition biological information by molecule.Both measure protein-protein Indirect Interaction by the change of the physical features of fabricated chip caused by interaction.Single quartz crystal slice is handled with acceptor/antibody interacted with target ligand etc. in QCM.This chip is vibrated by microbalance and the frequency of chip is recorded.Target protein is allowed through chip and causes the frequency shift of thin slice with the interaction of molecules of combination.By the change of the condition of the interaction of part and chip, the binding characteristic of molecules of interest can be determined.

Apparent molecular weight is also a kind of physicochemical characteristicses, and it can be used for determining the similitude between the present invention and those protein produced with selectively mode or chimeric molecule.

As used herein, term " molecular weight " is defined as the summation of the atomic weight of composed atom in molecule, is directed to sometimes " molecular mass " (Mr).Molecular weight can add up to determine by the atomic mass to composed atom in molecule in theory.Term " apparent molecular weight " is defined as the molecular weight determined by one or more analytical technologies such as SDS page or ultracentrifugation, and dependent on the relation between molecule and detecting system.The apparent molecular weight of protein or its chimeric molecule can be measured with any of a series of experiments method.The analysis method of molecular weight for determining protein includes, and is not limited to, exclusion chromatography (SEC), gel electrophoresis, Rayleigh light scattering, analytical ultracentrifugation and, in a way, time-of-flight mass spectrometry (TOFMS).

Gel electrophoresis is the assay method of some physicochemical characteristicses (particularly apparent molecular weight and DI) of protein and on molecule to be separated into the dielectrophoresis of isoform, so as to provide the information of protein product posttranslational modification.Specifically, electrophoresis is to force charged molecule (such as protein or DNA) to be divided a word with a hyphen at the end of a line the method by gel-type vehicle (most of common polyacrylamides or agarose), passes through the use of the current potential through colloid.Most common electrophoretic type for protein is isoelectric focusing, non denatured, and sds polyacrylamide gel electrophoresis.Protein is placed in the polyacrylamide gel with pH gradient therebetween through in isoelectric focusing.Albumen will migrate to a position in gel, the net charge that it is zero that albumen, which has, in this place, so as to provide the isoelectric point of albumen.

Sugared quality fingerprinting (GMF) is a kind of method, through this method, and the oligosaccharides feature of one of protein or its isoform is accredited by electrophoresis and subsequent special mass-spectrometric technique.Sample protein matter is purified by the 1D SDS-PAGE that are determined for total protein or for the 2D gel electrophoresises of special isoform characteristic.Protein band/spot cuts off and decolourized to remove pollutant from gel.Carbohydrate and by chemistry or enzyme process release and using nanoflow LC systems desalination/separation then identify the oligosaccharides being present in sample.LC streams, which can directly be expelled to Electrospray Mass instrument, (is used for quality measurement and then discriminating amount is present in sample) its feature or fingerprint for providing each isoform, quantitative technique such as Dionex analyses can be combined, to determine total component of tested molecule.

Primary structure can be assessed by the physicochemical characteristicses for the protein or chimeric molecule for determining the present invention.

The primary structure of protein or its chimeric molecule can use one or more following systems to be analyzed.

The information of the primary structure of protein or its chimeric molecule can be constituted with mass spectrum (MS), DNA sequencing, amino acid, the combination of protein sequencing and peptide quality fingerprinting is measured.

In order to determine the sequence of amino acid backbone, N- terminator sequencing chemistries, tandem-mass spectrometry sequencing, or both combination can use.N- terminator sequencing chemistries utilize Edman chemistry (Edman P. " Sequence determination " Mol Biol Biochem Biophys 8：211-55,1970), peptide bond described in it between protein N-temiinal amino acid and the amino acid of 2 peptide bond more every other than in sequence is weak.By using intermediate acidity's condition, -terminal amino acid is removed, and derivative is the retention time with fluorophor (FTIC) and measure in reversed-phase HPLC post, and which kind of amino acid is compared with standard sample to determine is.This method can determine the actual primary structure of molecule but not be quantitative.Optional, nanoflow liquid chromatograies tandem-mass spectrometry can be used (LC-MS/MS).In this method, protein is hydrolyzed to peptide using special endo protease and the molecular weight of peptide is determined.Then it is broken peptide bond with energetic encounter gas such as nitrogen or argon and measures the quality of the peptide of acquisition.By the change for the quality for calculating peptide, the sequence of each peptide can be determined (every kind of amino acid has unique quality).Then by using different protease, peptide can overlap the order to determine them and thus determine the complete sequence of protein.

Clearly, enzymic digestion, chemically derived, liquid chromatogram (LC)/MS and series winding MS combination provide a kind of very effective instrument, for AA sequence analyses.For example, the detailed construction of recombinant soluble CD4 acceptor is characterized by the combination of method, have determined the primary structure of the 369AA glycoprotein more than 95% and have been found that the complete characteristic at N- and C- ends, correct configuration (the Carr et al.J BiolChem 264 (35) of the attachment position of polysaccharide, polysaccharide structure and disulfide bridge bond：21286-21295、1989).

Mass spectrum (MS) is a kind of method that deduction by the behavior of molecule in conductive environment under vacuum measures molecular mass.MS is highly useful in stability study and quality control.This method requirement first carries out (trypsase with proteolytic enzyme, V8 protease, chymotrypsin, subtilopeptidase A, and clostripain) treatments of the sample (Franks et al.Characterization of proteins, Humana Press, Clifton, NJ, 1988；Hearn et al.Methods in Enzymol 104：190-212,1984) then, passes through the separating digesting sample of RP chromatography (RPC).With Trypsin Induced combination LC-MS, peptide mapping can be used for detecting genetic stability, the homology of product batch, and fermentation, and purification, formulation prepares the stability with protein in storage process.

Before quality analysis, some modes are used to HPLC being connected to mass spectrograph：1) direct liquid injection；2) supercritical fluid；3) conveyer belt system；4) thermal spraying.Eluent in post is transported to the sample probe joint of mass spectrometric chamber for Caprioli ' the s HPLC/MS connections operated using Fused-silica capillary column.When sample solution appears in capillary tube connector, probe joint is continuously bombarded with the Xe atoms of high energy, causes sample solution to sputter.Then quality passes through Instrumental Analysis (Caprioli et al.Biochem Biophys Res Commun146：291-299、1987).

MS/MS and LC/MS connections have been expanded MS and potentially applied.MS/MS allows direct discriminating (the Carr et alAnal Chem 63 of part, deacylation amine site and the isomerization of more than 25AAs peptide complete sequence：2802-2824、1991).RPC or Capillary Electrophoresis (CE) are combined with MS can carry out high sensitive analysis (Figeys and Aebersold, Electrophoresis 19 of protein：885-892、1998；Nguyen et al.J ChromatogrA 705：21-45、1995).LC/MS allows the LC methods of the isolated peptides before MS is entered, the constant current FAB being for example connected with micropore HPLC (Caprioli et al.1987, as above)." connection " below allows the sequencing of the single peptide from complex mixture：Selected peptide is broken by first time MS, is then passed through the ion cloud of collision cell：CID (collision induced dissociation).Collision produces the characteristic set of fragment, it is possible thereby to sequence is inferred to, without knowing other information, such as cDNA sequence.In single MS experiments, those of mixture (for example, from enzymic digestion) injection and the quality of leading ion with being expected by cDNA sequence that are not classified of peptide are compared.Fast atom bombardment (FAB)-MS analysis and proteolysis of the sequence of RhIL-2 by CNBr have been verified (Fukuhara et al.J Biol Chem260：10487-10494、1985).

Electro-spray ionization MS (ESI-MS) is inserted in pin under high voltages using proteolytic aerosol, produces a series of electric charge peak of identical molecules with a variety of electric charges.Because the peak for being each produced from different charge species produces the estimation of molecular weight, these estimations can combine to increase total accuracy of molecular weight estimation.Matrix assisted laser desorption ionization MS (MALDI-MS) uses high concentration chromophore.Higher-strength laser pulse by Matrix absorption and the energy evaporation section matrix absorbed and almost can bring protein example into gas phase completely.Then the MS flight time of the ion of acquisition is analyzed.The ionization of moderate can strengthen the ability that this method provides quaternary structure information.MALDI-MS can easily be run within 15.It does not need fragment chemoattractant molecule and when PAGE gel densitometric scan, as a result easily explains, for more than mass range 100kDa.

Amino acid sequence can be determined by determining the DNA of encoding proteins matter or its chimeric molecule.But, actual protein sequence is probably different once in a while.Generally, PCR reaction of the DNA sequencing reaction to replicating (DNA denaturation, duplication) DAN is similar.By DNA clone technology, the gene can be cloned and nucleotide sequence is determined.

The amino acid sequence of one or more following network analysis protein or chimeric molecule can be used.

Description to protein or the complete sequence of its chimeric molecule usually requires that the description product.Amino acid sequencing includes：Trypsin Induced is carried out on gel, fraction is carried out to the peptide of digestion with RPC-HPLC, the peptide peak with most symmetrical Absorption Characteristics is screened by MALDI-TOF MS, with the peptide of edman degradation first (N- ends).The primary sequence data that Ai Deman is chemically derived are the classical ways for determining protein on a molecular scale.MALDI-TOF MS can be used for N- terminal sequence analysis.But, all enzymic digestions and peptide sequencing for HPLC recommend to first pass through MS or MS/MS protein and differentiate to reduce time-consuming and reduce cost.After the endopeptidase digested protein separated from SDS-PAGE of the Lysyl by Trypsin Induced in situ or in matrix, separation is carried out with HPLC can obtain the amino acid sequence of inside.

Recommend that the inside of standard peptide is sequenced and is analyzed sample and runs together to make instrument maintain peak performance.Eukaryotic protein more than 80%, which is reported, closing amino-terminal end, hinders direct amino acid sequencing.When running into the eukaryotic protein of closing, the presence of internal standard can ensure that instrumentation is normal.

Edman degradation can be used to N-terminal direct Sequencing by chemical process, methods described derives N-terminal amino acid to discharge amino acid, exposes the amino terminal of next amino acid.Ai Deman sequencings include：1) N-terminal sequence analysis is repeated by Ai Deman chemical cycles by micropore HPLC in, and each Ai Deman chemical cycles can identify an amino acid.2) polypeptide generated in gels or after the digestion of PVDF associated proteins with HPLC separating digestings, internal sequential analysis of protein is carried out by edman degradation chemical method.

Peptide mixer is analyzed and purified using micropore HPLC and capillary HPLC and using RPC-HPLC methods.Use sample and PVDF samples in different pillar purifying gels.N-terminal analysis can be carried out using mastrix-assisted laser desorption ionization time of flight mass spectrum (MALDI-TOFMS) analysis of Matrix-assisted after HPLC segmentation separation.Selection standard is：1) apparent purity of HPLC fractions.2) quality of peptide and the length thus estimated.Peptide quality information is assigned with using to confirmation Ai Deman sequencing amino acid, and useful to the possible detection of translation or posttranslational modification.

(In-gel) digestion on gel is suitable for the purifying in high sensitivity HPLC system.Internal sequential analysis of protein carries out enzymic digestion by sds page (SDS-PAGE) first.Albumen in SDS-PAGE microgels can be reliably only by trypsase in digested in-gel.Purify fragments of peptides with RPC-HPLC, then with MALDI-TOF MS analyses, screening is adapted to the peptide of Ai Deman sequencing analysis.Albumen can only be analyzed with internal sequence analytic approach in gel, but can obtain very accurate peptide quality, and this, which can be provided, extra refers to the useful information of fixed sum data library searching to amino acid.

PVDF associated proteins are suitable to N-terminal and inside Ai Deman sequencing analysis.PVDF associated proteins for example hydrogenate Triton X-100 by suitable enzymic digestion (trypsase, interior protease Lys-C, interior protease Glu-C, clostripain, interior protease, Asp-N, thermolysin) and non-ionic detergent.In PVDF associated proteins, the mastrix-assisted laser desorption ionization time of flight mass spectral analysis for the detergent that the peptide of digestion discharges from film to can interfere with to Matrix-assisted.Before enzyme is added, cystine (Cys) is reduced with dithiothreitol (DTT) (DTT), and carboxyamidomethylation cystine is generated with iodoacetamide subsequently, can be identified in N-terminal sequencing analysis.

In order to determine that the amino acid of protein or its chimeric molecule is constituted, strength hydrochloric acid (HCl) the acid condition hydrolyzation sample being catalyzed in the gas phase with phenol.Once hydrolysis is completed, derive the amino acid of release with a kind of fluorophor compound, make reversed nature special on the molecular band of combination.Anti-phase high speed liquid chromatography (RP-HPLC) separates derivative amino, and is detected with fluorescence detector.Using outwardly and inwardly standard, the quantity of each amino acid in sample is calculated by the peak area observed.This information can be used for identification sample and to the protein quantification in sample.For example, the deviation of theoretical and actual result can be used for the possibility for initially determining that a desamidation position.It is combined with Monosaccharide analysis, it may determine that glycosylation percentage by weight composition and acidoglycoprotein percetage by weight.The limitation of this method is that it can provide the skeleton actual sequence information that inherent variation is produced because of the occasional breakage of environmental pollution and amino acid.Such as this method can not possibly detect the point mutation of sequence.

Peptide mass fingerprinting (PMF) is another method for being able to confirm that protein or its chimeric molecule.Its process is included initially with electrophoresis (1 dimension or 2 dimensions) separation sample, and point/band is cut from gel and is digested with special endo protease (typically with Porcine trypsin).Peptide is eluted from gel fragment and the quality of peptide existed is determined with mass spectral analysis.Then the Theoretical Mass debris database of the peptide quality of generation and the protein of all announcements is compared (or theoretical peptide quality of the implementation sequence built).The fact that this technology is unique dependent on " fingerprint " (i.e. its peptide mass combination) of protein.It can reliably identify that (degree of accuracy more than 90%) is small to 4 peptides and 30% sequential covering.Modification, such as fat part and desamidation can be identified in the PMF stages of analysis.Further analyzed by tandem mass spectrometry (MS-MS) with the peak that the protein of identification is not consistent, MS-MS technologies carry out colliding the energy of generation interrupting PTM weak bond using collision gas.Then quality is carried out to the molecule and original peptide that newly discharge to analyze to identify posttranslational modification and the fragments of peptides that it adheres to again.

It is different patterns to be divided HPLC according to the special component of size, electric charge, hydrophobicity, function or target biological molecules.Generally, a kind of protein is purified using two or more chromatographies.Above all to consider the characteristic of protein and sample solvent when selecting chromatogram mode

Their secondary structure can be evaluated by characterizing the protein of the present invention or the characteristic of its chimeric molecule.

Following one or more systems can be used to carry out the secondary structure of analysing protein or its chimeric molecule.

In order to study Secondary structure, it should use and relatively more the most frequently used several spectroscopic approach.Electromagnetic energy can be determined according to the size and shape of ripple with radiation continuous wave.Different spectroscopic approach uses different electromagnetic energy.

Wavelength is the length the distance between (two continuous wave maximum) of the single ripple of radiation.When radiant increase, wavelength shortens.Relation between frequency and wave number is：

Wave number (cm^-1)=frequency (s^-1)/the light velocity (cm/s)

Absorption of the molecule to electromagnetic radiation includes vibration and rotational transition and electron transition.The most popular method of measure molecular vibrational energy for identifying secondary structure is infrared (IR) and Raman spectrum.But their method is different with molecule absorption is determined.

Scatter emittance and be less than stokes line incident radiation.Scatter emittance and be more than anti-Stokes line incident radiation.Excite the base electronic state vibrational energy interval of the energy and molecule increased or decreased relevant.Therefore, Stokes and anti-Stokes line wave number are that the direct detection of molecular vibrational energy is measured.

Stokes shift (Stokes shift) is only observed in Raman spectrum.The wave number of stokes line is less than (or wavelength is more than) exciting light.High-energy excitaton source (such as laser) can be used to strengthen Raman scattering efficiency.Excitaton source should be monochromatic, because we are to exciting and the capacity volume variance (wave number) of stokes line is interested.

In order that vibration has infrared active (IR active), the dipole moment of molecule must change.Therefore, the symmetrical stretching, extension of carbonic anhydride is without infrared-active, because dipole moment does not change.It is that dipole moment is changed that asymmetry, which stretches the reason for having infrared active,.In order that vibration has Raman active (Raman active), the polarizability of molecule must occur vibration and change.The symmetry of carbonic anhydride, which stretches, Raman active, because the polarizability of molecule is changed.Therefore, Raman spectrum supplements infrared spectrum (Herzberg et al.Infrared andRaman Spectra of Polyatomic Molecules, Van Nostrand Reinhold, New York, NY, 1945).For example, in default of dipole moment motion, it is infrared to detect same core diatomic molecule, but Raman spectrum can be detected, because key stretches and shrinking is changed the polarizability of molecule, the interaction between this exoelectron and core is also changed.

For the polyatomic molecule (such as benzene) of the high degree of symmetry with upset center, it is possible to have bands of a spectrum activity in infrared spectrum and there is no bands of a spectrum activity in Raman spectrum, vice versa.Symmetry is low or chiral molecular in, it is possible to it is all active in infrared and Raman spectrum.

Infrared spectrum Detection wavelength and sample infrared absorption intensity.Infrared energy can make molecular vibration be energized into higher energy level.Infrared and Raman spectrum all detects the vibration of bond distance and bond angle.

It is infrared to characterize molecular vibration by detecting the light absorbs corresponding to molecule particular energy of the vibrational excitation of (or higher) state from v=0 to v=1.There are some to determine the selection of the ability of infrared spectrum detection molecules rule, infrared ray can not excite the vibration (Herzberget al.1945, as above) of all normal modes.

Infrared spectrum energy provides the qualitative and quantitative information of secondary protein structure, such as α spirals, β-pleated sheet, β-bend and irregular structure.Most useful infrared band in protein analysis is acid amides I (1620-1700cm^-1), acid amides II (1520-1580cm^-1) and acid amides III (1220-1350cm^-1).Acid amides I is the most strong absorption band of protein.Its stretching vibration comprising C=O (70-85%) and C-N groups (10-20%).Definite band position is determined by bone framework image and hydrogen bonding pattern.Acid amides II is more complicated than acid amides I.Acid amides II N-H in plane bend (40-60%), C-N (18-40%) and C-C (10%) stretching vibration and determined.Acid amides III bands use less (Krimm andBandekar, Adv Protein Chem 38：181-364、1986).Most of FTIR amide I bands B foldable structures are usually located at 1629cm^-1Left and right, minimum 1615cm^-1, maximum 1637cm^-1, secondary part may be in 1696cm^-1Neighbouring (minimum 1685cm^-1) display peak, α spirals are mainly in 1652cm^-1。1680cm^-1It is nearby β-bend.

The principle of Raman scattering is different from infrared absorption.The wavelength and intensity of the inelastic scattering light of Raman spectrum detection molecules.Raman scattering, molecular vibration energy makes lambda1-wavelength change, and produces Raman diffused light.

In order to there is Raman active, vibrate for inelastic scattering, its key is that polarizability changes in vibration.In symmetrical stretch, electronics bond strength is different between minimum and maximum nuclear separation.Therefore, polarizability changes in vibration, and this vibration mode scattering Raman light, and this vibration has Raman active.In asymmetric stretching, extension, the electronics that the electronics in the key of stretching, extension is easier in polarization, the key of compression is more difficult to polarization.Polarizability does not change generally, and asymmetric stretching, extension is no Raman active (Herzberg et al.1945, as above).

Circular dichroism can be used to detect any unsymmetric structure, such as protein.The different amounts of dextrorotation of optically-active chromophore absorption and left-handed rotation, this different absorption cause positive and negative absorption spectrum (right avertence vibrational spectrum generally, is subtracted from left-hand polarization spectrum).Generally, far ultraviolet or acid amides region (190-250nm) are mainly contributed by peptide bond, the information of amido link carbonyl environment is provided, therefore the information of secondary protein structure is provided, α spirals generally show two negative peak (Holzwarth et al J Am Chem Soc 178 at 208,222nm：350,1965), β-pleated sheet shows a negative peak in 196nm, and random coil shows a negative peak in 218nm.The peak of near ultraviolet region (250-350nm) by fragrant chromophore (Phe, Tyr, Trp) ambient contribution.Disulfide bond makes the minimum CD band increases near 250nm.

The three-dimensional structure that circular dichroism is generally folded with the height of side-chain structure tightly.Most protein is denatured Free up Memory steric hindrance, and denaturation degrees increase, circular dichroism spectra weakens.For example, hGH side chain circular dichroism spectra is very sensitive to adding partial denaturation caused by denaturant.Some reversible molecular chemistry changes, such as disulfide bond reduction or alkalimetric titration will change side chain circular dichroism spectra.For hGH, the change of the response of circle two of chromophore or the specific chromophore of influence is removed completely can cause these spectral differences, but denaturation or conformational change do not cause spectral difference (Aloj et al.J BiolChem 247：1146-1151,1971).

Ultra-violet absorption spectrum is to detect one of most efficient method of protein characteristic.It can provide the information of protein concentration and chromophore direct environment.Protein function group, such as amino, alcohol (or phenol) hydroxyl, carbonyl, carboxyl or sulfydryl can be converted into strong chromophore.It can be seen that be used to monitor two kinds of chromophore with near-ultraviolet spectrum：Metalloprotein (being more than 400nm) and the protein (260-280nm) containing Phe, Trp, Tyr residue.Ultraviolet or fluorescence signal change can be negative or positive, depend on protein sequence and solution properties.

Fluorescent detection molecules are radiated the energy launched after excited state.The aromatic amino acid of many protein is excited in 250 to 300nm, the fluorescence in the range of transmitting 300 to 400nm.Only the protein with Phe, Trp, Tyr residue can be detected, and intensity sequence is Trp》Tyr》Phe.Fluorescence spectrum can reflect the micro-loop environment information influenceed by protein folding.For example, the Trp of embedment is generally in hydrophobic environment, fluorescence is launched in the range of 325 to 330nm, but the residue or free amino acid of exposure launch fluorescence at 350 to 355nm.A kind of common agents for detecting protein stretching, extension are Bis-ANS.Bis-ANS fluorescence is pH dependent forms.Although its signal in water is weak, the hydrophobic site of the exposure that it can be stretched by being attached in protein increases signal (James and Bottomley Arch BiochemBiophy 356：296-300,1998).

In unfolded protein Tyr and Trp be effectively quenched cause protein stretch after signal dramatically increase.A kind of simple solute can also cause this change.In order that detection sensitivity is maximized, signaling rate can be used.For example, in research rFXIII stretches, using ratio (the Kurochkin et al.J Mol Biol248 of 350nm and 330nm fluorescence intensities：414-430,1995).Can be by the technique study conformational change that excitation energy is changed between fluorogenic donor and absorption acceptor, because conversion efficiency relies on the distance between both chromophores (Honroe et al.Biochem J 258：199-204,1989).Antitrypsin conformation (K won and Yu, Biophim Biophys Acta1335 are detected with fluorescence：265-272,1997), to determine HAS Tm (Farruggia et al.Int J BiolMacromol 20：43-51,1997), and detect that MerP stretches interaction (Aronsson etal.FEBS Lett.411：359-364,1997).

Under neutral ph, fluorescence emission spectrum intensity sequence is Trp ＞ Tyr.At acidic, because conformational change destroys ability conversion, Tyr fluorescence is better than Trp.Fluorescence experiments also confirm the intermediate in protein stretching, extension transformation caused by guanidine.

The three-level and quaternary structure of the protein of the present invention or the physics and chemistry form of chimeric molecule are to finding out that its function is also important.

Protein or the three-level and quaternary structure of their chimeric molecule can be determined using one or more following systems.

NMR and X ray diffractive crystal analysis are the most common techniques of research protein 3D structures.The method for detecting tertiary protein structure of other summaries includes two grades of derivatives of CD, ultraviolet spectra (Ackland et al.J Chromatogr 540 of near ultraviolet region：187-198,1991) and fluorescence.

NMR is one of main method of research molecular structure and intermolecular interaction in structure biology.In addition to studying protein structure, NMR can be used for the protein in the research present invention or the carbohydrate structure in chimeric molecule.Chemist studies the structure of chemical substance usually using simple one-dimensional NMR spectral technology.Two dimensional technique can be used to determine the structure of more complicated molecule.Time domain NMR be used to detect molecules in solution dynamics.Solid state NMR be used to determine solid-state molecular structure.NMR can be used for the structure and dynamics for studying protein, the nucleotides low molecular weight compound related to medical science to a variety of biological, pharmacology.But, such as not every core all has the appropriate characteristic that can be read by NMR, and not every core all has a spin, required for spin is NMR.Spin causes core to produce NMR signal, plays the function of small magnetic field.

The crystalline texture of one or more following system measurement protein or their chimeric molecule can be used.

X ray diffractive crystal analysis is a kind of experimental technique, and X-ray energy is by crystal diffraction, and the X-ray with suitable wavelength (in angstrom scope ,~10-8cm) is by the electron cloud institute diffraction of the atom of suitable size.The molecule or the diffraction pattern of atom X-ray diffraction combined according to the cycle in crystal, electron density can be reconstructed.Additional phase information can be obtained from diffraction data or in mending diffraction experiment, to complete reconstruct.Experimental electron density model is progressively then set up, according to data-optimized, very accurate molecular structure is as a result obtained.

X ray diffractive crystal analysis has developed into the structure with the stateful material of any ray research institute, and such as ion, electronics, epithermal neutron and proton, the distance between wavelength and atom to be measured or molecular structure are similar.

Light scattering spectrum is based on such a simple principle, and big particle is more than the light that small particles are scattered.In 310-400nm regions, based on a big KPT Scatter just inclined baseline, such as aggregation Schmid et al.Protein structure in solution, a practicalapproach, Creighton Ed., IRI Press, Oxford, England, 1989).

Light scattering spectrum can be used to assess protein molecular weight, and it is a kind of simple tool for monitoring quaternary structure of protein or protein aggregate.Protein aggregation degree can be characterized with simple Turbidity measurement.The pharmaceutical solutions finally produced carries out transparency inspection, because cloud and lacteous is presented in most of collectins.Quasi-elastic light scattering spectrum (QELSS), sometimes referred to as photon correlation spectroscopy (PCS) or dynamic light scattering (DLS), are a kind of non-intrusion type exploration technologies of macromolecular (protein, polysaccharide, synthetic polymer, micelle, colloidal solid and aggregation) complex fluid diffusion.In most examples, light scattering spectrum directly produces scattering class interdiffustion coefficient.When being applied to the monodisperse liquor of dilution, the diffusion coefficient that QELSS is obtained can estimate size.In polydisperse system, it estimates the width of molecular weight distribution.For accurate measure, using 200-500mW laser energies, widely used conventional Ar+/Kr+ gas lasers (Phillies Anal Chem62：1049A-1057A, 1990).Protein aggregate (Liet al.Biochemistry 34 are have detected with mankind's relaxin：5162-5772,1995).

The stability of protein or its chimeric molecule is also an important determinant of function.The analysis method of this characteristic includes DSC, TGA and freeze-drying cryomicroscope, analysis freeze thaw resistance and protease resistant.

The protein or chimeric molecule of the present invention is freezed can be more stable after (freeze-drying).Lyophilized stability and/or the shelf life that be used to increase product, because product is stored in powder form, rather than liquid form.Its process includes starting frozen samples, then removes liquid by being dehydrated under vacuo.Final result is a kind of protein of drying and " pie " of auxiliary material (the other materials composition used).The uniformity of the pie finally given is crucial to successful reconstitution.Freeze-drying process can cause protein to change, especially by the aggregated forms of crosslinking, also desamidation and other modifications.These can lose, the immune response of activity or induction for aggregation be reduced, so as to reduce effect.It is using stabilizer (such as mannitol, trehalose, Tween 80, human serum albumins) that protein is lyophilized by formula in order to detect lyophilized stability.If the activity of protein can be determined by suitable bioassay method, the amount of the protein recovered is detected after freezing with ELISA.HPLC or western blot analysis detection protein aggregate can be used.

Before lyophilized, it should determine the Tg or Te (defining Tg or Te) of composition, come set dry first in product maximum permissible temperature.Equally, the crystallinity of composition or amorphicity information help more reasonably to design lyophilized circulation.These thermal parameters product informations can be obtained using differential scanning calorimetry (DSC), thermogravimetry (TGA) or lyophilized cryomicroscope

Differential scanning calorimetry (DSC) is a kind of physics heat analysis method, and detection, the thermal characteristic of sign and analysis of material simultaneously determine thermal capacitance, melt enthalpy and corresponding transition point.DSC scans a temperature range with linear ratio.According to " power back-off zero balance " principle, individual other thermal source in instrument is respectively sample and provides heat with reference to disk.During physical transformation, energy absorption or the imbalance for causing the amount of energy for being supplied to sample bomb is sent.The different thermal behavior of sample is relied on, energy will be removed or spread from sample, and temperature difference will be detected to be transmitted to computer telecommunication number.As a result, the adjust automatically of heater makes the temperature of sample bomb equal with reference to holder.Compensate the electric energy needed and calorimetric effect is equal.

The purity of organic matter can be estimated with DSC according to shape and the DSC temperature for melting heat absorption.Under the same conditions, power back-off DSC is compared with hot-fluid DSC very high resolution ratio is provided.Power back-off DSC produces certainty and accuracy preferably melts regional area, is obscured because melting regional area not as secondary resolution factor hot-fluid DSC in a narrow temperature interval.Power back-off DSC can inherently produce more preferable local melting region, therefore can carry out more preferable purity analysis.By StepScan DSC help, using traditional and time-proven method, power back-off DSC can provide direct thermal capacitance detection, it is not necessary to which deconvolution extracts sinusoidal amplitude.

Thermogravimetry (TGA) detects sample weight loss and rate of weight loss, is used as temperature or the function of time.

In DSC, lyophilized low-temperature device can be rapidly achieved a wide temperature range.At present, as preplanning and planning experimental tool, platform of the lyophilized circulation there is provided best small-scale research protein component thermal parameters is simulated in lyophilized low-temperature device.The influence of component and process factors during lyophilized microscopes prediction is freezed and dried.Low temperature test only needs to 2-3mL samples, this technology is turned into that a valuable research is rare, is difficult to the instrument of medicine that obtains.It is refrigerating effect, ratio, the index of aridity, a kind of good instrument of thawing rate in the lyophilized circulation of research.Lyophilized cryomicroscope experiment can help to annealing research.Because the extensive use of freeze drying technology, and to extremely expensive medicine (such as protein and gene therapy medicament) stabilized wilderness demand, it is desirable to pharmaceuticals industry is in the near future by the Microobservation in implementation process.

Resistance is melted in the freezing that protein or its chimeric molecule can be determined using the one or more in following systems.

In translation or posttranslational modification, for example, glycosylate, freeze/thaw cycle repeatedly can be subjected to protected protein matter.In order to detect it, the carrier-free homologue that can be produced with the chimeric molecule and Escherichia coli of comparison protein or the present invention.Protein or its chimeric molecule are diluted in suitable medium (such as cell growth medium, PBS or similar other materials), then freezed with a variety of methods, for example, freezed at once in liquid nitrogen, be positioned over -70 DEG C of slow freezings or the snap frozen in dry ice.Although fast melt or slow melting samples at 4 DEG C at room temperature.Then some samples are freezed again, this process repeats some circulations.The albumen quality existed is detected with ELISA, experienced staff selects suitable biological detecting method to determine activity.The value of activity/remaining protein is compared with original material, to determine the resistance after many freezing/thaw cycles.

Protein or the chimeric molecule of the present invention can change thermal stability in the solution.In vitro can be by the following thermal stability for determining the present invention.

Protein or the chimeric molecule of the present invention can be mixed into buffer solution, such as containing carrier protein (such as human serum albumins) phosphate buffer, and are incubated the specific time (such as 37 DEG C, 7 days) at a certain temperature.The remaining quantity of protein or its chimeric molecule after being handled with ELISA measure, and want to compare with the material for being stored in -70 DEG C.Association area experienced person carries out suitable biological detection and determines the protein of remaining or the bioactivity of its chimeric molecule.

The protease resistant of one or more of following system measurement protein or its chimeric molecule can be used.

In order to compare protease resistant, the protease that the solution of the solution of the chimeric molecule containing protein or the present invention and the homologue containing Bacillus coli expression can and be selected is incubated (such as unpurified haemocyanin enzyme, the protease of purifying, recombinant protease) different period.The protein content of remaining is determined using suitable ELISA (such as capturing and detect that the identification epitope of antibody is split by proteolytic cleavage site), the suitable biological detecting method selected using experienced person determines remaining protein or the activity of its chimeric molecule.

One or more following system measurement protein or the bioavilability of its chimeric molecule can be used.

Bioavilability is administration latter medicine or other materials by the available degree of destination organization.Bioavilability is dependent on the half-life period of medicine or the ability of its arrival destination organization.

The mixture of chimeric molecule containing protein or the present invention is injected using subcutaneous or intramuscular.Egg then determines the level of protein or its chimeric molecule in blood with ELISA or radiocounting.Or, the suitable biological detecting method selected with experienced person detects the protein active in blood sample, for example, stimulating the propagation of specific targeted cell population.Because sample comes from blood plasma or serum, it is thus possible to there is some other molecule to have an impact the activity detected.This can be controlled by using the neutralizing antibody of testing protein.Therefore, any remaining bioactivity is all caused by other serum compositions.

Stability or the half-life period of one or more following system measurement protein or its chimeric molecule can be used.

Protein or the chimeric molecule of the present invention may have the half-life period of change in serum and blood plasma.In vitro can be by the following half-life period for determining the present invention.The mixture of chimeric molecule containing protein or the present invention can be mixed into human serum/blood plasma and be incubated special time (such as 37 DEG C, 4 hours, 12 hours etc.) at a certain temperature.The amount of remaining protein or its chimeric molecule after being handled with ELISA measure.The suitable biological detecting method selected using association area experienced person determines remaining protein or the bioactivity of its chimeric molecule.The serum of selection may come from different human blood groups (such as A, B, AB, O).

The half-life period of protein or its chimeric molecule can also be determined in vivo.The mixture of chimeric molecule containing protein or the present invention, it can be marked with radioactive tracer or other methods, can be by intravenous, subcutaneous, rear eye socket, tail vein, intramuscular or intraperitoneal injection into the species of experimental selection, such as mouse, rat, pig, primate, people.Different time points obtain blood sample after injection, analyze the protein existed or its chimeric molecule (precipitating radiocounting with ELISA or with TCA).With the control of the mixture containing Escherichia coli or the CHO protein produced or its chimeric molecule as a comparison.

, in vivo, can be to injecting a kind of protein or its chimeric molecule in male Wag/Rij rats or other suitable animals ivs in order to determine the half-life period of protein or chimeric molecule of the invention.

Before material is given, 200 μ l EDTA blood of sampling are used as negative control.Different time points, 200 μ l EDTA blood are sampled using constructed from animal after injection.After last time blood sampling, animal is killed.Sample is centrifuged 15 minutes at room temperature in 30 minutes after collection.The concentration of protein or the chimeric molecule of the present invention in each plasma sample is determined with special ELISA.

Protein or the chimeric molecule of the present invention can pass through blood-brain barrier.

Whether human brain epithelial cell can be combined using following determination method vitro detection protein or the chimeric molecule of the present invention.

The ability of the protein of energy detection of radioactive labels or the chimeric molecule combination human brain capillary epithelium cell of the present invention.The protein of separation or the chimeric molecule of the present invention are manually combined into upper radioactive label using methods known in the art, certain specific activity is realized, such as chloramine-T method is marked¹²⁵I or³H。

The original cuiture of human brain epithelial cell can grow in flat 96 orifice plate, gently be fixed with acetone within 5 days after covering with.Cell is dissolved, and is transferred on glass fibre membrane.Liquid scintillation counter detection of radioactive labels albumen or the chimeric molecule of the present invention can be used.

The chimeric molecule combination human brain epithelial cell of protein or the present invention can be detected in vivo using following determination methods.

The combination of human brain tissue section detection human specific protein being cut into slices using (not by fixing) of fresh food frozen, in cryostat, being positioned on sheet glass and fixed with acetone or the chimeric molecule and human brain capillary of the present invention.Using in quantitative autoradiography detection brain section³The combination of H- protein or the chimeric molecule of the present invention.

The Tissue distribution and blood clearance of human specific protein or the chimeric molecule of the present invention can be detected in vivo in primate system.

In two male cynomologus monkeys or other suitable primates, the chimeric molecule of a protein or the present invention are used for determining¹⁴C- mark albumen or the present invention chimeric molecule Tissue distribution and blood remove, protein or the present invention chimeric molecule and³The reference protein matter of H marks is given the animal with intravenous catheter simultaneously.In experimentation, collect blood sample to determine protein from the removing in circulation.24 hours after injection, kill animal and select organ, collect representational tissue to determine the distribution of isotope and remove naturally.In addition, according to Triguero et al. (J of Neurochemistry 54：1882-1888,1990) method to from brain different zones sample carry out capillary reduce test.The method eliminates the vascular system more than 90% from brain tissue homogenate (Triguero etc. is quoted above).

It is consistent that time-dependent redistribution of the chimeric molecule of radiolabeled protein or the present invention from capillary portion to substantial portion migrates across blood-brain barrier with the time-dependent of protein or the chimeric molecule of the present invention.

Protein or the chimeric molecule of the present invention can promote or suppress angiogenesis.

Methods known in the art can be used to determine the latent effect of the angiogenesis of protein or the chimeric molecule of the present invention.Angiogenesis degree is determined for example, can be sprouted in angiogenesis model by capilary.In this detection, by Shepherd et al. (ArteriosclerThromb Vase Biol 24：898-904,2004) method separation rat fat microvessel fragment (RFMFs), epididymal adipose tissues pad, chopping blend compounds protoenzymes digestion are collected from the animal of execution.RFMFs and separate cell are being isolated from fat and fat cell by the method for centrifugation, and are being suspended in 0.1%BSA PBS.It is continuous to filter RFMF suspensions to remove the fragment of tissue in fragment, unicellular and red blood cell.With 15,000RFMF/ml suspended in the neutral big rat-tail type i collagen of cold, pH (such as 0.25ml/ holes) in RFMFs, and the hole being layered in 48 orifice plates cultivated.After collagen polymerization, the DMEM containing 10%FBS of equal volume is added in each glue.After gel is formed, the blood vessel extended characteristic of angiogenesis progressively occurs on the 4th day in culture.In default of roughening, smooth muscle with form, it is easy to distinguish these newly-generated blood vessels and parent blood vessel fragment.Energy protein or the chimeric molecule of present invention processing RFMF 3-D cultures, culture determine newly-generated length of vessel on the 5th and 6 day.

Guedez et al. (Am J Pathol 162 can also be used：1431-1439,2003) description body vessel generation detection method determine protein or the present invention chimeric molecule angiogenesis latent effect.This detection method is included in subcutaneous transplantation semi-closed (semiclosed) silicone cylinder (vascular reaction device, angioreactor) in nude mice.The extracellular matrix of premixing or non-pre-mixed proteins matter or the chimeric molecule of the present invention is filled with vascular reaction device.Fluorescein isothiocynate (FITC)-glucan is injected intravenously before recovery to quantify the vascularization in vascular reaction device, then carries out fluorescence spectrophotometry detection.Fluorescence spectrophotometry detection is carried out to vascular reaction device can show the cell of different developmental phases and the new vessels of intrusion.

Protein or the chimeric molecule of the present invention can have the immunoreactivity feature that different available immunoassays are determined, including the interaction with one or more antibody directly against molecule.The example of immunoassay includes enzyme crosslinking immuno absorbence detection (ELISA), Dot blot and immunochromatography detection, such as flow test or strip test.

Immunoassay process can be used to determine protein or the level of its chimeric molecule, for example, a kind of ELISA kit of commercial distribution.Because the specificity of the antibody provided in immune reagent kit, the chimeric molecule of protein or the present invention can have different immunoreactivity features to the protein or its chimeric molecule of non-human cell's expression.For example, the capture of immunoassay and/or detection antibody can be the specific human protein expressed directly against non-human cell or the antibody of its chimeric molecule.

In addition, the incorrect folding of the human protein or its chimeric molecule of non-human cell's expression can cause the unexposed epitope exposure in the human protein or its chimeric molecule of the people's cell expression correctly folded.Incorrect folding can be by, for example, and foreign preteins is excessively produced in non-human cell's endochylema to produce, (Baneyx Current Opinion inBiotechnology, 70 for example, Escherichia coli：411-421,1999).Further, the human protein or its chimeric molecule of non-human cell's expression can have different protein or chimeric molecule posttranslational modification form of the invention.For example, the human protein or its chimeric molecule of non-human cell's expression can have the quantity of exception and/or sugared structure, phosphate radical, sulfate radical, fat or the other residues of type.This may cause the exposure of the unexposed epitope in protein or the chimeric molecule of the present invention.Opposite, the change of posttranslational modification pattern may cause to lack exposure in the epitope in the chimeric molecule of protein or the present invention, human protein or its chimeric molecule that the epitope is expressed in non-human cell.

Any one or the above-mentioned factor combined may cause such as following incorrect detection：

(a) human protein naturally-produced in laboratory sample or people's tissue, or

(b) in laboratory sample, recombinant human protein's matter that people is organized or people's cell is expressed in human embryo stem cell (hES) culture medium or its chimeric molecule

The human protein of people's cell expression or the immunoreactivity feature of its chimeric molecule determined using suitable immunoassay can provide the sign of the immunogenicity of protein in human body, as described below.

Most biological products cause the antibody response for them of certain level.Antibody response can cause possible serious side effects and/or loss effect in some cases.For example, some receive the recombinant protein of non-human cell's expression or the patient of its chimeric molecule treatment there may be neutralizing antibody, especially in long-term treatment in use, simultaneously therefore weakening the effect of protein and/or facilitating side effect.The human protein or chimeric molecule of people's cell expression are less likely to produce neutralizing antibody, therefore the human protein or its chimeric molecule expressed with non-human cell are compared and enhance curative effect.

The immunogenicity of one or more following system measurement protein or its chimeric molecule can be used.

Most biological products cause the antibody response for them of certain level.Antibody response can cause possible serious side effects and/or loss effect in some cases.For example, some receive recombinant epo treatment patient there may be neutralizing antibody, and with the EPO cross reactions of patient itself.In this example, they can occur simple erythroid aplasia, and produce resistance to EPO treatments, result in the need for frequent dialysis.

Immunogenicity is to cause the ability of immune response in organism.Immunogenic portion depends on the size of the material, and partly depends on the dissimilar degree of it and host molecule.Because with new physiochemical properties, protein or its chimeric molecule may have the immunogenicity changed.For example, the glycosylation structure of protein or its chimeric molecule may shield or weaken identification of the antibody to epitope, therefore prevent or weaken antibody binding to protein or its chimeric molecule.Or, some antibody can recognize the non-existent glycopeptide epitope in non-glycosylated protein matter.

The protein or the ability of its chimeric molecule of plysiochemical form of the patient's sample identification with characteristic can be determined by different method of immunity, as described here.The appropriate method of immunity of design includes consideration directly suitable detection, quantitative and sign antibody response.Some are listed in Mire-Sluis et al. JImmunol Methods 289 (1-2) to immunoassays design and the suggestion optimized：1-16, in 2004, is incorporated herein by reference document.

The protein or its chimeric molecule that one or more following system measurements can be used to be used in therapeutic engraftment.

Present invention extension operates stem cell using protein or its chimeric molecule.One main stem-cell therapy application is tissue, cartilage or bone regeneration.In one embodiment, the cell in the 3-dimensional matrix of bio-compatible is possible to be introduced in human body.Transplanting will include cell mixture, skeleton, growth factor and necessary composition, such as biodegradable polymer, proteoglycans.Intend to mix protein or its chimeric molecule into these matrix in building process to adjust the behavior of cell.This transplanting can be used for bone formation, nerve from growth of progenitor cells and other application.Protein derived from people's cell can reduce the quantity and/or kind of the allogene protein under stem cell cultivation conditions, and therefore reduce the risk of inhuman pathogenic infection.

The present invention protein or chimeric molecule can be different with matrix generations for forming graft interaction, and adjust cell mix graft in.It is expected that the protein or chimeric molecule and graft composition of the present invention, which is used in combination, will cause one or more following pharmacology characters, such as high proliferation, promote differentiation, the maintenance of desired differentiation state, stronger differentiation lineagespecific, increase the secretion of matrix components, form more preferable three-dimensional structure, strengthen signal, more preferable structural behaviour, reduce toxicity, reduce side effect, reduce inflammation, reduce immunocyte infiltration, reduce injection, the graft duration is longer, graft function is more permanent, graft peripheral cell is preferably stimulated, more preferable regeneration, more preferable organ dysfunction, more preferable tissue remodeling.

The effect that one or more following system measurement protein or its chimeric molecule can be used to express different genes.

The gene expression difference of the cell exposed to protein or its chimeric molecule can be analyzed.

Microarray technology can determine the mRNA expression of almost all of gene in an organism genome simultaneously.The method uses gene " chip ", and the oligonucleotides of correspondence different genes sequence is attached on the solid carrier in " chip ".Using the obtained marks of the mRNA extracted from cell or tissue interested cDNA and chip be incubated, allow cDNA and attachment complementary sequence hybridization.Also need to, using control, compare both signals after then being hybridized and being cleaned.Which gene upregulation or downward are determined using special software or which expression does not change.Each chip can analyze thousands of genes.For example using Affymetrix technologies, human genome U133 (HG-U133) gathers, including two GeneChip (registration mark) arrays, containing about 45000 probe set, more than 39000 transcripton extracted from about 33000 human genes well confirmed is represented.GeneChip (registration mark) mouse genome 4302.0 contains more than 39000 transcripton in individual array.

It is such to analyze the change for disclosing Global mRNA expression profiles, therefore it can be found that the expression change of the unknown gene regulated and controled by particular stimulation thing.Therefore this technology is suitable for the gene expression of the analysis induction related to the protein or chimeric molecule of the present invention.

It is determined that by particular stimulation thing regulate and control known to and new gene will be helpful to confirm important biochemical pathway in the protein or chimeric molecule bioactivity of the specific present invention.These information will be useful to determination novel therapeutic target spot.

The product that the system can be used for observing with can purchase compares the protein of the present invention or the changes in gene expression of chimeric molecule induction.

The binding ability effect of one or more following system measurement protein or its chimeric molecule can be used.

The protein of the present invention or the binding ability of chimeric molecule and different material can be studied, the material includes extracellular matrix, artificial material, heparin sulfate, carrier or co-factor.

Following determination methods can be used to determine the effect of the specified protein combination extracellular matrix in protein or its chimeric molecule.

The pan coating extracellular matrix protein in suitable buffer solution, including but not limited to collagen, vitronectin, fibronectin, laminin.Uncombined site can be coated with methods known in the art, for example, being incubated with BSA solution.Surface is cleaned, for example, with PBS solution, then adding the solution containing albumen to be detected (protein or chimeric molecule of the invention) on surface.After coating, cleaning surface and and identification of protein or its chimeric molecule antibody incubation.The antibody that then detection is combined, for example, using a kind of enzyme-linked secondary antibody for recognizing primary antibody.By the way that the combining antibody visualization of color change reactions is incubated and observed with suitable material.Not glycosylated protein is compared, and glycosylation albumen stronger can be adhered on extracellular matrix protein.

Or, by the protein or chimeric molecule of (being specified with ELISA concentration or biologicall test active unit) present invention of equal amount, or the protein of the invention or chimeric molecule homologue expressed with non-human cell, it is incubated together with the coated aperture of matrix, then cleaning aperture, combined amount is detected with ELISA.The decline indirect determination combined amount of ELISA reactivity after being incubated by sample and coating surface.

One or more following system measurement protein or the ability of its chimeric molecule combination artificial material can be used.

In order to determine the ability of protein or its chimeric molecule combination artificial material, surface is coated with artificial material, including but not limited to metal, support in suitable buffer solution.Surface is cleaned, for example, with PBS solution is used, then adding the solution containing albumen to be detected (protein or chimeric molecule of the invention) on surface.After coating, cleaning surface and and identification of protein or its chimeric molecule antibody incubation.The antibody that then detection is combined, for example, using a kind of enzyme-linked secondary antibody for recognizing primary antibody.By the way that the combining antibody visualization of color change reactions is incubated and observed with suitable material.

Or, by the protein or chimeric molecule of (being specified with ELISA concentration or biologicall test active unit) present invention of equal amount, with the protein of the invention or chimeric molecule homologue expressed with non-human cell, it is incubated together with the coated aperture of artificial material, then cleaning aperture, combined amount is detected with ELISA.The decline indirect determination combined amount of ELISA reactivity after being incubated by sample and coating surface.

There may be biology effect with reference to the ability of artificial surfaces, such as in support coating.Or, the support for being coated with a kind of protein of the invention or chimeric molecule is used for repopulating cell.The then growth and differentiation of monitoring cell, and compared with the not coated or different support of coating.

One or more following system measurement protein or the ability of its chimeric molecule combination heparin sulfate can be used.

Due to the protein or the physics and chemistry form of chimeric molecule of the present invention, it is contemplated that they and heparin sulfate have different interactions.It is expected that these difference are obvious in the experimental models such as cell propagation, differentiation, migration.Protein or its chimeric molecule and the pre- pharmacology character in respect of characteristic of heparin sulfate are used in combination in designated treatment.It is probably extended serum half lives, bioavilability increase, reduces immune related removing, stronger effect, the advantage for reducing dosage reduction side effect and correlation.

The ability of one or more following system measurement protein or its chimeric molecule combination carrier or co-factor can be used.

When protein or its chimeric molecule are present in blood plasma, they will combine other molecules.These molecules can be named as " carrier " or " co-factor ", and will influence the bioavilability or serum half-life of these factors.

It is incubated with the plasma proteins of purifying and analyzes the interaction that resulting solution can determine the protein or chimeric molecule and their binding partner of the present invention with molecular-exclusion chromatography.If protein or its chimeric molecule combine a kind of co-factor, obtained compound causes elution time to change by with bigger molecular weight.The bioactivity of compound, external or Half-life in vivo and bioavilability can be compared.

The effect of one or more following system measurement protein or its chimeric molecule to biologicall test can be used.

The protein of a variety of bioassary method detection present invention or the activity of chimeric molecule can be carried out, including determines cell propagation, cell differentiation, Apoptosis, cell size, cell factor/cytokine receptor adhesion, cell adherence, cell dispersion, cell movement, migration and infiltration, chemotaxis, ligand receptor combination, receptor activation, signal transduction and isoform rate of change.

One or more following system measurement protein or the effect of its chimeric molecule cell proliferation can be used.

Cell, in specific embodiments, the cell of exponential growth are incubated in the growth medium of the protein containing the present invention or chimeric molecule.This can be carried out in arrow-necked bottle or 96 orifice plates.Cell growth then counts cell with the method for direct (such as cell count) or indirect (MTT, MTS, tritiated thymidine) for a period of time.Control only containing culture medium, which compares, determines increasing or decreasing for propagation.The protein or its chimeric molecule of different concentration can be used to obtain dose response feature in parallel serial experiment.ED50 and ED100 (producing the dosage required for the maximum and maximum response effect of half) can be determined with it.

One or more following system measurement protein or its chimeric molecule can be used to maintain the effect of undifferentiated state to cell differentiation or cell.

Cell is incubated in the protein or the growth medium of chimeric molecule that there are the present invention.After suitable a period of time, cell analysis is carried out to differentiation indicant.It can be the expression of cell surface special sign thing, the expression of endochylema mark, the change of cell size, shape or kytoplasm feature.Mark potentially includes protein, sugared structure (such as glycosaminoglycan, such as heparin sulfate, chondroitin sulfate), fat (glycosphingolipid or lipid bilayer composition).Multiple technologies can be used to detect that these change, the technology includes microscope, protein blot, FACS dyeing or forward direction/lateral scattering feature.

The effect of one or more following system measurement protein or its chimeric molecule to Apoptosis can be used.

Apoptosis is defined as programmed cell death, different with other cell death ways such as necrosis.It is characterized by the cellular change determined, and the activation (such as Fas, TNFR) of such as signal path causes a protease subgroup for being referred to as caspase (caspases) to be activated.Starting the activation of caspase causes the activation of lethal caspase, and it cuts various kinds of cell albumen, causes nuclear fragmentation, and nuclear lamins is broken, and cytoplasm bubbles and destroys cell.Apoptosis can be induced by protein ligands, such as FasL, TNFa, lymphocytotoxin or by signal induction, for example, ultraviolet light and cause the material of DNA damage.

Cell is incubated in the growth medium of the reagent containing protein or its chimeric molecule or other suitable detections.For example, it may be desirable to which transcription (actinomycin D) can be blocked or the reagent of (cycloheximide) is translated.After suitable a period of time is incubated, cell quantity is determined with suitable method.Cell quantity, which declines, might mean that apoptosis.Other apoptosis can be obtained by cell dyeing to characterize, for example, annexin or the trapezoidal DNA form of observation characteristic.The expression of apoptosis mark can be prevented by being incubated with Premeabilisation of cells Caspase inhibitors (such as z-VAD FMK), then detection apoptosis mark, further to confirm apoptosis.

The protein or chimeric molecule of the present invention may provide survival signaling to prevent apoptosis by cells survival path (such as Bc12 or Akt paths).Can be expressed by cell Bcl2 increases or detects that Akt activates form (phosphorylation) increased protein blot and confirms the activation of these paths using the phospho-specif iotac antibodies directly against Akt.

For these detections, cell culture is under the conditions of Survival Factor (such as IL-3 and certain immunocyte) is contained or not contain.The apoptosis under Extending culture of the part cell under the conditions of Survival Factor is lacked is cultivated, cultivating the cell under the conditions of sufficient amount Survival Factor will survive or breed.The activation for the cell pathway for being responsible for these effects can be determined by protein blot, immunocytochemistry and facs analysis.

One or more following system measurement protein or its chimeric molecule can be used to suppress the effect of apoptosis.

Have detected the present invention protein or chimeric molecule protect in vitro rat, mouse and people's cortical neural cell oxygen content it is low and lack glucose condition under there is the activity of cell death.Therefore, being extracted cell culture from rat embryo.In order to determine the protein of the present invention or the effect of chimeric molecule, cell is containing 30mM Glucose Medium without serum and 95% wetness air/5%CO₂(oxygen content is normal) or without Glucose Medium without serum and 95% wetness nitrogen/5%CO₂Under the conditions of (hypoxemia and glucose lack), under the conditions of the protein or chimeric molecule of the absence or presence present invention, maintained 48 hours at 37 DEG C in the module culture storehouse in water jacketed mcubator.Cell culture returns to less than 24 hours and then oxygen content normal condition lower 24 hours under the conditions of hypoxemia and glucose shortage.With the blue fluorescence analysis cytotoxicities of Alamar, this method determines cell viability by metabolic activity.

In another method, neuronal cell cultures thing is under oxygen content normal condition, under the conditions of the protein of the invention or chimeric molecule of absence or presence various concentrations, exposed to 1mM Pidolidones or alpha-amido -3- hydroxyl -5- methyl oxazole -4- propionic acid (AMPA) 24 hours.With the blue fluorescence analysis cytotoxicities of Alamar, this method determines cell viability by metabolic activity.

Protein or its chimeric molecule can influence growth, apoptosis, development or the differentiation of various kinds of cell.In other detectable parameters, these changes can be developed the cell size change caused by intracellular organelle and the change of kytoplasm complexity reflects.For example, the culture induction keratinocyte differentiation that suspends, shows as surface marker (such as beta 1 integrin, β 1integrins) downward, cell becomes big, the increase of kytoplasm complexity.One or more following system measurement protein or its chimeric molecule can be used to cell size or the effect of kytoplasm complexity.

FACS determines the amount of light scattered after beam of laser is incided on cell.The argon laser for being 488nm usually using offer wavelength.Cell is bigger, and it is more that forward direction light beam is blocked, therefore preceding levels of scatter is related to cell size.In order to determine the change of cell size, the cell handled with the protein or chimeric molecule of the present invention is diluted in sheath fluid (sheath fluid) and is injected into flow cytometer (FACSVantage SE, Becton Dickinson).Untreated cell is used as control.Cell is by light beam, and the forward scattering quantity of light is related to cell size.

The change (kytoplasm complexity) of intracellular organelle growth and development can also be determined with FACS.Intracellular organelle is to side-scattered light.Accordingly, it is capable to the change for the light side-scattered quantitative measurement kytoplasm complexity that cell is caused in aforementioned manners, and the light side-scattered level that can be sent by determining cell determines the level of complexity and cell granulations degree level of intracellular organelle.

FACS can be used to determine protein or its chimeric molecule to cell size or the effect of kytoplasm complexity, come the signal of the untreated cell transmitting of comparing signal characteristic and equal amount that the cells of such as 20000 processing are sent.By comparing the signal of different disposal cell mass, the relative change of cell size and kytoplasm complexity can be detected.

One or more following system measurement protein or its chimeric molecule cell growth, apoptosis, development or the effect of differentiation can be used.

It can be marked with dyestuff such as propidium iodide (propidium iodine) and handle the DNA of cell to determine the apoptosis and cell growth or cell cycle change that protein is induced, the excitation wavelength of propidium iodide is launched in 620nm regions in 488nm regions.The cell DNA for occurring apoptosis is condensing, and size and granularity are also different.These factors give positive size scattering signatures and fluorescence signal, and therefore distinguish the positive cell mass and normal cell for occurring apoptosis.DNA quantity in cell also reflects which that cell is in the cell cycle in stage.For example, G₂The DNA quantity of phase cell is G₀Twice of phase.This can pass through G₂The fluorescence signal that phase cell sends twice is reflected.The signal of the untreated cell transmitting of fluorescence signal and equal amount that the cell that FACS can be used to compare such as 20000 processing is sent, determines protein or the effect of its chimeric molecule.

The protein or chimeric molecule of the present invention can also change the expression of multiple proteins.The protein or chimeric molecule of one or more following system measurement present invention can be used to the effect of protein expression.

In order to determine the increase and reduction of protein expression in whole cell, it can fix and permeabilized cells, the antibody incubation then and using proteins of interest matter epitope being crosslinked as the fluorescein of target spot.A variety of fluorescence labelings can be used in argon laser system.Often using FITC, Alexa Fluor 488, Cyanine 2, Cyanine 3 these fluorescence molecules in this experiment.By marking on cell surface, only the epitope of exposure can be by the non-permeabilized cells of antibody labeling, and the method can also be used to determine surface marker and protein expression change.The signal of the untreated cell transmitting of fluorescence signal and equal amount that the cell that FACS can be used to compare such as 20000 processing is sent, determines protein or the effect of its chimeric molecule.

The effect that one or more following system measurement protein or its chimeric molecule can be used to adhere to ligand/receptor.

Compared with known physics and chemistry form before those, protein of the invention or chimeric molecule there may be lower or more high-adhesiveness.Interacting can be and protein acceptor, because sugared structure (such as selects albumen, such as L- selects albumen and CD62P) and extracellular matrix components (such as fibronectin, collagen, vitronectin and laminin) or with non-protein component such as glycan molecule (heparin sulfate, other glycosaminoglycans).

Protein or its chimeric molecule can also be different with non-biological material such as tissue culturing plastic, medicine equipment composition (such as support or other grafts) or dental material generation interaction.For medicine equipment, this may change graft immigration rate, graft and specific cell type or the interaction between body connection type

Any suitable protein adherence detection method can be used.For example, in certain embodiments, the binding partners on protein or the solution of chimeric molecule and an immobilization surface containing the present invention are incubated.After incubation, the quantity of the protein or chimeric molecule in solution is detected with ELISA, the remaining quantity variance between parent material is bonded to the amount on gametophyte.For example, the interaction between protein or chimeric molecule and extracellular matrix proteins can be determined with the aperture of coated 96 orifice plate of ECM protein (such as fibronectin) first layer.Then it is incubated with BSA solution and prevents non-specific binding.After cleaning, the protein or its chimeric molecule solution of a kind of concentration known are added up to the time of determination.The amount of remaining protein or its chimeric molecule in subsequent removal solution, measure solution.Determine the amount being attached on ECM protein by using the antibody incubation aperture for protein or its chimeric molecule, then with a kind of suitable system (or a kind of mark secondary antibody or with used in biotin-avidin multienzyme complex such as ELISA) detected.

Determining the method for the amount for being attached to other surfaces can include, from inertia graft surface hydrolysis protein or its chimeric molecule, then determining the amino acid in solution.

The effect of one or more following system measurement protein or its chimeric molecule to cell adherence can be used.

Cell adherence is at least partly mediated to matrix (such as extracellular matrix components such as fibronectin, vitronectin, collagen, laminin) by integrin molecule.Integrin molecule includes α and beta subunit, and α and the unique combination physical efficiency increase of beta subunit are directed to the binding specificity of particular ligand (such as a2b1 integrins incorporating collagen, a5b1 binding fiber associated proteins).There is integrin subunit big extracellular domain to be used for binding partner, and shorter endochylema region domain is used for and cytoskeleton interaction.When there is part, endochylema region domain is responsible for inducing signal transduction event (signal transduction in outer).Extracellular signal event can regulate and control compatibility of the integrin to their part, subsequently result in the change (internal/external signal transduction) of integrin cytoplasmic tail.

It is incubated with the protein or chimeric molecule of the present invention and can change cell adherence by certain methods.First, it can qualitatively change the expression of specific integrin subgroup, cause the change of binding ability.Secondly, thus it is possible to vary the expression quantity of specific integrin, the combination of cell and its target matrix is caused to change.3rd, the affinity (internal/external signal transduction) of integrin can be changed in the case where not changing the surface expression of specific integrin.All these changes can change combination of the cell to a pedigree part, or change the combination to certain specific part.

Cell-ECM adhesion detection method the detections generally carried out in 96 orifice plates protein or chimeric molecule of the invention can be used.Aperture is coated with matrix, is then coated with BSA in hole and is not associated with site.By coated hole and the cell incubation of quantification, uncombined cell is then washed away, the cell of hatching combination under conditions of protein or its chimeric molecule is contained or not contain.The quantity of cell is determined by indirect method such as MTT/MTS.Or, with radioactively labelled substance (for example⁵¹Cr cell) is marked, the radioactivity (i.e. cell) of dose known amounts is added in each hole.Measure combination radioactivity, calculates the ratio for accounting for loading quantity.

Cell is also adhered on other cells, such as on another cell of a group cell adherence to individual layer.In order to detect this situation, the labeled upper radioactivity of suspension cell is simultaneously added on cell monolayer.Subsequent cell is incubated under conditions of presence or absence of protein or its chimeric molecule.Uncombined cell is washed away, the mixing with cells colony of remaining is dissolved and detects radioactivity.

The effect of one or more following system measurement protein or its chimeric molecule to Cell expansions can be used.The protein or chimeric molecule of the present invention has different effects to Cell expansions.Cell starts to spread the committed step for being cell mobility and infiltration behavior.Cell can start diffusion in many ways in vitro.Suspension cell, which is layered on ECM compositions, to cause the adhesion by integrin and ligand binding.This starts signal transduction event, causes the activation of the small GTPases families of Cdc42, Rac and Rho.The activation of these protein causes actin polymerization and piece foot (1amellipodium) extension, causes cell gradually to flatten and their acceptor touches more integrins.Final cell is put down flat and forms focal adhension (big structure containing integrin and signal protein) completely.The cell that can also be adhered to by using factors stimulated growth carrys out active cell extension, also causes the activation of Cdc42/Rac/Rho protein and the formation of focal adhension.

Can by after protein or its chimeric molecule are stimulated different time points detect a large amount of cells Cell expansions quantified.Image analysis software can be used to determine the region of each cell, it is possible to be compared Cell expansions percentage and Cell expansions degree and time.The stronger activation of Cdc42/Rac/Rho paths, which can start, faster to be extended, in addition, it is temporary, the qualitative and quantitative differences that they are activated can be determined when having the protein or chimeric molecule of the present invention.The change of this protein for reflecting the present invention successively or the signal event of chimeric molecule induction.

One or more following system measurement protein or its chimeric molecule can be used to cell mobility, migration and the effect infiltrated.

It is not that static holding is anchored on a site to adhere to the cell on tissue culture dishes, but lasting stretching, extension and shrinks their cell body.When being observed with time lapse photography, it is observed that cell is strolled about in plate, the individual cells or cell colony of separation are not always the case.This motion both can be " random walk " (i.e. not towards specific direction) or orientation.Two kinds of motion can be strengthened by adding growth factor.The total distance and general direction of time lapse photography quantitative cell covering in preset time section can be used.

In directional migration, cell will be moved by experiencing chemical gradient and orienting their migration machine towards the source of chemoattractant.In many cases, chemoattractant is growth factor.Can be by providing chemoattractant source (such as by long pipette) and then cell being imaged to the migration in source with time lapse photography, to quantitative determine directional migration.

Another system for determining directional migration is Boyden chamber detection.In this detection, cell is placed in by splitting in the upper chamber that aperture is connected with lower room in film.Growth medium is all added in two rooms, but only adds chemoattractant in lower room, causes there is diffusion gradient between two rooms.Cell is attracted by growth factor source and migrates across the aperture in segmentation film, enters the lower surface of film.After a few hours, take film away and counting measure is carried out to the cell for moving to film bottom.

Cellular infiltration process has used many and migration identical component.Cellular infiltration multi-layer cellular epimatrix can be used to be used as cellular infiltration model.For example, matrigel is the mixture (ECM compositions, growth factor etc.) of basement membrane components, it is liquid at 4 DEG C, but gel is quickly formed at 37 DEG C.The upper surface of Boyden chamber can be utilized to be coated with, chemoattractant is added in lower floor.For by the cell to film lower surface, they must use enzyme (such as clostridiopetidase A and matrix metalloproteinase (MMP)) to carry out matrix degradation glue and be oriented migration towards chemoattractant.This detection simulates the various procedures needed for cellular infiltration.

The effect of one or more following system measurement protein or its chimeric molecule to chemotaxis can be used.

Cell can be determined in Boyden chamber in vitro towards the migration in chemoattractant direction.The protein or chimeric molecule of the present invention is placed to lower room, and suitable target cell group is placed into upper chamber.The migration by one layer of cells can be detected, to simulate the extracorporeal procedures of migration of the immunocyte from blood to inflammation part.With the upper surface in the hole of the one layer of cells cladding Boyden chamber covered with, the cell such as epithelium, endothelium or fibroblast, this is by blocking immunity cell Direct Transfer by the hole in hole, and immunocyte will need to adhere on cell monolayer and migrate across it to reach at detected albumen.Only there is the chemotactic sexuality that cell indicates protein or chimeric molecule in the Boyden chamber lower surface or lower opening culture medium in the hole handled with protein or its chimeric molecule.In order to show that this effect is that protein or its chimeric molecule are specific, albumen and neutralizing antibody can be allowed to be incubated together in lower room.

In addition, in order to detect that a kind of material (chemical substance, protein, sugar) prevents the ability of chemotaxis, the solution by the material and containing known chemotactic sexuality (can be the conditioned medium of the cell of specific chemokine, cell derived or a series of chemotactic factor (CF)s of secretion) is incubated together in the lower room of Boyden chamber.Permissive cell group then is added in upper chamber, detection is proceeded as described above.

The effect of one or more following system measurement protein or its chimeric molecule to ligand receptor-combination can be used.

The protein or chimeric molecule of the present invention may have different ligand-receptor binding abilities.Ligand-receptor can be determined by many kinds of parameters to combine, for example, dissociation constant (Kd), dissociation rate constant (k^-), association rate constant (k⁺).The difference that ligand-receptor is combined may be relevant with activation with the different signal time limits, causes different biological results.

Ligand-receptor combination can be determined and analyzed by scatchard plot method or other methods such as Biacore methods.

For scatchard plot method, with such as radioactive label (for example¹²⁵I) come the protein or its chimeric molecule that mark, under conditions of the "dead" protein or the competitor of its chimeric molecule that there is varying number, and expression respective ligand or the cell of acceptor or its extract are incubated together.Determine the protein or the quantity of its chimeric molecule of the mark of specific binding, and calculations incorporated parameter.

For Biacore methods, the restructuring part corresponding with protein or its chimeric molecule or acceptor and detection unit match.Solution containing selected protein or its chimeric molecule then determines combination by detecting cell, and by detecting the change of unit character.By by the solution containing protein or its chimeric molecule by detect cell until recorded fixed reading (when all permissions site all it is occupied sometimes) determine association rate constant.The solution of protein or chimeric molecule is not contained by cell, protein is disintegrated down from respective ligand or acceptor, gives dissociation rate constant.

The effect of one or more following system measurement protein or its chimeric molecule to receptor activation can be used.

Interaction between protein or its chimeric molecule and corresponding part or acceptor can be contrasted by the difference in the protein induced signal event of cellular endogenous.Protein or its chimeric molecule can be used, the interaction time limit is accurately characterized with combination/dissociation rate constant or dissociation constant.

The acceptor of activation is taken the photograph in cell is frequent.Subsequent receptor/ligand complex dissociation (for example, the low pH in cell follicles bubble causes part to depart from), and acceptor recirculates to cell surface.In addition, the compound may also turn into the target spot of degraded.In the process, acceptor will effectively be lowered and can not produce further signal, but then can repeating signal process when they recirculate.Different acceptors is combined or activation may cause acceptor to be changed into the path that recirculates from degraded, causes stronger biological response.

The effect of one or more following system measurement protein or its chimeric molecule to signal transduction can be used.

Part or acceptor, which are attached on protein or its chimeric molecule, to send signal by a variety of plasmosins, wherein potentially including reverse signal.When the part of film combining form is by combining its solubility or film combining form acceptor come conducted signal, reverse signal occurs.Reverse signal may also occur in by after a kind of antibody binding film combination part.These signal events (including reverse signal event) cause the change of gene and protein expression.Therefore, the protein or chimeric molecule of the present invention can induce or suppress different signal transductions or other signal transduction events in different paths, such as JAK/STAT paths, Ras-erk paths, the activation of AKT paths, PKC, PKA, Src, Fas, TNFR, NFkB, p38MAPK, c-Fos activation, by protein recruitment to acceptor, receptor phosphorylation, is taken the photograph in vivo, receptor cross-talk or secretion.

It may be unique to the protein or chimeric molecule of the present invention to raise protein or part or acceptor on its chimeric molecule, because induction of the different conformations of part or acceptor.A method for determining these differences is come immunoprecipitation part or acceptor with a kind of antibody being linked on sepahrose globules.After immunoprecipitation and cleaning, by protein loading 2D gel power supplys, com-parison and analysis dot pattern.Discrepant point can be cut and Mass Spectrometric Identification is used.

The effect that the upper mediation of one or more following system measurement protein or its chimeric molecule to surface marker can be used to lower.

Cell may have a variety of responses to the protein or chimeric molecule of the present invention.There are a series of protein to be responsible for the communication between cell and extracellular matrix in cell surface.By regulating and controlling encytosis and exocytosis process, multiple proteins are transported to and transported out of cell surface.The Representative Western found in cell surface includes acceptor, associated proteins, modulin and signaling molecule.The change of protein expression and degradation rate also changes the level of cell cortex protein.Some protein are also stored in intracellular container, transport of the distinctive signal energy inducible protein between these containers and cell membrane.

Cell is incubated the suitable time in the culture medium of the protein containing the present invention or chimeric molecule, is compared with the cell not contained exposed to identical in the protein of the present invention or the culture medium of chimeric molecule.Protein on cell membrane can be dissolved and by centrifugation and cell separation.Specific protein expression level can be determined with Western blot.The antibody labeled cells that can also be crosslinked with fluorescein, are visualized with Laser Scanning Confocal Microscope system or are counted by Fluorescence-activated cell sorting (FACS).This will detect any change of protein expression and distribution on cell.The change of protein dependent interaction can also be studied using Laser Scanning Confocal Microscope and immunoprecipitation by using multiple antibody.Similar, these experiments can be expanded in internal animal model.Cell can be extracted from the animal privileged site handled with the protein or chimeric molecule of the present invention, be detected with identical method.

Different marks will be expressed by adding the protein of the present invention or the cell of chimeric molecule induction differentiation in vitro or in vivo, the mark is separated by these cells and for processing cellular regions.Some cells, for example, progenitor cells or stem cell, can be divided into a variety of isoforms, are distinguished by their surface marker.One protein or chimeric molecule of the invention may stimulate progenitor cells to be divided into a variety of isoforms according to a specific ratio.

The protein and its chimeric molecule of the present invention is possible to effective to cellular rejection.

Protein or its chimeric molecule are convenient cellular rejection detection methods in regulating cell and neure growth and the effect pointed to.

Interaction between destruction subunit and the other components of protein is a kind of method for suppressing protein or the biological effect of its chimeric molecule.The compound for suppressing this biological effect is identified by a variety of methods.

High flux screening project, which produces lead compound using small chemical entities storehouse (compound or peptide), is used for clinical development.Some detection methods can be used to screen the ability of the biological related end of the final point of the compounds affect in storehouse.Each potential compound in storehouse is detected in an independent aperture in one-time detection, the effect of compound is determined.The embodiment of some detections is provided below.

To this detection, cell is taped against in micro plate (96 orifice plates, 384 orifice plates etc.).The reading mechanism that cell will be activated with protein or its chimeric molecule.This can include detection cell growth, the stimulation (such as technology based on FRET) for detecting specific passageways, the induction (such as CAT, beta galactosidase, fluorescin) of examining report gene, detection apoptosis and detection differentiation.Subsequent cell is being exposed in the protein of the present invention or chimeric molecule under conditions of existing or lacking specific small molecule.Medicine can be added before, after, or during protein or its chimeric molecule is added.After the suitable period, using suitable method to indivedual hole readings (such as the cell quantity in the induction of the fluorescence or fluorescin of FRET, MTT, betagalactosidase activity etc.).The control wells of any medicine or cell factor are added without as comparing.Any molecule that can suppress acceptor/cytokine complex will provide the readings different with compareing.Further experiment is also needed to show the specificity of suppression.In addition, medicine may influence detection method (false positive) by non-cytokine, non-receptor mechanism.

The part or acceptor of protein or its chimeric molecule are immobilized in the surface of solids.It is subsequently added protein or its chimeric molecule and compound to be detected.Protein or its chimeric molecule can be first added, compound is subsequently added；Compound is first added, protein or its chimeric molecule is subsequently added；Or compound and protein or its chimeric molecule are added together.Then by suitably detecting protein or chimeric molecule that antibody test is combined.Antibody can be detected with a kind of enzyme (such as alkaline phosphatase or horseradish peroxidase for colorimetric determination) or a kind of fluorescent tag label for fluoroscopic examination.Furthermore, it is possible to a kind of suitable technical mark (such as biotin, radioactive label) protein or chimeric molecule and with a kind of suitable technology (for example, for biotin labeling, streptavidin and colorimetric assay system；For radioactive label, dissolve compound and count) detection.The suppression that protein is combined is determined by comparing the decline of reading with control wells.

The soluble ligand or acceptor of protein or its chimeric molecule are combined on globule.This association reaction both can be that a kind of adsorption process relates to they being chemically crosslinked onto plate.Globule and protein or chimeric molecule and a kind of candidate compound are incubated in a suitable aperture.Protein or chimeric molecule can be first added, compound is subsequently added；Compound is first added, protein or chimeric molecule is subsequently added；Or add compound and protein or chimeric molecule together.Be subsequently added fluorescence labeling recognizes a kind of protein or the detection antibody of its chimeric molecule.Remove uncombined antibody and globule is passed through into FACS.If compound suppresses the interaction of protein or its chimeric molecule and its acceptor, the amount of fluorescence of detection will decline.

For multiple interactions between screening protein and its respective ligand/acceptor and a kind of inhibiting compound, it is necessary to use the ability of FACS Instrumental Analysis scattering signatures.The ball of larger diameter will have the scattering signatures different with smaller ball, it is possible to be separated for analyzing (" gating ").

Some different protein, one of which is the protein or chimeric molecule of the present invention, is all linked on the ball with special diameter.The ligand/receptor mixture of above-mentioned protein is then added in the ball mixed liquor containing a kind of candidate compound.The ligand/receptor combined is then detected with the secondary antibody of a species specific fluorescence labeling.Antibody can all mark identical to detect fluorophor.The ability that compound prevents protein from combining its ligand/receptor is then determined by the Run sample on FACS instruments and to the ball gating of each known dimensions.Then analysis individually each combines result respectively.The key benefit of this analysis method is each compound that can be screened with some protein Parallel testings, consequently reduces screening time.

Protein or its chimeric molecule can also be characterized by its crystalline structure.The physics and chemistry form of protein or its chimeric molecule can provide unique 3-dimensional crystal structure.Further, it is also possible to produce the crystal structure of protein-ligand/receptor complex using the protein or chimeric molecule of the present invention.Because the invention provides the protein essentially similar with naive existence form or its chimeric molecule, the compound is likely to be the more suitably representative of the internal structure of naturally occurring protein-ligand/receptor complex.Once obtaining crystal structure, the interaction between protein or its chimeric molecule and the compound for potentially suppressing this interaction just can determine that.

Once determine potential compound by high flux screening or from protein-ligand/receptor complex crystal structure, it is possible to proceed by design and rational pharmaceutical procedures.

Carried out generally using some steps in analogies design according to the compound with design specified characteristic.First, it is determined that compound privileged site crucial and/or important in characteristic needed for determining.For peptide, above-mentioned work can be completed by the amino acid residue in systematic change peptide, for example, replace each residue successively.This peptide motif is often refined using Alanine-scanning.These parts or residue constitute the active site of compound, and the active site is referred to as its pharmacophoric group.

Once it is found that pharmacophoric group, its structure is modeled using a series of data in sources according to its physical characteristic, the physical characteristic such as spatial chemistry, bonding, size and/or electric charge, data such as spectroscopy techniques, X ray diffracting data and the nuclear magnetic resonance in the series source.In this modeling process calculating analysis, similitude can be used to do figure (electric charge and/or volume-based model of pharmacophoric group, rather than interatomic bonding) and other technologies.

In a variant of this method, the 3 d structure model of part and its binding partners is made.This changes conformation when combining for part and/or binding partners and is particularly useful, and modeling can be allowed to consider this problem in design simulation thing.Modeling can be used to produce the inhibitor interacted with linear order or a kind of 3-dimensional conformation.

The template molecule that then selection can will simulate the chemical group transplanting of pharmacophoric group up.Template molecule can easily be selected and chemical group up is transplanted, therefore analogies are easily synthesized, it may be possible to it is pharmacologically acceptable and non-degradable in vivo, remain the bioactivity of lead compound.Or, when analogies are to be based on peptide, further stability can be obtained by the way that peptide is cyclized, increase its rigidity.The analogies that find by this method can be then screened to observe whether they have target property, or they show the target property of much degree.Then further it can be optimized or be modified, one or more final analogies are entered into internal or clinical detection.

The target of rational drug design is generation biologically active polypeptide interested or the analogue of small molecule, they and these polypeptides or small molecular phase interaction (such as activator, antagonist, inhibitor or reinforcing agent), in order to form medicine, for example, more active or more stable polypeptide form, or, for example strengthen or disturb the function of polypeptide in vivo.See, such as Hodgson (Bio/Technology 9：19-21、1991).In a procedure, first by x ray crystallography, by microcomputer modelling or it is most typical, be combined by a variety of methods and to determine the three-dimensional structure of protein interested.The useful information on polypeptide structure can also be obtained by the structural modeling based on homologous protein.The example of one rational drug design is exploitation hiv protease inhibitor (Erickson et al.Science 249：527-533、1990).Furthermore, it is possible to analyze target molecule (Wells, MethodsEnzymol 202 by Alanine-scanning：2699-2705、1991).In this technology, an amino acid residue is replaced by Ala, and determines its influence to peptide activity.Each amino acid residue of analytical peptide determines the important area of peptide in this way.

It is also possible to isolate target specificity antibody, is selected by functional analysis, and then parses its crystal structure.In principle, this method produces a drug core, and follow-up drug design can be carried out based on it.Bak protein crystallography is omitted completely possibly through anti-idiotype is produced to a functional, the antibody for having pharmacological activity.The binding site of anti-idiotype therefore is expected for the analog of original acceptor as the mirror image of mirror image.Therefore anti-idiotype can be used to identify and isolated peptides chemically or in biogenic peptide storehouse.Then drug core is used as with the peptide of selection.

On the one hand, protein of the invention or chimeric molecule are used as immunogen to produce antibody.The protein of the present invention or the physics and chemistry form of chimeric molecule can increase the antibody for protein or chimeric molecule；The antibody of the glycopeptide of protein or chimeric molecule for the present invention；Directly against in protein or its chimeric molecule another translation in or posttranslational modification peptide antibody.

The protein or its chimeric molecule of the present invention may have in vivo the epitope of not accessible (it is possible that presence) under normal circumstances.For example, it is possible to there is a region under normal circumstances with the part contact of a heteropleural acceptor in receptor domain.The monoclonal antibody for intersecting response with endogenous recipient can be produced with these epitopes.This antibody can block a receptor element and another interaction, and therefore prevent signal transduction.This can be used in the overexpressing cells factor or acceptor as treatment.Antibody can also acceptor overexpression and do not need part just produce signal when used as treatment.

Antibody can be also used for the level (for example, serum levels to determine half-life period) of detection protein or its chimeric molecule in treatment disease.

In addition, antibody can be used for protein or chimeric molecule of the diagnostic assay with the presence or absence of the present invention in specific sample.

" antibody " being previously mentioned includes the form of ownership for the antibody being previously mentioned, and includes but is not limited to：It is complete anti-(such as containing complete Fc regions), including such as monoclonal antibody；Antigen binding antibody fragment, including such as Fv, Fab, Fab ' and F (ab ')₂Fragment；Humanized antibody；Human antibodies (for example, in transgenic animals or the antibody produced by display technique of bacteriophage)；With the immunoglobulin derived peptide produced by technique for gene engineering.With other differences refered in particular to, term " antibody " and as described here including complete anti-and its antigen-binding fragment.

Unless otherwise noted, the present invention represents that the antibody substantially only combines its target antigen, the appreciable combination without unrelated protein on the specificity of antibody.It is, however, possible to which an antibody will be designed or select to combine two or more related proteins.Related protein includes different same proteins or comes from the splice variant or fragment of the homologous protein of different plant species.This antibody is also believed to have specificity to those protein, and is included in the present invention.Term " substantially " means in this context to be not above non target antigen the detectable combination of substrate level, i.e., non-specific.

The antibody of the present invention can be prepared by well known method.See, e.g., MonoclonalAntibodies, Hybridomas：A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980)；And Antibodies：A Laboratory Manual, Harlow and Lane (eds.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY, (1988).

Producing a method of the antibody of the present invention includes a protein or chimeric molecule or its immunogenic portion of the invention being produced with animal (for example, one include receptor binding domain peptide, antibody is by receptor binding domain come with reference to the polypeptide or immunogenic portion in a protein or its chimeric molecule) non-human animal, such as one mouse or a transgenic mice is immunized.There are a variety of methods to increase the antigenicity of specified protein or its chimeric molecule, for example, give adjuvant or conjugated antigen, including for antigen and another element that required antibody is responded, these methods are all known in the art and are to use.Typical immunization includes a series of initial immune and subsequent enhancings and is immunized.Blood can be taken to animal and the antibody titer in serum is analyzed.Animal can be strengthened, the plateau until reaching titre.With protein fusion conjugant can be prepared in recombinant cell culture thing.Equally, aggregating agent such as alum is applied to enhancing immune response.

Polyclonal and monoclonal antibody can be prepared by this method.The method for obtaining two types antibody is well known in the art.Polyclonal antibody uses less but relatively easy preparation, suitable animal is injected with the protein of the invention or chimeric molecule or its immunogenic portion of effective quantity, serum is collected from animal and protein or the antibody of its chimeric molecule is directed to using the separation of any of immunoadsorbent technics is specific.The antibody produced by the method is using relatively fewer, because product has potential inhomogeneity.

The reason for significantly being had a preference for using monoclonal antibody is that they can largely be produced, and product has homogeney.Monoclonal antibody can be produced by conventional method.

Term " monoclonal antibody " used herein refers to the antibody obtained from substantial amounts of homogeneous antibody, i.e. by the independent antibody that identical colony constitutes in addition to the possible naturally-produced mutation existed on a small quantity.Monoclonal antibody is high specific, directly against single antigen site.In addition, and the polyclonal antibody preparations that are typically include for the different antibodies of different antigenic determinants (epitope) it is different, each monoclonal antibody is directly against antigenic determinant single on antigen.Modifier " monoclonal " refers to that antibody is characterized in be obtained from abundant homologous antibody colony, rather than is construed to need to produce antibody by any specific method.For example, Kohler et al. Nature 256 can be passed through according to monoclonal antibody used in the present invention：It is prepared by the hybridoma method that 495 (1975) are announced first, or can be prepared by recombinant DNA method (see, for example U.S. Patent number 4,816,567)." monoclonal antibody " can also be separated from phage antibody library, use such as Clackson et al. Nature 352：624-628,1991 and Marks et al. J Mol Biol 222：581-597, the method described in 1991.

A kind of method of hybridoma cell line is produced present invention contemplates a kind of, including a kind of non-human animal, such as mouse or transgenic mice is immunized with the protein or chimeric molecule of the present invention；Splenocyte is harvested from immune animal；The splenocyte of harvest and myeloma cell line fusion are produced into hybridoma；And identify that production can combine the hybridoma cell line of a kind of protein or the monoclonal antibody of its chimeric molecule.

This hybridoma cell line and the monoclonal antibody that they are produced are included in the present invention.The monoclonal antibody secreted by routine techniques purified hybrid oncocyte system.The monoclonal antibody of hybridoma or their generations can further be screened to identify the monoclonal antibody with required particular characteristics, for example, suppress the ability of its signal by cytokine receptor.

Can be used for the protein or its chimeric molecule or its immunogenic portion of immune animal in the initial period of the antibody of the production present invention should come from people's expression source.

The antigen-binding fragment of the antibody of the present invention can be produced by conventional method.The example of this fragment includes but is not limited to：Fab、Fab′、F(ab′)₂With Fv fragments, including Single-Chain Fv Fragment of Murine (being named as sFv or scFv).The stable Fv fragments (dsFv) of the antibody fragment and derivative produced by genetic engineering, such as curing, single-stranded Variable domain (Abs), according to the present invention, it is also contemplated that miniantibody and binary are used to using.

Characteristic needed for this fragment and derivative directly against protein or the monoclonal antibody of its chimeric molecule can be prepared by known technology and is screened, the technology includes detection described herein.These detections provide the method for the fragment and derivative of identification energy conjugated protein or the antibody of the invention of its chimeric molecule, and can identify those active fragments and derivative for also retaining the signal for suppressing protein or its chimeric molecule.These technologies necessarily include the DNA of the polypeptide chain (or its part) in separation coding mAb interested, and operate these DNA by recombinant DNA technology.Can be by DNA being fused on another DNA interested or other methods (such as by mutagenesis or other routine techniques) increase, delete or replaced one or more amino acid residues.

Can be from the DNA (such as weighing or light chain, only variable region or total length) of separation encoding antibody polypeptide in the B cell of the immune mouse of the protein or chimeric molecule of the present invention.Conventional program can be used to separate DNA.Phage display is the example of another known technology, and the derivative of antibody can be prepared by it.In one approach, expressed in any suitable recombinant expression system as a kind of polypeptide of a part for antibody interested, and the polypeptide expressed can be assembled to form antibody molecule.

Heavy chain and light chain variable district (Fv areas) fragment can be connected to form single-chain antibody by amino acid bridge (small peptide linker), form a single polypeptide chain.This scFv s (scFvs) is prepared by the DNA of the fusion coded polypeptide linker between the DNA of two variable domain polypeptides (VL and VH) is encoded.According to the length of flexible connection between two variable domains, gained antibody fragment can form dimer or tripolymer (Kortt et al. ProteinEngineering 10：423、1997).The technology for the production single-chain antibody that developed includes United States Patent (USP) 4,946,778；Bird(Science 242：423rd, 1988), Huston et al. (Proc Natl Acad Sci USA 85：5879th, 1988) and Ward et al. (Nature334：544th, the 1989) technology.The single-chain antibody derived from antibody provided herein is included in the present invention.

In one embodiment, the invention provides can combine the present invention protein or chimeric molecule and suppress protein or the signal of its chimeric molecule antibody antibody fragment or chimeric molecule, recon or synthesized form.

Known technology can obtain the antibody of different subclass or isotype from antibody interested, i.e. subclass is changed.It may be thus possible, for example, to obtain IgG1 or IgG4 monoclonal antibodies from IgM monoclonal antibody, vice versa.This technology can produce the new antibodies for the antigenic binding property for possessing specified antibody (female antibody), but the biological characteristics that also the display antibody isotype different with female antibody or subclass have.Recombinant DNA technology can be used.The clone DNA of encoding particular antibodies polypeptide can be used in the process, such as the DNA of the constant region of isotype antibody needed for encoding.

Above-mentioned monoclonal production process, such as mouse, to produce monoclonal antibody can be used in animal.The conventional antibodies obtained from these animals, such as mouse antibodies, are typically considered to not be suitable for human body, because they can cause immune response.Therefore, this antibody may need modification to be applied to human body.The process for preparing chimeric molecule and/or humanized antibody is known in the art, be will be described in further detail below.

Monoclonal antibody herein particularly including " chimeric " antibody, wherein heavy chain and/or light variable domains are identical or homologous with the corresponding sequence for the antibody for coming from non-human species (such as mouse), and the remainder in chain is identical or homologous with the corresponding sequence for the antibody for coming from human body, the fragment of this antibody is also that so, therefore they show required biological activity (United States Patent (USP) 4,816,567；With Morrison et al. .Proc Natl Acad Sci USA81：6851-6855、1984).

" humanization " form of inhuman (such as mouse) antibody is chimeric antibody, wherein containing a small amount of sequence for coming from non-human immunoglobulin.For most part, humanized antibody is human immunoglobulin(HIg) (recipient's antibody), wherein the complementary determining region (CDRs) of recipient's antibody is replaced by the corresponding CDRs of non-human species' (donor antibody), and the non-human species for example possess mouse, rat, rabbit or the non-human primate of required characteristic (such as specificity and compatibility).In some instances, the framework residues of people's immune globulin are replaced by corresponding non-human residues.Also, humanized antibody may be embodied in undiscovered residue in acceptor antibody or donor antibody.These modifications are carried out further to refine the performance of antibody.In a word, humanized antibody will basically comprise it is all at least one, and typical case is the variable domains of two, wherein all or essentially all of complementary determining region corresponds to non-human immunoglobulin, and all or generally all of framework residues are human immunoglobulin sequences.Humanized antibody will also optionally contain at least one of constant region for immunoglobulin (Fc), typically human immunoglobulin(HIg).Further details are referring to Jones et al. Nature 321：522-525、1986；Reichmann et al. Nature 332：323-329、1988；Presta、Curr Op Struct Biol 2：593-596、1992；Liu et al. Proc Natl Acad Sci USA 84：3439、1987；Larrick et al. Bio/Technology7：934、1989；With Winter and Harris, TIPS 14：139、1993.

In a further embodiment, the invention provides immunity detection reagent, the level of the human protein expressed by the people's cell in a kind of biological products can be detected, the biological products include the biological products for including naturally-produced people's albumen.

The biological products that the energy of the present invention is detected using immunity detection reagent include but is not limited to laboratory sample, cell, tissue, blood, serum, blood plasma, urine, excrement, saliva and phlegm.

The immunity detection reagent of the present invention includes a kind of solid phase support matrix, is not limited to but including a kind of film, dipstick, pearl, gel, pipe or porous, flat, round bottom or the microtest plate at v-shaped bottom, such as 96 orifice plates；Directly against the antibody preparation (capture antibody) of human protein interested；It is coated with pharmaceutical solutions (such as BSA or casein)；Secondary antibody preparation (detection antibody), is conjugated directly against human protein interested and with suitable detection molecules (such as alkaline phosphatase)；A kind of Chromogenic Substrate Solution (such as nitro blue tetrazolium)；One kind addition substrate solution (the chloro- 3- indolyl phosphates of the bromo- 4- of such as 5-)；A kind of substrate buffer solution mother liquor (such as 0.1M Tris-HCL (pH 7.5) and 0.1M NaCl, 50mM MgCl₂)；The protein of the invention of concentration known (standard) or the preparation of chimeric molecule；And operation instructions.

Suitable detection molecules can be selected to be attached on including but not limited to staphylococcal protein A or streptococcal protein G equimolecular from the list including reagents such as enzyme, dyestuff, fluorescence molecule, chemiluminescence agent, isotope or collaurums.

In a specific embodiment, capture and detect that antibody is monoclonal antibody, it, which is prepared, includes the protein with the present invention or chimeric molecule immunizing non-human animals, such as mouse or transgenic mice, and standard method is then carried out as described above.Monoclonal antibody can be produced in the method with restructuring of alternative, as described above, it is possible to including people or chimeric antibody part or domain.

In another embodiment, capture and detect that antibody is polyclonal antibody, it, which is prepared, includes the protein with the present invention or chimeric molecule immunizing non-human animals, such as mouse or rabbit, sheep or horse, and standard method is then carried out as described above.

The component of detection kit is provided by predetermined ratio, and the change of the relative populations of different reagents suitably maintains the reagent concentration in solution, allows detection sensitivity substantially to maximize.Especially, reagent can be provided in the way of dry powder, typically freezed, including auxiliary material, the dissolving each reagent solution of relief is for biological products to be measured with suitable concentration

The application method of the immunity detection reagent of the present invention can be discussed in detail in operation instructions.For example, operation instructions can introduce the method that solid phase support matrix is coated with suitable concentration with the capture antibody-solutions prepared, such as 4 DEG C overnight.The method for being coated with nonspecific protein binding site with the coating solution prepared can be described in further detail in operation instructions；(such as 37 DEG C 1 hour or room temperature 2 hours) adds sample and the incubation of the protein containing the present invention being serially diluted or chimeric molecule under suitable conditions, then serial cleaning, such as 0.1M PBS (pH 7.2) solution containing 0.05% polysorbas20 are carried out using suitable buffer solution known in the art.In addition, specification can be provided, using being incubated under suitable conditions after detection antibody preparation, for example, 37 DEG C of 1 hour or room temperatures 2 hours, then carry out a series of cleanings.The working solution of detection buffer solution is prepared using the detection substrate and substrate buffer solution of offer, is subsequently added in each hole, is kept under suitable conditions, from room temperature 1 hour 5 minutes to 37 DEG C.Can be by adding 1N NaOH or 2N H₂SO₄Terminate chromogenic reaction.

In another alternate embodiment, operation instructions can provide any combinations for any or all said components to be added that can be added simultaneously in reservation ratio, the change of the relative populations of different reagents suitably maintains the reagent concentration in solution, allow compound formed produced by detectable signal substantially maximize.

ELISA Plate Readers or spectrophotometer can be used, under suitable optical density (OD), or it is used as exciting light, using spectrophotometer, fluorescence photometer or flow cytometer, under suitable wavelength, or radioactive counter is used, under suitable power spectrum, or by opacimeter, or by carrying out visual comparison, detection color products or fluorescence or chemiluminescence or radioactivity or other levels by combining, being conjugated the signal that connecting detection reagent is produced with chart or index.With the serial dilutions of above-mentioned sample Parallel testing standard preparation.Standard curve or chart are produced, the level of protein or its chimeric molecule in sample is calculated according to standard curve or chart interpolation (interpolated).

Present invention also offers people's derived protein or its chimeric molecule, as the standard protein in immune detection.The present invention extends further a kind of method for determining the level of people's albumen of people's cell expression or its chimeric molecule in biological products, including the suitable determination method for determining people's albumen or chimeric molecule, the detection method includes (a) and is combined biological products and one or more antibody directly against people's albumen or its chimeric molecule, and (b) determines the level of people's albumen or chimeric molecule in the antibody or each antibody binding biological products；(c) it will be combined between standard people albumen or a kind of chimeric molecule sample and one or more for the antibody of people's albumen or chimeric molecule；(d) level of people's albumen or chimeric molecule in the combination biological products of the antibody or each antibody is determined；(e) level of people's albumen or chimeric molecule and the level of the antibody or each antibody binding standard people albumen or chimeric molecule sample in the antibody or each antibody binding biological products are compared；

Especially, standard people albumen or chimeric molecule sample are the preparations of a kind of protein comprising the present invention or chimeric molecule.

Biological products include but is not limited to laboratory sample, cell, tissue, blood, serum, blood plasma, urine, excrement, saliva and phlegm.As described above or by methods known in the art, biological products can combine one or more capture antibody.For example, being coated with solid phase support matrix (for example, 4 DEG C are stayed overnight) under suitable conditions with the capture antibody-solutions prepared first；The binding site of nonspecific protein is then closed with the coating solution prepared；It is subsequently added the series of diluted samples of the protein containing the present invention or chimeric molecule and is incubated (such as 37 DEG C 1 hour or room temperature 2 hours) under suitable conditions, then carries out series cleaning (such as 0.1M PBS (pH 7.2) solution containing 0.05% polysorbas20) using suitable buffer solution known in the art.

Then, biological products and one or more detection antibody for being combined with suitable detection molecules as described here are combined.For example, be incubated under suitable conditions after application detection antibody preparation (for example, 37 DEG C 1 hour or room temperature 2 hours), a series of cleanings are then carried out.

Level can be combined as described above or by methods known in the art measure.For example, preparing the working solution for detecting buffer solution using detection substrate and substrate buffer solution, it is subsequently added in each hole, keeps under suitable conditions, from room temperature 1 hour 5 minutes to 37 DEG C.Can be by adding 1N NaOH or 2N H₂SO₄Terminate chromogenic reaction.

In a specific embodiment, the present invention contemplates the protein or chimeric molecule known clearly one and separated as described above.

In a particular embodiment, to next or multiple physicochemical property (P_x) and pharmacological characteristics (T_y) characterize the present invention TNF-α feature, it includes：

- apparent molecular weight (P₁) it is about 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, in a specific embodiment, for 10-30kDa；

-pI(P₂) scope is about 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 4-8.5 in a specific embodiment；

- it there are about 2 to 50 isoform (P₃), such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, in a specific embodiment, it is 10-40 isoform；

- carbohydrate percetage by weight (P₅) it is about 1 to 99%, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, in a specific embodiment, for 0-10%；

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) it is about 8 to 30kDa；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 8 to 25kDa, it is 10 to 20kDa in a specific embodiment；

- immune response feature (T₁₃) it is different from the humanTNF-α expressed in nonhuman cells' system, in a specific embodiment, when determining the protein concentration of TNF-α of the present invention with the ELISA kit of the humanTNF-α expressed in containing nonhuman cells' system, the protein concentration of TNF-α of the present invention is underestimated.

In a particular embodiment, to next or multiple physicochemical property (P_x) and pharmacological characteristics (T_y) characterize the present invention LT- α feature：

- apparent molecular weight (P₁) it is about 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, in a specific embodiment, 32kDa is arrived for 15；

-pI(P₂) scope is about 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 5 to 11 in a specific embodiment；

- it there are about 2 to 100 isoform (P₃), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 isoforms, in a specific embodiment, for 7-33 isoform；

- carbohydrate percetage by weight (P₅) it is about 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, in a specific embodiment, for 0 to 42%；

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) it is about 10 to 30kDa, it is 12 to 25kDa in a particular embodiment；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 10 to 25kDa, it is 12 to 23kDa in a specific embodiment；

- immune response feature (T₁₃) the people LT- α expressed in nonhuman cells' system are different from, in a specific embodiment, LT- α of the present invention protein concentration is relatively low when being determined with ELISA kit, because ELISA kit contains the people LT- α expressed in nonhuman cells' system.

In a particular embodiment, to next or multiple physicochemical property (P_x) and pharmacological characteristics (T_y) characterize the present invention TNFR I-Fc feature, it includes：

- apparent molecular weight (P₁) it is about 5 to 120, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, in a specific embodiment, 75kDa is arrived for 45；

-pI(P₂) scope is about 2 to 12, such as 2,3,4,5,6,7,8,9,10,11,12, it is 5.5-9.5 in a specific embodiment；

- it there are about 2 to 20 isoform (P₃), such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 isoforms are 8-16 isoform in a specific embodiment；

- carbohydrate percetage by weight (P₅) it is about 10 to 90%, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90%, in a specific embodiment, for 0-36%；

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) it is about 35 to 65kDa, it is 36 to 60kDa in a specific embodiment；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 35 to 65kDa, it is 36 to 60kDa in a specific embodiment；

Percentage (the P of-acidity contents of monosaccharides₈) it is about 0-50%, such as 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50, in a specific embodiment, it is 0-10%；

- contents of monosaccharides (P₉) and sialic acid content (P₁₀), during with GalNAc as standard, for 1: 0.1-8 trehalose, 1: 7-27 G l cNAc, 1: 1-14 galactolipin, 1: 2-17 mannose and 1: 0-3 NeuNAc, in a specific embodiment, the trehalose for being 1: 1-4.5,1: 10-18 GlcNAc, 1: 3-9 galactolipin, 1: 4-11 mannose and 1: 0.1-2 NeuNAc；During with 3 times of mannoses as standard, for 3: 0.01-3 trehalose, 3: 0.01-3 GalNAc, 3: 1-17 GlcNAc, 3: 0.1-5 galactolipin and 3: 0-3 NeuNAc, in a specific embodiment, for 3: 0.1-1.5 trehalose, 3: 0.1-1 GalNAc, 3: 3-11 GlcNAc, 3: 1-2.5 galactolipin and 3: 0-2 NeuNAc；

- sulphates content (P₁₁), during with Ga1Nac as standard, the sulfate for being 1: 0.1-21, in a specific embodiment, the sulfate for being 1: 1.5-14；During with 3 times of mannoses as standard, the sulfate for being 3: 0.1-6, in a specific embodiment, the sulfate for being 3: 0.5-4；

- sulfation (P₅₉) it is expressed as the percentage of contents of monosaccharides in molecule, for 0-50%, such as 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50, in a specific embodiment, it is 10-16%；

Neutral percentage (the P of-N- connection oligosaccharides₁₃) it is about 30 to 100%, such as 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, in a specific embodiment, for 80 to 100%, in further specific embodiment, for 94 to 97%；

Acid percentage (the P of-N- connection oligosaccharides₁₄) it is about 0 to 50%, such as 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50%, in a specific embodiment, for 0 to 20%, it is 3 to 6% in further specific embodiment；

Neutral percentage (the P of-O- connection oligosaccharides₁₅) it is about 24 to 67%, such as 24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67%, in a specific embodiment, for 29 to 62%, it is 34 to 57% in further specific embodiment；

Acid percentage (the P of-O- connection oligosaccharides₁₆) it is about 10 to 80%, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80%, in a specific embodiment, for 38 to 71%, in further specific embodiment, for 43 to 66%；

Position (the P of-N- connection oligosaccharides₂₁) include N-299 (being numbered from the section start of signal sequence), give after PNGase, identified by PMF.

In a particular embodiment, to next or multiple physicochemical property (P_x) and pharmacological characteristics (T_y) characterize the present invention TNFRII-Fc feature, it includes：

- apparent molecular weight (P₁) it is about 10 to 150, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, in a specific embodiment, 118kDa is arrived for 46；

-pI(P₂) scope is about 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 4 to 10 in a specific embodiment；

- it there are about 2 to 52 isoform (P₃), such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52, in a specific embodiment, it is 10 to 40 isoforms；

- carbohydrate percetage by weight (P₅) it is about 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, in a specific embodiment, for 0 to 56%；

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) it is about 40 to 100kDa, it is 46 to 87kDa in a specific embodiment；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 40 to 95kDa, it is 42 to 80kDa in a specific embodiment；

Percentage (the P of-acidity contents of monosaccharides₈) it is about 0-50%, such as 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50, in a specific embodiment, it is 1 to 10%；

- contents of monosaccharides (P₉) and sialic acid content (P₁₀), during with Ga 1NAc as standard, the trehalose for being 1: 0.01-3,1: 0.1-5 GlcNAc, 1: 0.1-3 galactolipin, 1: 0.1-3 mannose and 1: 0.01-3 NeuNAc；In a specific embodiment, the trehalose for being 1: 0.01-2,1: 0.1-3 GlcNAc, 1: 0.1-2 galactolipin, 1: 0.1-2 mannose and 1: 0.01-2 NeuNAc；During with 3 times of mannoses as standard, for 3: 0.01-3 trehalose, 3: 1-17 GalNAc, 3: 2-32 GlcNAc, 3: 1-9 galactolipin and 3: 0.1-3 NeuNAc, in a specific embodiment, for 3: 0.1-2 trehalose, 3: 3-11 GalNAc, 3: 5-21 GlcNAc, 3: 3-6 galactolipin and 3: 0.1-2 NeuNAc；

- sulphates content (P₁₁), during with GalNac as standard, the sulfate for being 1: 0.1-6, in a specific embodiment, the sulfate for being 1: 1-4；During with 3 times of mannoses as standard, the sulfate for being 3: 4-29, in a specific embodiment, the sulfate for being 3: 9-19；

- sulfation (P₅₉) it is expressed as the percentage of contents of monosaccharides in molecule, for 10-90%, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90%, in a specific embodiment, for 27 to 41%；

Neutral percentage (the P of-N- connection oligosaccharides₁₃) it is about 10 to 100%, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, in a specific embodiment, for 69 to 89%, in further specific embodiment, for 74 to 84%；

Acid percentage (the P of-N- connection oligosaccharides₁₄) it is about 0 to 80%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80%, in a specific embodiment, for 11 to 31%, in further specific embodiment, for 16 to 26%；

Neutral percentage (the P of-O- connection oligosaccharides₁₅) it is about 5 to 90%, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, in a specific embodiment, for 17 to 54%, in further specific embodiment, for 22 to 49%；

Acid percentage (the P of-O- connection oligosaccharides₁₆) it is about 5 to 99%, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, in a specific embodiment, for 46 to 83%, in further specific embodiment, for 51 to 78%；

The structure of-one or more N- glycan is listed in (P in the ratio of the N- of table 37 (a) connection oligosaccharides₁₉)；

The structure of-one or more O- glycan is listed in (P in the ratio of the O- of table 37 (b) connection oligosaccharides₂₀)；

- bioactivity is distinguished in the people TNFRII-Fc expressed in nonhuman cells' system, and in a specific embodiment, TNFRII-Fc of the present invention neutralizes the cell (T of TNF-α induction in L-929 cells₃₀) toxicity people TNFRII-Fc of the ability than being expressed in E.coli cell ability it is high 8-18 times.

In a particular embodiment, to next or multiple physicochemical properties (Px) and pharmacological characteristics (T_y) characterize the present invention OX40-Fc feature, it includes：

- apparent molecular weight (P₁) it is about 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, in a specific embodiment, 75kDa is arrived for 46；

-pI(P₂) scope is about 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 4 to 9 in a specific embodiment；

- it there are about 2 to 50 isoform (P₃), such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, in a specific embodiment, it is 8-16 isoform；

- carbohydrate percetage by weight (P₅) it is about 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, in a specific embodiment, for 0 to 36%；

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) it is about 40 to 75kDa, it is 44 to 72kDa in a specific embodiment；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 38 to 75kDa, it is 41 to 70kDa in a specific embodiment；

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) it is about 46 to 65kDa；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 46 to 65kDa；

- contents of monosaccharides (P₉) and sialic acid content (P₁₀), during with GalNAc as standard, the trehalose for being 1: 0.01-3,1: 1-4 GlcNAc, 1: 0.1-3 galactolipin, 1: 0.1-3 mannose and 1: 0-3 NeuNAc；In a specific embodiment, the trehalose for being 1: 0.1-1,1: 2-3 GlcNAc, 1: 0.5-2 galactolipin, 1: 0.5-1 mannose and 1: 0-2 NeuNAc；During with 3 times of mannoses as standard, for 3: 0.1-3 trehalose, 3: 1-7 GalNAc, 3: 3-15 GlcNAc, 3: 2-9 galactolipin and 3: 0-3 NeuNAc, in a specific embodiment, for 3: 0.5-2 trehalose, 3: 3-5 GalNAc, 3: 6-10 GlcNAc, 3: 4-5 galactolipin and 3: 0-2 NeuNAc；

- sialic acid content (P₁₀) it is expressed as the percentage of contents of monosaccharides in molecule, about 0 to 50%, such as 0,1st, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50%, in a specific embodiment, it is 0-10%；

- sulphates content (P₁₁), during with GalNac as standard, the sulfate for being 1: 0-3, in a specific embodiment, the sulfate for being 1: 0.30-2；During with 3 times of mannoses as standard, the sulfate for being 3: 0.1-7, in further specific embodiment, the sulfate for being 3: 1-5；

- sulfation (P₅₉) it is expressed as the percentage of contents of monosaccharides in molecule, for 0-50%, such as 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50, in a specific embodiment, it is 9 to 15%；

Neutral percentage (the P of-N- connection oligosaccharides₁₃) it is about 69 to 100%, it is 74 to 100% in a specific embodiment, is 79 to 195% in further specific embodiment；

Acid percentage (the P of-N- connection oligosaccharides₁₄) it is about 0 to 31%, it is 0 to 26% in a specific embodiment, is 5 to 21% in further specific embodiment；

Neutral percentage (the P of-O- connection oligosaccharides₁₅) it is about 20 to 100%, it is 40 to 90% in a specific embodiment, is 45 to 80% in further specific embodiment；

Acid percentage (the P of-O- connection oligosaccharides₁₆) it is about 0 to 80%, it is 10 to 60% in a specific embodiment, is 20 to 55% in further specific embodiment；

Position (the P of-N- connection oligosaccharides₂₁) include N-160 to N-298 (being numbered from the section start of signal sequence), give after PNGase, identified by PMF.

In a particular embodiment, to next or multiple physicochemical property (P_x) and pharmacological characteristics (T_y) characterize the present invention BAFF feature, it includes：

- apparent molecular weight (P₁) it is about 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, in a specific embodiment, 22kDa is arrived for 10；

-pI(P₂) scope is about 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 4 to 8 in a specific embodiment；

- it there are about 2 to 50 isoform (P₃), such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, in a specific embodiment, it is 5 to 10 isoforms.

- carbohydrate percetage by weight (P₅) it is about 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, in a specific embodiment, for 0 to 25%.

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) it is about 8 to 22kDa, it is 10 to 22kDa in a specific embodiment；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 8 to 22kDa, it is 10 to 22kDa in a specific embodiment；

- bioactivity is distinguished in the people BAFF expressed in nonhuman cells' system, in a specific embodiment, and the ability of BAFF induction RPMI 8226 cell propagation of the present invention is 1.1-2.4 times of the people BAFF expressed in E.coli cells.

In a particular embodiment, to next or multiple physicochemical property (P_x) and pharmacological characteristics (T_y) characterize the present invention NGFR-Fc feature, it includes：

- apparent molecular weight (P₁) it is about 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, in a specific embodiment, 105kDa is arrived for 55；

-pI(P₂) scope is about 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 3: 6 in a specific embodiment；

- it there are about 2 to 50 isoform (P₃), such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, in a specific embodiment, it is 8 to 16 isoforms；

- carbohydrate percetage by weight (P₅) it is about 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, in a specific embodiment, for 11 to 53%.

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) for 45 between 100kDa, be 48 to arrive 90kDa in a specific embodiment；

Actual measurement molecular weight (P after-N- connections and O- connections oligosaccharides deglycosylation₇) it is about 45 to 95kDa, it is 48 to 85kDa in a specific embodiment；

In a particular embodiment, to next or multiple physicochemical property (P_x) and pharmacological characteristics (T_y) characterize the present invention Fa s parts characteristic, it includes：

- apparent molecular weight (P₁) it is about 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, in a specific embodiment, 35kDa is arrived for 15；

-pI(P₂) scope is about 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14；

- it there are about 2 to 50 isoform (P₃), such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms；

- carbohydrate percetage by weight (P₅) it is about 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, in a specific embodiment, for 0 to 51%.

Actual measurement molecular weight (P after-N- connection oligosaccharides deglycosylations₆) for 10 between 28kDa, be 12 to arrive 21kDa in a specific embodiment；

Position (the P of-N- connection oligosaccharides₂₁) include N-184 (being numbered from the section start of signal sequence), give after PNGase, identified by PMF.

In one embodiment, protein of the invention or chimeric molecule are in N- crosslink parts (P₁₉) in contain at least one following structure.In these charts, " u " or "" different header structure is represented for a or b, and/or crosslinking sites are 2,3,4, and/or 6.

XX glycan structures

Glycan structures Gal (1-)GlcNAc(1-)[Ga l(1-)GlcNAc(1-)]Man(a1-3)[Gal(1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-6)][GlcNAc(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc+″+3xGal(1-)GlcNAc(1-)″

Glycan structures Gal (1-)GlcNAc(1-)[G al(1-)GlcNAc(1-)]Man(a1-3)[Gal(1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-6)][GlcNAc(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc+″+3xGal(1-)GlcNAc(1-)+Fuc(1-)″

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-)[Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-6)]Man(a1-)]Man(b1-4)GlcNAc(b1-4)GlcNAc+″+3xGal(b1-4)GlcNAc(b1-3)″

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-)[Gal(b1-4)GlcNAc(b1-2)[Ga l(b1-4)GlcNAc(b1-6)]Man(a1-)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc+″+3xGal(b1-4)GlcNAc(b1-3)″

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-)[Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-6)]Man(a1-)]Man(b1-4)GlcNAc(b1-4)GlcNAc+″+3xGal(b1-4)GlcNAc(b1-3)+Gal(b1-3)GlcNAc(b1-3)″

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-)[Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-6)]Man(a1-)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc+″+3xGal(b1-4)GlcNAc(b1-3)+Gal(b1-3)GlcNAc(b1-3)″

Glycan structures Gal (1-)GlcNAc(1-)[Ga l(1-)GlcNAc(1-)]Man(a1-3)[Gal(1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-6)][GlcNAc(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc+″+4xGal(1-)GlcNAc(1-)″

Glycan structures Gal (1-)GlcNAc(1-)[Ga l(1-)GlcNAc(1-)]Man(a1-3)[Gal(1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-6)][GlcNAc(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc+″+4xGal(1-)GlcNAc(1-)+Fuc(1-)″

Glycan structures Gal (1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-3)[Gal(1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-6)][GlcNAc(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc+″+5x Gal(1-)GlcNAc(1-)″

Glycan structures Gal (bl-4) { GlcNAc (b1-3) Gal (b1-4) } kGlcNAc (b1-2) Man (a1-3) [Gal (b1-4) { GlcNAc (b1-3) Gal (b1-4) } jGlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ " Where j+k=14＆j, k ＞=1 "

Glycan structures NeuAc (a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}jGlcNAc(b1-2)Man(a1-)[Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}kGlcNAc(b1-2)Man(a1-)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ " Where j+k=14＆j, k ＞=1 "

Glycan structures NeuAc (a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}kGlcNAc(b1-2)Man(a1-3)[NeuAc(a2-) Gal (b1-4) { Gl cNAc (b1-3) Gal (b1-4) } jGlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ " Where j+k=14＆k, j ＞=1 "

Glycan structures Gal (b1-4) { GlcNAc (b1-3) Gal (b1-4) } kGlcNAc (b1-2) Man (a1-3) [Gal (b1-4) { GlcNAc (b1-3) Gal (b1-4) } jGlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ " Where j+k=14＆j, k ＞=1 "

Glycan structures NeuAc (a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}jGlcNAc(b1-2)Man(a1-)[Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}kGlcNAc(b1-2)Man(a1-)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ " Wherej+k=14＆j, k ＞=1 "

Glycan structures NeuAc (a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}kGlcNAc(b1-2)Man(a1-3)[NeuAc(a2-) Gal (b1-4) { GlcNAc (b1-3) Gal (b1-4) } jGlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ " Where j+k=14＆j, k ＞=1 "

Glycan structures Gal (b1-4) { GlcNAc (b1-3) Gal (b1-4) } kGlcNAc (b1-2) Man (a1-3) [Ga l (b1-4) { GlcNAc (b1-3) Gal (b1-4) } jGlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ " Where j+k=14＆j, k ＞=1 "

Glycan structures NeuAc (a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}jGlcNAc(b1-2)Man(a1-)[Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}kGlcNAc(b1-2)Man(a1-)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ " Wherej+k=14＆j, k ＞=1 "

Glycan structures NeuAc (a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}kGlcNAc(b1-2)Man(a1-3)[NeuAc(a2-) Gal (b1-4) { GlcNAc (b1-3) Gal (b1-4) } jGlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ " Where j+k=14＆j, k ＞=1 "

Glycan structures Gal (b1-4) { GlcNAc (b1-3) Gal (b1-4) } kGlcNAc (b1-2) Man (a1-3) [Gal (b1-4) { GlcNAc (b1-3) Gal (b1-4) } jGlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ " Wherej+k=14＆j, k ＞=1 "

Glycan structures NeuAc (a2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}jGlcNAc(b1-2)Man(a1-)[Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}kGlcNAc(b1-2)Man(a1-)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ " Where j+k=14＆j, k ＞=1 "

Glycan structures NeuAc (a 2-)Gal(b1-4){GlcNAc(b1-3)Gal(b1-4)}kGlcNAc(b1-2)Man(a1-3)[NeuAc(a2-) Gal (b1-4) { GlcNAc (b1-3) Gal (b1-4) } jGlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ " Where j+k=14＆j, k ＞=1 "

Glycan structures GlcNAc (b1-2) Man (a1-6) [Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures GlcNAc (b1-4) Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures GlcNAc (b1-2) Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-4)] [Man (a1-6)] Man (b1-4)

GlcNAc(b1-4)GlcNAc

Glycan structures Fuc (a1-6) [GlcNAc (b1-4)] GlcNAc

Glycan structures Man (a1-6) Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures GlcNAc (b1-2) Man (a1-6) Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Man a1-3 Man al-6 Man b1-4 GlcNAc b1-4 GlcNAc

Glycan structures Man (a1-3) Man (a1-6) Man (b1-4) GlcNAc (b1-4) GlcNAc

NeuAc a2-u Galb1-4 G1cNHc b1-2 Man a1-3 Man b1-4 GlcNAc

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)Man(a1-3)Man(b1-4)GlcNAc

Glycan structures HSO3 (- 4) GalNAc (b1-4) GlcNAc (b1-2) Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures GlcNAc (b1-2) [GlcNAc (b1-4)] Man (a1-3) [GlcNAc (b1-4)] [Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures GlcNAc (b1-2) [GlcNAc (b1-4)] Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures GlcNAc (b1-2) [GlcNAc (b1-4)] Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures GlcNAc (b1-2) [GlcNAc (b1-4)] Man (a1-3) [GlcNAc (b1-2) [GlcNAc (b1-6)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures HSO3 (- 4) GalNAc (b1-4) GlcNAc (b1-2) Man (a1-3) [HSO 3 (- 4) GalNAc (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)Man(a1-3)[Man(a1-6)]Man(b1-4)GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc (b1-4) Gl cNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Fuc (1-)[Gal(1-)]GlcNAc(1-)Man(a1-)[Man(a1-)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Man (a1-3) Man (a1-6) [Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [GlcNAc (b1-2) Man (a1-3)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Gal (1-)GlcNAc(1-)Man(a1-)[GlcNAc(1-)Man(a1-)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-)]GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [GlcNAc (b1-2) Man (a1-3)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [GlcNAc (b1-2) [GlcNAc (b1-4)] Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures NeuAc (a2-6) GalNAc (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures HSO3 (- 4) GalNAc (b1-4) GlcNAc (b1-2) Man (a1-3) [NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [GlcNAc (b1-2) Man (a1-3)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Gal (1-)GlcNAc(1-)Man(a1-)[GlcNAc(1-)Man(a1-)][GlcNAc(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [NeuAc (a2-6) GalNAc (b1-4) GlcNAc (b1-2) Man (a1-3)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [NeuAc (a 2-6) GalNAc (b1-4) GlcNAc (b1-2) Man (a1-3)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ "+2xMan "

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)Man(a1-3)[Man(a1-3)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Fuc (a1-2) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Fuc (1-)[Gal(1-)]GlcNAc(1-)Man(a1-)[Gal(1-)GlcNAc(1-)Man(a1-)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Fuc (a1-2) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Fuc (a1-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures HSO3 (- 6) [NeuAc (a2-3)] Gal (b1-4) GlcNAc (b1-2) Man (a1-)[NeuAc(a 2-)Gal(b1-4)GlcNAc(b1-2)Man(a1-)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-2) Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) Man (a1-3)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-2) Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) Man (a1-3)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Fuc (a1-2) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) Man (a1-3)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-2) Man (a1-3)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-2) Man (a1-3) [Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures NeuAc (a 2-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-3)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Fuc (a1-2) [GalNAc (a1-3)] Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Fuc (a1-2) [GalNAc (a1-3)] Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Fuc (a1-2) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Fuc (a1-2) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) Man (a1-3)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Fuc (a1-2) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Fuc (a1-2) Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (1-)GlcNAc(1-)[GlcNAc(1-)]Man(a1-)[Gal(1-)GlcNAc(1-)Man(a1-)][GlcNAc(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc+ "+NeuAc "

Glycan structures Gal (b1-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc+ "+2x NeuAc "

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)[NeuAc(a2-)Gal(b1-4)GlcNAc(b1-4)]Man(a1-3)[NeuAc(a2-)Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) Man (a1-3)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Fuc (a1-6) [Gal (b1-4)] GlcNAc (1-2)Man(1-6)]Man(1-4)[Fuc(a1-3)Fuc(a1-3)]GlcNAc+″+NeuAc(2-6)″

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Man (a1-3) [Man (a1-6)] Man (a1-6) [Man (a1-2) Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Man (a1-3) [Man (a1-6)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)Man(a1-3)[Man(a1-3)[Man(a1-6)]Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ "+Fuc (a1-3) "

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)Man(a1-6)[Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-4)]Man(a1-3)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-4)[Gal(b1-4)GlcNAc(b1-2)]Man(a1-3)[Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-4)]Man(a1-3)[Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Gal (1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-)[Gal(1-)GlcNAc(1-)Man(a1-)]Man(b1-4)GlcNAc(b1-4)GlcNAc+″+NeuAc(a2-6)″

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-2) [NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ "+3x NeuAc (a2-)″

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) Man (a1-3)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-4) [Gal (b1-4) GlcNAc (b1-2)] Man (a1-)[Gal(b1-4)GlcNAc(b1-2)Man(a1-)]Man(b1-4)GlcNAc(b1-4)GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-4) [Gal (b1-4) GlcNAc (b1-2)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ "+Fuc (a1-2) "

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ "+Fuc (a1-3) "

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-4) [NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-4) [Gal (b1-4) GlcNAc (b1-2)] Man (a1-3) [NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ "+NeuAc (a2-3)+NeuAc (a2-6) "

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) [Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-4)] Man (a1-3) [NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcNAc (1-)[Gal(b1-4)GlcNAc(1-)]Man(a1-)[Gal(b1-4)GlcNAc(1-)Man(a1-)]Man(b1-4)GlcNAc(b1-4)GlcNAc+″+Fuc+2x NeuAc(a2-)″

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-4) [NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2)] Man (a1-3) [NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures NeuAc (a 2-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-4) [NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2)] Man (a1-3) [NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-4) [Gal (b1-4) GlcNAc (b1-2)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ "+3x NeuAc (a2-)″

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ "+HSO3 (- 6)+2x NeuAc (a2-3)+NeuAc (a2-6) "

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ "+2x HSO3 (- 6)+2x NeuAc (a2-3)+NeuAc (a2-6) "

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (al-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc+ "+Gal (b1-2) GlcNAc (b1-3)+3x NeuAc "

Glycan structures Gal (a1-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ "+NeuAc (a2-)″

Glycan structures Ga l (a1-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ "+NeuAc (a2-3)+NeuAc (a 2-6) "

Glycan structures Gal (a1-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ "+HSO3 (- 6)+2x NeuAc (a2-)″

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-4) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-3) [Fuc (a1-2) [Gal (b1-4)] GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ "+3x NeuAc (a2-)″

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-4) [Gal (b1-4) GlcNAc (b1-2)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Fuc (a1-3) [Gal (bl-4)] GlcNAc (b1-6) [Gal (b1-4) GlcNAc (b1-2)] Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-4) [Gal (b1-4) GlcNAc (b1-2)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ "+3x NeuAc (a 2-)″

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ "+3x NeuAc (a 2-)″

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-2) [NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-6)] Man (a1-6) [NeuAc (a 2-3) Gal (b1-4) GlcNAc (b1-4) [NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2)] Man (a1-3)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

Glycan structures NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)[NeuAc(a2-)Gal(b1-4)GlcNAc(b1-4)]Man(a1-3)[NeuAc(a2-)Gal(b1-4)GlcNAc(b1-2)[NeuAc(a2-)Gal(b1-4)GlcNAc(b1-6)]Man(a1-6)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-4) [Gal (b1-4) GlcNAc (b1-2)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ "+4x NeuAc (a2-)″

Glycan structures Gal (b1-4) GlcNAc (1-)[Gal(b1-4)GlcNAc(1-)]Man(a1-3)[Gal(b1-4)GlcNAc(1-)[Gal(b1-4)GlcNAc(1-)]Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc+″+2x Fuc″

Glycan structures Gal (1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-3)[Gal(1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-6)][GlcNAc(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc

Glycan structures Gal (1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-3)[Gal(1-)GlcNAc(1-)[Gal(1-)GlcNAc(1-)]Man(a1-6)][GlcNAc(1-4)]Man(b1-4)GlcNAc(b1-4)[Fuc(1-6)]GlcNAc+″+Fuc″

Glycan structures Gal (b1-4) GlcNAc (b1-4) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-4) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Glycan structures Gal (a1-3) Gal (b1-4) GlcNAc (b1-2) [NeuAc (a2-)Gal(b1-4)GlcNAc(b1-4)]Man(a1-3)[Gal(a1-3)Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures Gal (a1-3) Gal (b1-4) GlcNAc (b1-4) [NeuAc (a2-)Gal(b1-4)GlcNAc(b1-2)]Man(a1-3)[Gal(a1-3)Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)[Fuc(a1-6)]GlcNAc

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc+ "+2x Gal (b1-4) GlcNAc (b1-3)+2x NeuAc "

Glycan structures Gal (b1-4) GlcNAc (1-)[Gal(b1-4)GlcNAc(1-)]Man(a1-3)[Gal(b1-4)GlcNAc(1-)[Gal(b1-4)GlcNAc(1-)]Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc+″+Gal(b1-4)GlcNAc(1-)+4xNeuAc(a2-)″

Glycan structures Gal (b1-4) GlcNAc (b1-)Gal(b1-4)GlcNAc(b1-6)[Gal(b1-4)GlcNAc(b1-2)]Man(a1-6)[Gal(b1-4)GlcNAc(b1-2)[Gal(b1-4)GlcNAc(b1-4)]Man(a1-3)]Man(b1-4)GlcNAc(b1-4)GlcNAc+″+5x NeuAc(a2-)″

Glycan structures Gal (bl-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ "+Gal (b1-4) GlcNAc (b1-3) "

Glycan structures Gal (b1-4) GlcNAc (1-)[Gal(b1-4)GlcNAc(1-)]Man(a1-3)[Gal(b1-4)GlcNAc(1-)[Gal(b1-4)GlcNAc(1-)]Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAc+″+2x Fuc+Gal(b1-4)GlcNAc(1-)″

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-)[Gal(b1-4)GlcNAc(b1-2)Man(a1-)]Man(b1-4)GlcNAc(b1-4)GlcNAc+″+2x Gal(b1-4)GlcNAc(b1-3)+Gal(b1-3)GlcNAc(b1-3)″

In one embodiment, protein of the invention or chimeric molecule comprise at least a following structure (P in O- crosslink parts₂₀).In these charts, " u " or "" different header structure is represented for a or b, and/or crosslinking sites are 2,3,4, and/or 6.

Fuc

Glycan structures Fuc

Glc u1-u Fuc

Glycan structures Glc (1-)Fuc

GlcNAc

Glycan structures GlcNAc

GalNAc

Glycan structures GalNAc

NeuAc a2-6GalNAc

Glycan structures NeuAc (a2-6) GalNAc

GlcNAc b1-3GalNAc

Glycan structures GlcNAc (b1-3) GalNAc

Glycan structures GlcNAc (b1-3) [NeuAc (a2-6)] GalNAc

Gal b1-3GalNAc

Glycan structures Gal (b1-3) GalNAc

Gal

Glycan structures Gal

NeuAc a2-3Gal

Glycan structures NeuAc (a2-3) Gal

Xyl u1-u Glc

Glycan structures Xyl (1-)Glc

NeuAc a2-3Gal b1-4Xyl

Glycan structures NeuAc (a2-3) Gal (b1-4) Xyl

Xyl u1-u Glc

Glycan structures Xyl (1-)Glc

Xyl u1-u Glc

+Xyl

Glycan structures Xyl (1-)Glc+″+Xyl″

NeuAc a2-3Gal b1-3GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-3) Ga lNAc

Glycan structures NeuAc (a2-3) Gal (b1-3) [NeuAc (a2-6)] GalNAc

Glycan structures Gal (b1-3) [NeuAc (a2-6)] GalNAc

Fuc a1-2Gal b1-3GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) [NeuAc (a2-6)] GalNAc

Glycan structures NeuAc (2-)Gal(1-)[Fuc(a1-)]GalNAc

Delta4,5GlcA b1-3GalNAc b1-4GlcA b1-3Gal b1-3Gal b1-4Xyl

Glycan structures delta4,5GlcA (b1-3) GalNAc (b1-4) GlcA (b1-3) Gal (b1-3) Gal (b1-4) Xyl

Glycan structures delta4,5GlcA (b1-3) [HSO3 (- 4)] GalNAc (b1-4) GlcA (b1-3) Gal (b1-3) Gal (b1-4) Xyl

Glycan structures HSO3 (-)[NeuAc(a2-)]GlcNAc(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

Glycan structures Gal (b1-3) [GlcNAc (b1-6)] GalNAc

Glycan structures Fuc (a1-4) GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-4) GlcNAc (b1-6) [GlcNAc (b1-6) Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-4) GlcNAc (b1-6) Gal (b1-3) [Fuc (a1-4) GlcNAc (b1-6)] GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Fuc a12Galb1-3GlcNAcb1-3GalNAc

Glycan structures Fuc (a1-2) Gal (bl-3) GlcNAc (b1-3) GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-3) GalNAc

Glycan structures Fuc (a1-2) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-3) GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-6) [GlcNAc (b1-3)] GalNAc

Glycan structures Gal (b1-3) GlcNAc (b1-3) [GlcNAc (b1-6)] GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-6) [GlcNAc (b1-3)] GalNAc

Gal b1-4GlcNAcb1-3Gal b1-3GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) Gal (b1-3) GalNAc

Glycan structures GalNAc (b1-4) [NeuAc (a2-3)] Gal (b1-3) GalNAc

Glycan structures GalNAc (b1-4) [NeuAc (a2-3)] Gal (b1-3) [NeuAc (a2-6)] GalNAc

NeuAc u2-u Gal u1-u GalNAcu1-u GalNAc

Glycan structures NeuAc (2-)Gal(1-)GalNAc(1-)GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-6) [NeuAc (a 2-3) Gal (b1-3)] GalNAc

Glycan structures Gal (b1-)GlcNAc(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-)GlcNAc(b1-6)[Gal(b1-3)]GalNAc

NeuAc a2-u Gal b1-u GlcNAcb1-u Gal u1-u GalNAc

Glycan structures NeuAc (a2-)Gal(b1-)GlcNAc(b1-)Gal(1-)GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] Ga1NAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) [Gal (b1-4) GlcNAc (b1-6)] GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] Ga1NAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-3) [HSO3 (- 6) GlcNAc (b1-6)] GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-6) [NeuAc (a 2-3) Gal (b1-3)] GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) [Fuc (a1-4)] GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-2) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-6) [Gal (b1-3)] GalNAc+ "+Fuc (a1-2) "

Glycan structures Fuc (a1-2) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) [Gal (b1-4) GlcNAc (b1-6)] GalNAc

Glycan structures Fuc (a1-2) Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc

Glycan structures NeuAc (2-3)Gal(1-3)[Fuc(1-4)]GlcNAc(1-3)Gal(1-3)GalNAc

Glycan structures Fuc (a1-2) Ga 1 (b1-4) [Fuc (a1-3)] GlcNAc (b1-3) Gal (b1-3) GalNAc

Glycan structures Fuc (a1-2) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc

Glycan structures Gal (b1-3) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) GlcNAc (b1-3) Gal (b1-3) [Gal (b1-4) GlcNAcb1-6)] GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Gal (b1-3) GlcNAc (b1-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) GlcNAc (b1-3) Gal (b1-4) G1cNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] Ga lNAc

Glycan structures Gal (b1-3) GlcNAc (b1-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) [Gal (b1-4) GlcNAc (b1-6)] Gal (b1-3) [Gal (b1-4) GlcNAc (b1-6)] Ga lNAc

Glycan structures Gal (b1-3) GlcNAc (b1-3) [Gal (b1-4) GlcNAc (b1-6)] Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Ga l (b1-3)] GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc

Glycan structures NeuAc (2-3)Gal(1-)GlcNAc(1-3)Gal(1-3)[Gal(1-4)GlcNAc(1-6)]GalNAc+″+Fuc″

Glycan structures Gal (b1-)GlcNAc(b1-)Gal(b1-4)[Fuc(a1-3)]GlcNAc(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

Glycan structures Fuc (a1-)[Gal(b1-)]GlcNAc(b1-)Gal(b1-4)GlcNAc(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

Glycan structures Fuc (1-)Gal(1-)[Fuc(1-)]GlcNAc(1-)Gal(1-)GlcNAc(1-)[NeuAc(2-)Gal(1-)]GalNAc

Glycan structures Gal (1-)GlcNAc(1-)Gal(1-)[Fuc(1-)]GlcNAc(1-)[NeuAc(2-)Gal(1-)]GalNAc+″+Fuc″

Glycan structures Fuc (1-)Gal(1-)[Fuc(1-)]GlcNAc(1-)Gal(1-)[Fuc(1-)]GlcNAc(1-)[NeuAc(2-)Gal(1-)]GalNAc

Glycan structures Gal (1-)GlcNAc(1-)Gal(1-)GlcNAc(1-)Gal(1-)GlcNAc(1-)[NeuAc(2-)Gal(1-)]GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-6) [Gal (b1-3) GlcNAc (b1-3)] GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-3) [Gal (b1-4) GlcNAc (b1-6)] GalNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-)GlcNAc(b1-3)Gal(b1-3)[NeuAc(a2-6)]GalNAc

Glycan structures Gal (b1-)GlcNAc(1-)[Gal(b1-)GlcNAc(1-)]Gal(b1-)GlcNAc(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc

Glycan knot Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-) Gal (b1-4) GlcNAc (b1-6) [NeuAc structures (a2-3) Gal (b1-3)] GalNAc

Glycan structures NeuAc (a2-6) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-)Gal(b1-4)GlcNAc(bl-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-)Gal(b1-4)GlcNAc(b1-)Gal(b1-4)GlcNAc(b1-6)[Gal(b1-3)]GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) GlcNAc (b1-3) [Gal (b1-4) GlcNAc (b1-6)] Gal (b1-3) [Gal (b1-4) GlcNAc (b1-6)] GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) [Fuc (a1-4)] GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-4) [Gal (b1-3)] GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Ga1 (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) [Fuc (a1-4)] GlcNAc (b1-3) [Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-6)] Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Ga1 (b1-3)] GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) [Fuc (a1-4)] GlcNAc (b1-3) [Gal (b1-4) GlcNAc (b1-6)] Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [NeuAc (a 2-3) Gal (b1-3)] GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-)GlcNAc(b1-3)[Fuc(a1-2)[Gal(a1-3)]Gal(b1-)GlcNAc(b1-6)]Gal(b1-4)GlcNAc(b1-3)Gal(b1-3)Gal(b1-3)[Gal(b1-4)GlcNAc(b1-6)]GalNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-)GlcNAc(b1-3)[Fuc(a1-2)[Gal(a1-3)]Gal(b1-)GlcNAc(b1-6)]Gal(b1-4)GlcNAc(b1-3)Gal(b1-3)Gal(b1-3)[Gal(b1-4)G lcNAc(b1-6)]GalNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-)GlcNAc(b1-3)[Fuc(a1-2)[Gal(a1-3)]Gal(b1-)GlcNAc(b1-6)]Gal(b1-4)GlcNAc(b1-3)Gal(b1-3)Gal(b1-3)[NeuAc(a2-3)Gal(b1-4)GlcNAc(b1-6)]GalNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-)GlcNAc(b1-3)[Fuc(a1-2)[Gal(a1-3)]Gal(b1-)GlcNAc(b1-6)]Gal(b1-4)GlcNAc(b1-3)Gal(b1-3)[Fuc(a1-2)[Gal(a1-3)]Gal(b1-4)GlcNAc(b1-6)]GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) G1cNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc+ "+Fuc (a1-3) "

Glycan structures Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] Ga lNAc+ "+2x Fuc (a1-3) "

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-)Gal(b1-4)GlcNAc(b1-)Gal(b1-4)GlcNAc(b1-6)[Gal(b1-3)]GalNAc

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-)Gal(b1-4)GlcNAc(b1-)Gal(b1-4)GlcNAc(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

Host cell can be modified by a variety of methods known in the art to obtain the protein of the present invention or the physics and chemistry form of chimeric molecule, a kind of one or more transgenosis for encoding enzyme or a variety of enzymes, producing required physics and chemistry form are including but not limited to introduced into host cell.This transgenosis includes polytype sialyltransferase, such as ST3Ga11, ST3Ga12, ST3Ga13, ST3Ga14, ST3Ga15, ST3Ga16, ST6Ga11, ST6Ga12, ST6GalNAc1, ST6GalNAc2, ST6GalNAc3, ST6GalNAc4, ST6GalNAc5, ST8Sial, ST8Sia2, ST8Sia3, ST8Sia4, ST8Sia5, ST8Sia6；Galactosyl transferase, such as GalT1, GalT2；Fucosyltransferase such as FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, FUT10, FUT11；Sulfurtransferase；GlcNAc transferases such as GNT1, GNT2, GNT3, GNT4, GNT5；Antenna nickase (antenna-cleaving enzymes) and endoglycosidase.

For example, the invalid terminal sialic acidization effect of N- glycan structures causes the protein sera half-life period of expression to reduce, the protein such as recombined human AchE, can improve this situation (JBiochem 336 by adding rat beta galactose glycosides α -2,6- sialyltransferase transgenosis into the cells of HEK 293：647-658、1998；J Biochem 363：619-631、2002).

Similarly, the sialyl Lewisx structures of the invalid structure such as N- glycan structures of specific Lewis x groups cause the reduction of expressing protein ligand binding, the expressing protein such as recombined human PSGL-1, can improve this situation (Fritz et al. PNAS95 by adding fucosyltransferase transgenosis into the cells of HEK 293：12283-12288、1998)

In a specific embodiment, α -2,3 or α -2, the transfer of 6 sialic acids or α -2,3 and α -2, the human cell line of 6 sialyltransferases (" sialylated albumen ") produce protein or its chimeric molecule using conversion.The example of sialylated albumen includes sialylated TNF-α, sialylated TNF-α-Fc, sialylated LT- α, sialylated LT- α-Fc, sialylated TNFRI, sialylated TNFRI-Fc, sialylated TNFRII, sialylated TNFRII-Fc, sialylated OX40, sialylated OX40-Fc, sialylated BAFF, sialylated BAFF-Fc, sialylated NGFR, sialylated NGFR-Fc, sialylated FasL, sialylated FasL-Fc.

Especially, with physical and chemical parameter (P_x) feature characterize the sialylated albumen of the present invention, including monose (P₉) and sialic acid content (P₁₀), it is 1: 0.1-100NeuNAc when using GalNAc as standard；When being constantly 3: 0.1-100NeuNAc using 3 times of mannoses as standard.Neutral percentage (the P of the N- connections oligosaccharides of the sialylated albumen of the present invention₁₃) it is 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%.Acid percentage (the P of the N- connections oligosaccharides of the sialylated albumen of the present invention₁₄) it is 1 to 100%, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.Neutral percentage (the P of the O- connections oligosaccharides of the sialylated albumen of the present invention₁₅) it is 0 to 99%, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98or 99%.Acid percentage (the P of the O- connections oligosaccharides of the sialylated albumen of the present invention₁₆) it is 1 to 100%, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

Compared with the protein of the invention of no transgenosis or the half-life period of chimeric molecule, the Half-life in vivo (T of sialylated albumen₁₁) increase.

In a specific embodiment, sialylated albumen contains at least one structural formula described herein, or at least one structural formula described herein, and wherein one or more NeuNAc crosslinkings are that the α 2,6 in N- crosslink parts is crosslinked.

In a specific embodiment, sialylated albumen contains at least one structural formula described herein, or at least one structural formula described herein, and wherein one or more NeuNAc crosslinkings are that the α 2,6 in O- crosslink parts is crosslinked.

In a specific embodiment, one or more physicochemical property (P below_x) and pharmacological characteristics (T_y) sialylated-TNFRI-Fc of the invention is characterized, it has：- apparent molecular weight (P₁) it is about 1 to 250, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250kDa, in a specific embodiment, 85kDa is arrived for 48；

-pI(P₂) scope is about 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, it is 5.5 to 8.5 in a specific embodiment；- it there are about 2 to 50 isoform (P₃), such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 isoforms, in a specific embodiment, it is 10-18 isoform；

In a specific embodiment, using the protein or its chimeric molecule that the present invention is obtained in the human cell line for having converted FUT3 (" marine alga glycated protein ").Marine alga glycated protein is for example including marine alga saccharification TNF-α, marine alga saccharification TNF-α-Fc, marine alga saccharification LT- α, marine alga saccharification LT- α-Fc, marine alga saccharification TNFRI, marine alga saccharification TNFRI-Fc, marine alga saccharification TNFRII, marine alga saccharification TNFRII-Fc, marine alga saccharification OX40, marine alga saccharification OX40-Fc, marine alga saccharification BAFF, marine alga saccharification BAFF-Fc, marine alga saccharification NGFR, marine alga saccharification NGFR-Fc, marine alga saccharification FasL, marine alga saccharification FasL-Fc.

Especially, with physical and chemical parameter (P_x) feature characterize the sialylated albumen of the present invention, including monose (P₉) and sialic acid content (P₁₀), it is 1 to 0.1-100NeuNAc when using GalNAc as standard；It is 3 to 0.1-100NeuNAc when using 3 times of mannoses as standard.

In a specific embodiment, marine alga glycated protein possesses more structure divisions containing Lewis structures (such as Lewis a, Lewis b, Lewis x or Lewis y) or sialyl Lewis structures (such as sialyl Lewis a or sialyl Lewis x).

In a specific embodiment, with the expression without transgenosis protein of the invention or the binding affinity of chimeric molecule is compared, and marine alga glycated protein possesses the ligand binding affinity of change.

Use the respective forward primer and reverse primer of the protein molecular selected from TNF-α, LT- α, TNFRI, TNFRII, OX40, BAFF, NGFR, FasL, by DNA of the polymerase chain reaction (PCR) using methods known in the art amplification coding GAP-associated protein GAP from EST, such as according to the PCR Super Mix High Fidelity (Cat.No. of Invitrogen companies：10790-020).Digest amplification is simultaneously connected in the corresponding restriction enzyme sites of suitable carrier, such as pIRESbleo3, pCMV-SPORT6, pUMCV3, pORF, pORF9, pcDNA 3.1/GS, pCEP4, pIRESpuro3, pIRESpuro4, pcDNA3.1/Hygro (+), pcDNA3.1/Hygro (-), pEF6/V5-His.Connection carrier is transformed into suitable e. coli host cell, such as XLGold, supercompetent cells (Strategene), XL-Blue, DH5 α, DHl0B or other.

For the generation of chimeric molecule, the DNA sequence dna of the Fc domains of immunoglobulin, such as IgG1, IgG2, IgG3, IgG4, IgGA1, IgGA2, IgGM, IgGE, IgGD are expanded by PCR using suitable forward and reverse primer from EST.Extension is cloned into the corresponding restriction enzyme sites of suitable carrier, for example, pIRESbleo3, pCMV-SPORT6, pUMCV3, pORF, pORF9, pcDNA3.1/GS, pCEP4, pIRESpuro3, pIRESpuro4, pcDNA3.1/Hygro (+), pcDNA3.1/Hygro (-), pEF6/V5-His.The DNA sequence dna of corresponding albumen is amplified and is cloned into the corresponding restriction enzyme sites of the respective Fc- carriers using Fc as framework.

In a specific embodiment, the complement activation area in Fc receptor binding domains or Fc regions can be by the insertion of one or more amino acid, deletion or the replacement of recombinant modified, including Fc region amino acid sequences.In addition, the complement activation area in Fc receptor binding domains or Fc regions can be modified by sulphation, its glycoforms are changed into, physically adds or removes carbohydrate moiety on amino acid backbone or after the common translation entity of any association or translation, add polyunsaturated fatty acid part or other parts based on fat.Fc regions can also be shortening form, be realized by a kind of enzyme digestion, and the enzyme includes the protease of papain, pepsin or other any locus specificities.Fc regions can promote spontaneous conformation to be formed by the stronger chimeric protein of the combination respective ligand or acceptor ability of the polymer of dimer, tripolymer or greater degree.

Carry out differentiating digestion using suitable Restriction Enzyme identifying/separating containing the bacterial clone with corresponding gene.Separate positive colony simultaneously -70 DEG C of glycerin storages.Clonal expansion to 750ml is then contained to 37 DEG C of shakes in ampicillin (100 μ g/ml) sterile LB fluid nutrient mediums to cultivate 16 hours.Plasmid is extracted according to methods known in the art, it is preferred that according to QiagenEndofree P1asmid Mega kits (Qiagen Mega Prep Kit#12381).

The human host cell for being adapted for introduction into the cloned dna sequence of the protein containing the present invention or chimeric molecule includes but is not limited to HEK 293 and its any redundant organism, the c18 of HEK 293, HEK293-T, the CEN4 of HEK 293, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene companies), 293A (Invitrogen companies), Hela cells and its any redundant organism, HepG2, PA-1 Jurkat, THP-1, HL-60, H9, HuT 78, Hep-2, Hep G2, MRC-5, PER.C6, SKO-007, U266, Y2 (Apollo companies), WI-38, WI-L2.

Host cell can be modified by a variety of methods known in the art to obtain the protein of the present invention or the physics and chemistry form of chimeric molecule, a kind of one or more transgenosis for encoding enzyme or a variety of enzymes, producing required physics and chemistry form are including but not limited to introduced into host cell.It can be incorporated into by being introduced into special DNA sequence dna to optimize cloned dna sequence in host cell gene group, different types of integrate includes but is not limited to locus specificity, targeting, the mediation of direct or enzyme integration.

The DNA of protein or its chimeric molecule can be incorporated into suitable host cell by a variety of transfection methods known in the art, for example, using chemical reagent for example diethyllaminoethyl glucan (DEAE-dextran), calcium phosphate, artificial liposome or by direct microinjection, electroporation, biologic grain transport infection or transfected virus structure, as described below.

DEAE-dextran is a kind of cationic polymer, and negatively charged nucleic acid is combined.Positive charge excessive on condensate can allow closer associate of compound to carry the cell membrane of negative electrical charge in DNA/ polymer composites.The absorption of compound relies on endocytosis by inference.The cationic polymer of the other synthesis used in transfection includes the poly- Methobromide of methylene (polybrene) of 1,5- dimethyl -1,5- phenodiazine 11, polyethyleneimine and dendrimers.

The instantaneous and stable transfection of various kinds of cell type can use coprecipitation of calcium phosphate.DNA and calcium chloride are mixed by certain controlled mode, be then added to the alkali metal salt of buffering/phosphate solution in, mixed liquor is incubated at room temperature.Produce precipitation and absorbed by cell by endocytosis or phagocytosis.

The most frequently used fat composition of liposome is the fat composition under physiology pH with net positive charge in liposome-mediated gene delivery.Generally cationic lipid and neutral fats are mixed, such as L-dioleoyl phosphatidyl-ethanolamines (DOPE).The negative electrical charge association of the cationic moiety and nucleic acid of fat molecule, causes to tighten in liposome/nucleic acid complexes amplifying nucleic acid.Compound is absorbed by endocytosis.

It will be a kind of effective but laborious technology in the cell or nucleus of DNA direct microinjections to culture, be not suitable for needing the situation of a large amount of transfectional cells.

Electroporation produces hole using a kind of electric pulse, allows nucleic acid to enter cell, is required for carrying out fine tuning and optimization to the duration of pulse and intensity for each type of cell this technology used.Commercially available electroporation device includes the Nucleofector kits (Amaxa Biosystems, Germany) of Amaxa Biosystems companies.

The method is dependent on nucleic acid microparticles rapid transit into recipient cell.

With virus or retroviral structural infection or transfection including the use of retrovirus, such as lentivirus, or DNA virus, such as adenovirus.Its process is entered in host cell including the use of a kind of virus or retroviral vector to transmit alien gene.

In some embodiments, protein or its chimeric molecule are produced by transient approach or from stably transfected cell line.Transiently transfected using adhesion or suspension cell line.For adherent cell system, cell grows in the culture medium (2-10% serum) containing serum, the culture medium such as DMEM, DMEM/F12 (JRH).The serum used can be hyclone (FCS), donor calf serum (DCS), newborn calf serum (NBCS) etc..Plasmid vector is incorporated into by cell by standard method known in the art.In a specific embodiment, the DEAE dextran or DNA of calcium phosphate precipitation transfected proteins matter or its chimeric molecule are used.After transfection, cell is transformed into the suitable albumen or its chimeric molecule collected and expression is collected in culture medium (such as serum-free DMEM/F12).

Plasmid vector can be introduced into by using the method for being briefly described above and carries out protein or its chimeric molecule from the transient expression in suspension cell.Suspension cell can grow in serum-containing media or serum free medium (such as Freestyle culture mediums (Invitrogen), CD293 culture mediums (Invitrogen), Exce11 culture mediums (JRH) etc.).It can be transfected under serum-free condition, by being transfected in a kind of suitable culture medium using suitable transfection method, for example, lipofectamine is in OptiMEM culture mediums.

Transient expression typically results in 2-3 days peak expressions after transfection.Episomal vector is replicated and continuous expression in cell.Therefore, in order to obtain substantial amounts of product, free expression vector is transfected into cell and amplifying cells.Protein or its chimeric molecule are expressed into culture medium, are collected after cell amplification several weeks.Expression culture medium can be that, containing serum or serum-free, cell can be adhesion or suspension.

Cell is entered by transfected expression vector and obtains stable clone, is then selected with suitable reagent, such as phleomycin, homomycin, puromycin, neomycin G418, methopterin.Stable clone can survive in selecting, because also containing resistant gene in addition to the gene of encoding proteins matter or chimeric molecule in plasmid.Introduce after gene 1 to 2 days, start to whole cells (stabilization pond) or the cell according to Clonal density bed board is selected.Non-transfected cell group is also selected to kill cell effect determine selective reagent.For adherent cell, cell is allowed to grow the clone until obtaining visible separation in tissue culturing plate.Then they are removed from flat board with pancreatin digestion or physical method and is taped against in tissue-culture well (each one clone in hole in 96 orifice plates).For suspension cell, limited dilution cloning is carried out after selection, clone is then expanded, and is then characterized and/or carried out the limiting dilution analysis of next round.

The stable clone being grown in the culture medium containing serum adapts to gradually decrease serum levels, then comes off and in the growth of low serum low suspension.Serum levels are then further reduced until serum-free state.Some culture mediums can make adaptation faster (for example, directly replacing with serum free suspension growth from the condition containing adhesion), and an example is the CD293 culture mediums of Invitrogen companies.

After being grown in serum free medium, clone can proceed by medium optimization.The industry characteristics of clone are detected in many different culture mediums, for example, complete vibrant cell quantity, until obtaining most suitable composition.This is dependent on the production method produced.For example, cell may be expanded in a culture medium, the additive of Enhanced expressing is then added before collection of products.

The albumen or chimeric molecule of overexpression may be accumulated in host cell.The recovery of intracellular protein includes handling host cell with lysis buffer, and the lysis buffer includes but is not limited to the buffer solution containing following composition：NP40, Triton X-100, Triton X-114, lauryl sodium sulfate (SDS), natrii tauroglycocholas, deoxysodium cholate, CHAPS, CHAPSO, Brij-35, Brij-58, Tween-20, Tween-80, octyl glucoside and Octylthioglucoside.Another cracking host cell method can include sonication, homogenate, the crushing of Freund cell and multigelation and handle cell with hypotonic solution.

Final product can be produced in a variety of different bioreactors, via non-limiting example, including stirring pool, gas-lifting type (airlift), packed bed perfusion, microcarrier, doughnut, bag technique, cell factory.Its method can be continuous culture, batch, stream plus culture (fed batch) or induce.Peptone class can be added in low blood serum medium to increase volume protein.

The purification strategy customized using protein or chimeric molecule specifically for the present invention purifies the protein or chimeric molecule of the present invention.Purification process includes but is not limited to：Tangential flow filtration (TFF)；Ammonium sulfate precipitation；Molecular sieve chromatography (SEC)；Gel filtration chromatography (GFC)；Affinity chromatography (AFC)；Purification that A albumen is affine；Receptor-mediated coordinate chromatograph (RMLC)；Dyestuff coordinate chromatograph (DLC)；Ion-exchange chromatography (IEC), including anion or cation-exchange chromatography (AEC or CEC)；Reverse-phase chromatography (RPC)；Hydrophobic interaction chromatography (HIC)；Metal chelate chromatography (MCC).

TFF is a kind of quickly and efficiently bio-molecular separation method, for concentrating, desalination or fractionation sample.TFF can concentrate hundreds of liters of samples for 10 milliliters.It is combined with suitable molecular weight mwco membrane, TFF can separate different size and the biomolecule of molecular weight (nominal molecular weight retention (NMWC) is 5KDa, 10KDa, 30KDa, 100KDa).Diafiltration process is then concentrated again including dilute sample, can be used for desalination or be replaced sample buffer.

Saltout or ammonium sulfate precipitation can be used for condensing protein dilute solution.It is additionally operable to fractionating proteins mixture.Similar charge repels decreased effectiveness between the ionic strength of solution of the increase containing protein causes protein molecule.It also reduces the power of Protein requirement surrounding molecules solvent shell.When these power decrease to degree, protein will be precipitated；Compared with hydrophilic protein, hydrophobin sinks to forming sediment compared with low salt concn.It is a kind of very effective partial purification method of protein by the classification separation for being stepped up ionic strength and carry out centrifugation progress protein compound.

SEC flows through porous matrix according to sample, passes through size separation protein.SEC with GFC principles are identical, and it be used to separate the molecule in liquid phase systems.In SEC, the molecule bigger than hole in packing material and solvent front are flowed out first together, are completely excluded.The molecule of middle size, excludes between reservation between complete, the hole in host material is passed through according to its size.The small molecule for freeing in and out hole is retained.Therefore, different size of protein has different elution volume and retention time.For the similar molecule of structure, molecular size is bigger, their flows it is more early.Before any sample is run, it should set up standard curve to determine working limit and refer to retention time.

When protein shape is identical, the molecular weight of post eluate can quickly be screened by UV absorption, fluorescence or light scattering according to the filler of different pore size in post.Photon correlation spectroscopy (PCs) is generally used in static sample and liquid chromatographic detection.Low angle laser light scattering is also coupled in chromatogram detection, direct detection molecules amount, and unrelated (Carr et al. the AnalBiochem 175 of the shape of protein：492-499,1988).SEC-HPLC is used for detection hGH degradeds and aggregation (Pikal et al. Pharm Res 8：427-436,1991).It is also used for pollution condition (the Yoshioka et al.Pharm Res 10 of the beta galactosidase of Estimation Study：103-108,1993).

AFC is according to the specific interaction between the chemical constitution between biomolecule and suitable affinity ligand purifying biological molecule.By complementary immobilization ligand specificity's and reversible adsorbed target molecule.Part can be a kind of inhibitor, substrate, analogies or co-factor, or it is a kind of can specific recognition target molecule antibody.Then, the molecule of absorption is washed away by competitiveness displacement, or conformational change is allowed by pH or ionic strength conversion.

A albumen affinity purifications are an affinity purification example of the affinity using certain bacterioprotein, the bacterioprotein energy broad incorporation antibody, regardless of specificity of the antibody to antigen.A albumen, G-protein, L albumen are provided with the clear antibody binding properties of solution.These three albumen recombinant production and conventionally used for from a variety of middle affinity purification key antibody types.A albumen, the genetic engineering recombinant forms of G-protein are referred to as albumin A/G, are also to use.These antibody binding proteins can be immobilized on supported matrix.The method has been modified for being connected with protein of interest in the recombinant protein in antibody A protein binding region (Fc regions).It is attached in physiological conditions on the A protein moleculars of immobilization, by changing pH or ionic strength elution.

RMLC is a kind of AFC of specific type, has used acceptor to the intrinsic compatibility of its related objective molecule.Acceptor molecule is immobilized on suitable chromatogram supported matrix by active amino, reactive hydrogen, carbonyl, carboxyl or mercapto groups.In a RMLC example, acceptor-Fc chimeric molecules molecule is fixed on A albumen Ago-Gel pearls by acceptor Fc parts to the affinity of A albumen.The method has directional at-tachment acceptor, and the advantage in its ligand binding site is exposed to its corresponding cell factor.Target molecule is allowed to be adsorbed onto on acceptor in physiological conditions, by changing pH or ionic strength elution.

DLC is a kind of ALC, has used reactive dye selectivity and reversible associated proteins ability.Dyestuff is typically Monochlorpheanmide compound.Active cl radical able person triasine dyes is readily immobilized on supported matrix, such as Ago-Gel (Sepharose) or agarose, and can be immobilized in recently on nylon membrane.

Being originally found for these dye-bond proteins comes from it was observed that the glucan blue (cibacron indigo plants FG-3A conjugated compound) as solvent resistant column void volume mark can delay the elution of some protein.Then some the specific researchs of dyestuff to specific protein have been carried out, majority uses the blue dyestuffs of prototype cibacron.These dyestuffs show maximally effective binding characteristic, albumen and the enzyme such as kinases and dehydrogenase, although other albumen such as serum albumin can also combine closely in terms of protein and enzyme of the nucleotides as co-factor is used in combination.It is believed that aromatic series triasine dyes structure is similar with the nucleotide structure of nicotinamide adenine (NAD), and the nicotinamide adenine in dyestuff and these albumen folds and occurs dependent interaction.In many cases, associated proteins can be eluted under competition model by substrate or nucleotide cofactors, and dyestuff has shown that the characteristic that substrate binding site is competed in free solution.Appearing these dyestuffs can be by electrostatic and hydrophobic interaction and " pseudo- affine " the interaction associated proteins in more specific and ligand binding site.(McGettrick et al. in many dehydrogenases of purifying and protease has been employed successfully in further to simulate part (plan ecofriendly dyes) by the specificity of modification increase dye ligand

Ion-exchange chromatography (IEC) has used delay of the albumen because of the electrostatic interaction between ion exchange column matrix and protein in pillar to carry out purifying protein.When mobile phase pH exceedes the p I of target protein, target protein is negatively charged, and will be interacted with anion-exchange column (AEC).When mobile phase pH is less than the pI of target protein, target protein positively charged should use cation exchange column (CEC).By using with target molecule identical electric charge target protein is eluted to increase the concentration of ion balance.

RPC is according to the hydrophobic interaction separation of biomolecules between molecule and chromatogram supported matrix.By controlling the pH in separation, under the neutral form of ionizable compound, they are easiest to analysis.Mobile Phase Additives, such as trifluoroacetic acid, albumen hydrophobicity, strong adsorption to stationary phase are increased by forming ion pair.By changing the polarity of stationary phase, biomolecule is eluted from the supported matrix of chromatogram.

HIC is similar with RPC, but with bigger nominal pore.In HIC, eluting solvent uses aqueous saline solution, instead of the aqueous phase or organic phase mobile phase used in RPC.Also, it is opposite that sample elution order is compared with RPC.Protein surface includes hydrophilic residue and hydrophobic " piece ", and stable protein is carried out in the inside that the latter is usually located at folded protein.When hydrophobic flakes are changed into being exposed in aqueous environment, they will destroy the normal solvent properties of albumen, be changed into thermodynamically unfavourable.In aqueous phase mobile phase, inorganic salts (such as ammonium sulfate) concentration is higher, and surface tension is bigger, therefore the intensity of the hydrophobic interaction between increase HIC resin hydrophobics group and albumen, adsorbs.But, when gradient reduces salinity, the reduction in surface tension of aqueous phase mobile phase, therefore cause hydrophobic interaction to reduce, cause albumen from desorption on pillar hydrophobic grouping.MCC is a kind of according to technology of the protein to the affinity protein isolate of chelated metal ions.Different metal ions includes but is not limited to be fixed on the Cu of chromatogram supporter stationary phase by covalently bound cheland (such as diglycinee)²⁺, Co²⁺, Zn²⁺, Mn²⁺, Mg²⁺Or Ni²⁺.The free coordination site of metal ion be used to combine different proteins and peptides.Eluted by using competitive molecular replacement albumen or by changing pH.For example, reduction pH of buffer causes the binding affinity of protein-metal ion complex to reduce, protein desorption.Or, reduce pH (using discontinuous gradient or linear gradient form) protein of elution of bound from pillar using gradient.

It can modify host cell by a variety of methods known in the art to obtain the present invention protein or the physics and chemistry form of chimeric molecule.

Present invention contemplates after protein or chimeric molecule expression and purifying, carbohydrate chemistry or enzyme are coupled on the peptide chain of protein or chimeric molecule.Chemistry and/or enzyme coupling process can be used to modify, increase or decrease quantity or feature that quantity or carbohydrate are obtained.Conjugation pattern used in relying on, sugar can be attached to (a) arginic amide groups, (b) free carboxyl group group, (c) mercapto groups, such as in cysteine those, (d) those in oh group such as serine, threonine, oxylysine, (e) those in aromatic residue such as phenylalanine, tyrosine or tryptophan, (f) those in the amide group of glutamine, or (g) amino such as histidine, arginine or lysine.It can be added by chemistry or Enzymology method.It is, for example, possible to use suitably recombinating glycosyl transferase continuous additional sugared unit on protein or its chimeric molecule.Glycosyl transferase increase can also be used to be covalently attached the sugar of substituted base.For example, the sialic acid with covalently additional polyethylene glycol (PEG) can be transferred to terminal galactose residues to increase molecular size and serum half-life by sialyltransferase.

A variety of functional groups, including phosphate radical, sulfate radical, hydroxyl, carboxylate radical, O- sulfate radicals and N- acetyl groups can be mixed with the carbohydrate side chain of chemistry or enzymatic modification protein or chimeric molecule.

The carbohydrate that can be gone with chemistry or enzyme process in isolating protein or its chimeric molecule.Trifluoromethanesulfonic acid or a kind of suitable compound can be used to carry out Chemical deglycosylation.This processing can cause it is most of or all sugared isolate, except combining sugar, and keep polypeptide complete.With other sugar or whole chain can be removed from protein or its chimeric molecule by a variety of endoglycosidases and exoglycosidase.

The chitosan component of modifying protein or chimeric molecule can be carried out by using sialidase, or remove with moderate acid treatment the sialic acid of residual；With it is circumscribed-inscribe-glycosidase come cut N- crosslinking oligosaccharide antenna or shorten O- connection oligosaccharides.Side base, such as trehalose and sulfate radical can also be removed with mycoside enzyme or sulfatase processing.Pseudo- glycan structures such as polyethylene glycol or glucan, or the sugared subunit of increase that the glycerine transferase cocktail with sugar-dUDP precursors can be used to be synthesized to glycan can be added on chemical normal direction amino acid backbone.

Present invention contemplates on chemistry or enzyme process coupling protein matter or its chimeric molecule to radionuclide.This protein or chimeric molecule can be selected from containing TNF-α, TNF-α-Fc, LT- α, LT- α-Fc, TNFRI, TNFRI-Fc, TNFRII, TNFRII-Fc, OX40, OX40-Fc, BAFF, BAFF-Fc, NGFR, NGFR-Fc, FasL, FasL-Fc list.

Can use iodination to protein or its chimeric molecule the additional iodine isotope of peptide chain (for example¹²³I).Especially, isotope can be attached on the phenol ring of (a) tyrosine of protein or the peptide chain of its chimeric molecule, or on the imidazole ring of (b) histidine.Can use chloramine-T (Chloramine-T), iodine monochloride, teriodide, electrolyte, enzyme, with reference to, the method for metallization removal, iodogen or iodine pearl carry out iodination.

Mtc labeled method can be used to use methods known in the art will^99mTc is attached on the protein of the present invention or chimeric molecule, for example, reduced by using go back original reagent (such as stannous chloride)^99mTcO₄ ^-, then carried out by bifunctional chelating agent (such as diethylenetriamine pentaacetic acid (DTPA))^99mTc labelled proteins or chimeric molecule.

It is coupled to present invention contemplates protein or its chimeric molecule chemistry or enzyme process on chemotherapeutant.It can use methods known in the art that suitable reagent (such as zoledronic acid) is attached on protein or its chimeric molecule, for example, the coupling reaction mediated by the enhanced carbodiimides of N-hydroxysulfosuccinimide.

It is coupled to present invention contemplates protein or its chimeric molecule chemistry or enzyme process on toxin.It can use methods known in the art that suitable toxin (including melittin, vanous toxin, Pseudomonas exotoxin, ricin (WA), gelonin and the diptheria toxin shortened) is attached on protein or chimeric molecule, methods described is for example acted on by maleimide or carbodiimides conjugation chemistry.

Can by allow separation protein or chimeric molecule and patient in target recipient or the method that comes in contact of part the protein of separation described herein or its chimeric molecule are transported in patient's body.In a specific embodiment, protein or its chimeric molecule are as in " pharmaceutical composition " transported patient.

On the other hand, present invention contemplates a kind of one or more protein or chimeric molecule and pharmaceutically acceptable carrier containing separation described above or the pharmaceutical composition of diluent.

The composition forms used suitable for injecting, which include aseptic aqueous solution (water soluble) and aseptic powdery, to be used for extemporaneous preparation of sterile and injects solution.It must be stable under production and condition of storage, and must keep avoiding the pollution of microorganism (such as bacterium and fungi).Carrier can be that a kind of solvent or diluent include, for example, water, ethanol, polyalcohol (for example, glycerine, propane diols, liquid polyethylene glycol etc.), their suitable mixture and vegetable oil.Suitable mobility can be maintained, for example, by using surfactant.Can be by a variety of antibacteriums and antifungal agent, such as metagin, anesin, phenol, sorbic acid, thirmerosal, to prevent the activity of microorganism.In many cases it is preferred to isotonic reagent, for example, sugar or sodium chloride.Can be by extending the absorption of Injectable composition using delayed absorption reagent in the composition, for example, aluminum monostearate and gel.

By mixing reactive compound in the suitable solvent with required active component and optional other active components of requirement, to prepare aseptic injectable solution, subsequent filtration sterilization or other suitable sterilizing methods are used.For the aseptic powdery for preparing aseptic injectable solution, suitable preparation method includes vacuum drying and lyophilized, and methods described produces the required composition that powdered active ingredient adds any addition.

When activating agent is suitably protected, it can be taken orally, for example; with inactive diluent or with assimilable edible carrier; it can either be encapsulated in hard or soft shell capsule or can be with boil down to tablet, or can be directly incorporated into the food of diet or be administered by breast milk.For oral therapeutic administration, active component can mix auxiliary material and be used in forms such as absorbable tablet, lozenge, capsule, elixir, suspension, syrup, wafers.This composition and preparation should contain the active component of at least 1% weight.The percentage of composition and preparation can be with, certainly, is change and can be with suitable between 5% to about the 80% of Unit Weight.The quantity of active agent is by the suitable dosage of acquisition in the composition of this treatment.In a specific embodiment, the composition or preparation according to the present invention are prepared, oral dosage unit form contains the conditioning agent between about 0.1 μ g and 200mg.Other dosage are included from about 1 μ g to about 1000mg, from about 10 μ g to about 500mg.These dosage can be each individual or per kilogram of body weight.Can administration per hour, daily, weekly, monthly or every year.

Tablet, lozenge, pill, capsule etc. can also contain the composition of following lists.Adhesive such as resin, Arabic gum, cornstarch, gelatin can be added；Auxiliary material such as Dicalcium Phosphate；Disintegrant is such as cornstarch, farina, alginic acid；Lubricant such as magnesium stearate；Sweetening agents such as sucrose, lactose or saccharin, or add fumet such as peppermint, wintergreen or cherry flavoring.When a dosage unit form is a capsule, it can be containing the above-mentioned type material and liquid-carrier.A variety of other materials can be used for coating or the in addition physical form of modification dosage unit.For example, shellac, sucrose or both together coating tablet, pill or capsule can be used.Syrup or elixir can contain reactive compound, and sucrose is used as preservative, dyestuff and flavoring such as cherry or citrus flavoring as sweetener, methanol and nipasol.Certainly, any material used in any dosage unit form is prepared should be pharmaceutical purity and be substantially nontoxic for the quantity used.In addition, reactive compound can be mixed in sustained release preparation and formulation.

Present invention further contemplates Topical dosage forms.In Topical dosage forms, active agent can suspend in emulsifiable paste or lotion or waxed dose or other liquid solutions, therefore topical application emulsifiable paste or lotion or waxed dose or liquid solution cause active agent to be introduced into the biological surface of patient.Active agent is selected from one or more TNFRI-Fc of the invention or TNFRII-Fc or its variant, homologue or the like.

In a particular embodiment, topical compositions include TNFRI and/or TNFRII and/or TNFRI or TNFRII chimeric molecule, and chimeric molecule is fused on the Fc of antibody parts or their function homologue directly or through one or more protein connexons by TNFRI or TNFRII and obtained.In other specific embodiments, topical compositions further include pharmaceutically acceptable topical vehicle.

Therefore, the invention provides pharmaceutical composition, it includes TNFRI-Fc polypeptides or its variant, homologue or the like and/or TNFRII-Fc polypeptides or its variant, homologue or the like, and pharmaceutically acceptable topical vehicle or diluent.

Although, from the angle of TNFRI polypeptides or its variant, homologue or the like and/or TNFRII polypeptides or its variant, homologue or the like and/or TNFRI-Fc or its variant, homologue or the like and/or TNFRII-Fc or its variant, homologue or the like, the topical preparation of the present invention has been illustrated herein, but, the present invention also extends to the pharmaceutical composition that the active agent worked as by function phase is constituted." active agent that function phase is worked as " for example including：The TNFRI or TNFRII (or including the segment of one or more extracellular domain) of other TNF binding reagents and the polypeptide portion for being blended in addition to Fc areas but substantially providing identical function

Also specifically contemplated " variant, the homologue or the like " of desired polypeptides of the present invention.Term " variant " or " homologue " include, compared with the amino acid sequence of TNFRI polypeptides and/or TNFRII polypeptides and/or TNFRI-Fc polypeptides and/or TNFRII-Fc polypeptides, and the polypeptide of insertion, missing or displacement occurs for one or more amino acid.

" analog " of desired polypeptides includes but is not limited to polypeptide of the side chain by modification, and artificial amino acid and/or synthesis polypeptide and the structure limitation using crosslinking agent and other methods enhancing polypeptide of its derivative are mixed in synthesis.

Side chain modification of the invention contemplated reacts with acetaldehyde for example including reproducibility alkyl, then uses NaBH₄Reduction；amidination with methylacetimidate；It is acylated with anhydrous aceticanhydride；Carbamylation is carried out to amino with cyanate；Trinitrobenzen effect is carried out to amino with 2,4,6- TNBs (TNBS)；Amino is acylated with succinic anhydride and tetrabydrophthalic anhydride；In NaBH₄After reduction, addition is carried out to lysine with pyridoxal 5-phosphate salt.

The guanidine group of arginine residues can be modified with the reagent formation heterocyclic condensation products of such as 2,3- ethylnorephinephrines, phenylglyoxal and glyoxal.

Carboxylic group can be modified by the activation of carbodiimides, by forming O- acyl isoureas, and then derivative is obtained, for example, form corresponding acid amides.

Mercapto groups can be modified by the following method, for example, occur carboxy methylation with iodoacetic acid or iodoacetamide；Performic acid oxidation obtains cysteic acid；With other sulfhydryl compounds formation mixed disulfide；Should with maleimide, maleic anhydride or other substituted maleimide hairs；With 4- chloromercuri-benzoates, 4- chloromercuribenzene sulfonates, mercury chloride benzene, 2- chloromercuri -4- nitrophenols and other mercurial form mercurial derivative；Carbamylation occurs at basic ph with cyanate.

Trp residue can carry out following modification, for example, being aoxidized or indole ring being alkylated with N-bromosuccinimide with 2- hydroxyl -5- Nitrobromobenzenes or sulfuryl halide.On the other hand, tyrosine can change to form 3- nitrotyrosine derivatives by the nitrification of tetranitromethane.

The imidazole ring of histidine modified, can be by below by way of completing：It is alkylated with iodoacetic acid derivatives or carries out N- acetylations with pyrocarbonic acid diethyl ester.

Artificial amino acid is mixed during synthetic peptide and closes derivative; for example include but is not limited to, utilize nor-leucine, 4-Aminobutanoicacid, 4- amino -3- hydroxyl -5- phenylvaleric acids, 6-aminocaprolc acid, t- bytyries glycine, norvaline, phenylglycine, ornithine, methyl amimoacetic acid, 4- amino-3-hydroxy-6-methylheptanoic acids, 2- thienyl alanines and/or Dextrorotatory amino acids.Artificial amino acid list as used herein is provided in table 5a.

In another embodiment, pharmaceutical composition is suitable to local application, and the nucleotides of its segment comprising coding TNFRI polypeptides or TNFRI-Fc polypeptides, and described nucleotide sequence is basic such as one or more SEQ ID NO：63rd, the nucleotide sequence shown in 65,67,69,71,73,75,77,79,81,83,85, or there is the nucleotide sequence of at least about 70% homogeneity, or the nucleotide sequence that can hybridize under low stringency condition with above-mentioned any sequence or their complementary type with any sequence listed above.

In another embodiment, pharmaceutical composition is suitable to local application, and its nucleotides comprising coding TNFRII polypeptides or TNFRII-Fc polypeptide fragments, and described nucleotide sequence is basic such as one or more SEQ ID NO：91st, the nucleotide sequence shown in 93,95,97,99,101,103,105,107,109,111,113,115,117,119,121, or there is the nucleotide sequence of at least about 70% homogeneity, or the nucleotide sequence that can hybridize under low stringency condition with above-mentioned any sequence or their complementary type with any sequence listed above.

In a specific embodiment, pharmaceutical composition is suitable to local application, and it is included by TNFRI polypeptides or TNFRI-Fc polypeptide fragments, and it has following amino acid sequences：Basic such as one or more SEQ ID NO：64th, the amino acid sequence shown in 66,68,70,72,74,76,78,80,82,84,86, therewith with least 70% similitude or its variant, homologue, analog；Or by the segment of TNFRII polypeptides or TNFRII-Fc polypeptides；Basic such as one or more SEQ ID NO：92nd, the amino acid sequence shown in 94,96,98,100,102,104,106,108,110,112,114,116,118,120,122, or there is at least 70% similitude or its variant, homologue, analog therewith.

TNFRI and/or TNFRI I and/or TNFRI-Fc and/or TNFRII-Fc can also be the product of modification after common translation or translation or addition, such as including carrying out glycosylated form on the amino acid backbone to them or after common translation or translation on the whole and/or to additional multi-section saturated fat acid moieties or other parts based on fat thereon.

Term " biological surface " as used herein contemplates any surface of organ outside or inside.For example, " biological surface " of the topical preparation of the present invention is likely to be suited for the biological surface such as skin surface for including body interior or outside, wound surface, interlesionalfissures, crack inside and outside and along alimentary canal, from anywhere in respiratory tract, intestines and stomach and genitourinary tract.

In addition to traditional emulsifiable paste, emulsion, paster or spray, reagent of the invention can also use a series of methods based on ionotherapy or electroporation (poration) by local and/or transdermal transport.

" ionotherapy " causes the ability of charging particle movement based on electric current.It is placed in a pair on skin close electrodes and a potential is established between skin and following capillary.In positive pole, positively charged drug molecule is dislodged skin surface to capillary.Opposite, skin will be passed through by promotion in the electronegative drug molecule of negative pole.Because electric current can be closed and changed, Iontophoretic device quickly can start and close, and medicament transport is highly controllable and is sequencing.

Electroporation technology has used high-frequency impulse energy, and (such as radio-frequency radiation, laser, heat or light) is of short duration in a variety of forms breaks cuticula, and cuticula is the skin layer for preventing most medicines from entering in blood flow.Be important to note that it is different with iontophoresis, the energy that electroporation technology is used be not used in transport medicine by skin, simply facilitate its movement.Electroporation provides one " window ", and drug substrate is compared with normal condition can be easier and quickly pass through.

In addition, pharmaceutically acceptable carrier can be with, although be not essential, exist in the form of pharmacological activity alkali.

Term " alkali " uses its traditional sense, that is, the material of hydroxide ion can be produced by being dissolved in aqueous liquid.Water is a typical aqueous solution, and can be the natural moisture of skin surface, or is present in paster or composition, and paster used or composition can include extra moisture, and/or are used in combination with a sealed protective layer.Similar, any liquid used or semisolid preparation are water into more preferably, or being used during preparation after the protective layer of one enclosed material composition of connection.The condition of any alkali that can be used is that its compound can produce free hydroxide ion in liquid, aqueous.Alkali can produce hydroxide ion in direct or indirect form, and therefore, alkali is it can be appreciated that " hydroxyl-release reagent ".Hydroxyl-release the reagent for directly producing hydroxide ion usually contains hydroxide groups, directly hydroxide ion is discharged into solution, the hydroxide of such as alkali metal.Hydroxyl-release the reagent for producing hydroxide ion indirectly is typically to send out Physicochemical in water environment to react and produce the compound of hydroxide ion, the carbonate or amine of such as metal.

The pharmacological activity alkali of the present invention is a kind of pharmaceutically acceptable inorganic or organic base.Preferred inorganic base includes inorganic hydroxide, inorganic oxide, the inorganic salts and its compound of weak acid.Preferred organic base is nitrogenous bases.

Highly basic is thought always for a long time, such as NaOH is not suitable as pharmacological activity alkali, because they can injured skin.But, in skin contacts preparation or paster, by skin in alkali or aqueous slkali, skin can be increased to the permeability of different pharmaceutical without causing skin injury.Using a variety of alkali or alkali concn, skin/solution is set to reach expected pH.Correspondingly, select alap pH it is not caused skin injury, but be enough to strengthen permeability of the skin to different activities reagent.So, the alkali content optimized in any paster or preparation is critically important, increases medicine by the flow of body surface and is preferably minimized the possibility of skin injury.In general, the pH for the body surface being in contact with the preparation or drug delivery system of the present invention tends to 8.0 to 13.0 or so scope, 8.5 to 11.5 or so more preferably, preferably 8.5 to 10.5 or so.In some aspects, scopes of the pH 9.5 to 11.5 or so, it is intended to 10.0 to 11.5 or so.

In a specific embodiment, the pH value of body surface is one of preparation factor, i.e. preparation of preparation or system to obtain the expection pH value of body surface.Anhydrous formulation and Transdermal System do not have measurable pH, therefore preparation said preparation or system reach a target pH value in body surface.Moisture can move to preparation or system from body surface, make alkali soluble solution, alkali is discharged into liquid, so as to reach expected target pH value in skin surface.In this case, preferred hydrophilic component.In addition, when using water formulation, preparation its pH after for skin may change with the time.For example, gel, liquid, emulsifiable paste etc., after for body surface, it may occur however that the net loss of moisture, that is, the moisture lost exceedes the moisture obtained from body surface.In this case, compared with preparing obtained pH, the pH of preparation may have occurred change.The water formulation is prepared, it is obtained a target pH in skin surface, so as to easily solve problem above.

In another specific embodiment of the present invention, scopes of the pH of pharmaceutical composition 3.0 to 13.0 or so in the pH or drug delivery system of preparation, it is intended to 3: 10.0 or so, 3.5 to 8.5 or so more preferably, preferably 4 to 7 or so.In one embodiment of the invention, the pH of preparation is higher than the pH of body surface.For example, during using a water formulation, the moisture from body surface can dilute preparation, therefore reach a different pH in body surface, and the generally pH than preparation in itself is low.

Typical inorganic base is inorganic hydroxide, inorganic oxide, the inorganic salts of weak acid, and its compound.Its aqueous solution of preferred inorganic base has high pH and is food or auxiliary pharmaceutical adjuvant acceptable.When being related to " alkali ", it is thus understood that while the form including hydration and without hydration.

Inorganic hydroxide includes, for example, ammoniacal liquor, monovalence alkali metal hydroxide and divalence alkali metal hydroxide, and its compound.Preferred inorganic hydroxide includes ammoniacal liquor；Monovalence alkali metal hydroxide such as sodium hydroxide and potassium hydroxide；Divalence alkali metal hydroxide such as calcium hydroxide and magnesium hydroxide；And its compound.

The consumption of inorganic hydroxide in invention formulation and system, it is typically expressed as accounting for topical compositions, or accounts for the drug reservoir of drug delivery system or the 0.3-7.0w/w% of paster or so, it is intended to 0.5-4.0w/w%, 0.5-3.0w/w% or so more preferably, preferably in 0.75-2.0w/w% or so.

Above-mentioned consumption is particularly suitable for use in these preparations and paster, active agent therein is (1) uncharged molecule, such as alkaline drug therein is the form of non-ionizing free alkali, (2) other species is not present in the basic salt of acidic drug, or (3) preparation or paster can occur the reaction or neutralization of any significance with inorganic hydroxide.

For such preparation and paster, medicine therein exists in the form of ackd salt, and/or there is other species in preparation or system can be with inorganic base (i.e. acid inactivation composition) reaction or neutralizing, the consumption of inorganic hydroxide tend to following sum：(1) consumption required for ackd salt and/or other species that can be neutralized by alkali is neutralized, plus (2) preparation or the 0.3-7.0w/w% of drug reservoir or so, tend to 0.5-4.0w/w%, 0.5-3.0w/w% or so more preferably, preferably in 0.75-2.0w/w% or so.So, for an ackd salt, synergist is enough to neutralize salt in there is certain consumption, plus an extra consumption (i.e., 0.3-7.0w/w% or so, tend to 0.5-4.0w/w%, 0.5-3.0w/w% or so more preferably, preferably in 0.75-2.0w/w% or so) pass through skin or the flow of mucosal tissue to increase medicine.The alkaline drug existed with the alkaline salt forms of neutral free alkali or acidic drug is generally free from the influence of alkali, therefore, for these medicines, and the consumption in (1) is typically required for neutralizing inactive acid ingredient.For paster, above-mentioned percentage is the gross weight relative to constituent and adhesive, glue or liquid reservoir.

By the speed and/or quantity for controlling alkali to discharge, especially in delivery process, the inorganic hydroxide of higher amount can be used.

Inorganic oxide includes, for example, magnesia, calcium oxide etc..

The consumption of invention formulation and the inorganic oxide of system is substantially higher than inorganic hydroxide consumption recited above, can be up to 20w/w%, and 25w/w% or higher can be up in some cases, but generally in 2-20w/w% or so scope.In view of the presence of any species that can be neutralized by alkali, these consumptions can be adjusted.

The inorganic salts of weak acid include ammonium phosphate (binary)；The alkali metal salt of weak acid such as sodium acetate, Boratex, sodium carbonate, sodium acid carbonate, sodium phosphate (ternary), sodium phosphate (binary), potassium carbonate, saleratus, potassium citrate, potassium acetate, potassium phosphate (binary), potassium phosphate (ternary)；The alkali metal salt of weak acid such as magnesium phosphate and calcium phosphate；Etc., and its compound.

The inorganic salts of preferred weak acid include, the alkali metal salt of ammonium phosphate (binary) and weak acid.

There is the nitrogen heterocyclic ring of amino, amide groups, oxime, cyano group, aromatic rings or non-aromatic, phosphoamide group, and its compound in the organic base used suitable for the present invention.More precisely, suitable organic base example is nitrogenous bases, including but not limited to primary amine, secondary amine, tertiary amine, acid amides, oxime, the species of cyano-containing (-- CN), the nitrogen heterocyclic ring of aromatic series or non-aromatic, urea, and its compound.

For nitrogenous bases, the consumption of reagent is expressed as accounting for topical preparation, or accounts for the drug reservoir of drug delivery system or the 0.5-4.0w/w% of paster or so, it is intended to which 0.5-3.0w/w% or so, 0.75-2.0w/w% or so are more preferably.In view of considering the presence to any species that can be neutralized by alkali, these consumptions can be adjusted.

Suitable nitrogenous bases can include following any or its compound：

- primary amine (-- NH₂) group；

- monosubstituted (secondary amine) amino group -- NHR, R therein is alkyl, typically alkyl or aromatic radical, such as relatively low alkyl or phenyl, it can also be replaced by one or more non-hydrocarbyl substituents, such as 1 to 3 halogen, hydroxyl, sulfydryl, or relatively low oxyl (it is this -- NHR groups include, such as methylamino, ethylamino, isopropylamino, fourth amino, cyclopropylamino, Cyclohexylamino, the own amino of n-, phenylamino, aminotoluene base, chloroethene enamino, hydroxyethylamino etc.)；

- two substitution (tertiary amine) amino group -- NR^aR^b, R therein^aAnd R^bCan with identical or different, can be R above definition (it is suitable -- NR^aR^bIncluding, such as dimethylamino, lignocaine base, diisopropylaminoethyl, dibutylamino, methylpropylamino, the own amino of methyl, methyl cyclohexane amino, ethyl cyclopropylamino, ethyl vinyl chloride amino, methylbenzene methylamino, methylphenylamino, methyl toluene amino, methyl-p- chlorobenzenes methylamino, methyl cyclohexane amino)；

- acid amides -- (CO) -- NR^cR^d, wherein R^cAnd R^dCan be able to be hydrogen or R with identical or different, wherein R be defined as above (including, such as R therein^cAnd R^dOne be H and another be methyl, butyl, benzyl etc. acid amides)；

- cyano group (-- CN)；

- aromatic nitrogenated heterocyclic, typically five or single six-membered rings substituent, or the fusion of two rings or five yuan and hexatomic ring be formed by connecting (such as pyrrole radicals, pyridine radicals, quinoline base, pyrrolidinyl, pyrimidine radicals, imidazole radicals, 1,2,4- triazolyls, tetrazole radical etc.)；And

- aromatic nitrogenated heterocyclic, typically quaternary or hexatomic ring, including lactams and acid imide, such as pyrroles, morpholine, piperazine, piperazine, N- phenyl-azetidinone, butyrolactam, ω-caprolactam, acetylimino, phthalimide, succinimide.

Primary amine, secondary amine and tertiary amine are typically according to molecular structure NR¹R²R³The N surrounded is classified, R therein¹、R²And R³Selected from H, alkyl, hydroxyalkyl, alkoxyalkyl, alkenyl, hydroxy alkenyl, alkoxyalkenyl, cycloalkyl, the alkyl of cycloalkyl substitution, monocyclic aryl and monocyclic alkyl-substituted alkyl, condition is at least one R¹、R²And R³It is not H.This amine for example includes but is not limited to diethanol amine, triethanolamine, isopropanolamine, triisopropanolamine, two butanolamines, three butanolamines, N- dodecyl monoethanolamines, N- (2- methoxyethyls) dodecyl amine, N- (2, 2- dimethoxy ethyls) dodecyl amine, N- ethyls-N- (dodecyl) monoethanolamine, N- ethyls-N- (2- methoxyethyls) dodecyl amine, N- ethyls-N- (2, 2- dimethoxy ethyls) dodecyl amine, dimethyl dodecyl amine-N- oxides, single lauroyl amino lysine, two palmityl lysines, dodecyl amine, octadecylamine, phenyl ethylamine, triethylamine, PEG-2 oleananes, PEG-5 oleananes, dodecyl 2- (N, N- dimethylaminos) propionate, two (2- ethoxys) oleyl amines and its compound.

Typical primary amine includes 2- ethylaminoethanols, 2-aminoheptane, 2- amino-2-methyls -1, ammediol, 2-amino-2-methyl-1-propanol, n- amylamines, benzene methanamine, Putriscine, n- butylamine, cyclohexylamine, ethamine, ethylenediamine, methylamine, Alpha-Methyl benzylamine, phenyl ethylamine, propylamine and trishydroxymethylaminomethane.

Typical secondary amine includes the compound containing following group, for example methylamino, ethylamino, isopropylamino, fourth amino, cyclopropylamino, Cyclohexylamino, the own amino of n-, phenylamino, aminotoluene base, chloroethyl amino, hydroxyethylamino, etc..Typical secondary amine includes diethanol amine, diethylamine, diisopropylamine and dimethylamine.

Typical tertiary amine includes the compound containing following group, for example the bright base amine of dibutylamino, dimethylamino, diisopropylaminoethyl, ethyl chloroethyl amino, ethyl cyclopropylamino, methylhexyl amino, methylcyclohexyl amine, methyl, methylbenzylamine, methyl-chlorphenyl amine, methylcyclohexyl amine, aminomethyl phenyl amine, methyl toluene acyl amine, etc..Typical tertiary amine includes N, N- diethylanilines, DMG, triethanolamine, triethylamine and trimethylamine.

Acid amides, is those of skill in the art's preference, with R⁴--(CO)--NR⁵R⁶Molecular structure, wherein R⁴、R⁵And R⁶It is generally selected from H, alkyl, cycloalkyl, alkyl, monocyclic aryl and the monocyclic alkyl-substituted alkyl of cycloalkyl substitution.Suitable Exemplary amides described here include but is not limited to four nitrogen pregnancy acid amides, four nitrogen pregnancy acrylamides, cyclohexyl lauramide, cyclohexyl palmitamide, N, N- dimethylformamides, N, N- dimethyl acetamides, N, N- dimethyloctanamides, N, N- dimethyl certain herbaceous plants with big flowers acid amides, dimethyl-m- toluamides, diethyl-m-toluamide and its compound.

The nitrogen heterocyclic ring described here for being adapted as pharmacological activity alkali includes, such as 2- pyrrolones, 1- methyl -2- pyrrolones, the pyrrolones of 5- methyl -2,1,5- dimethyl -2- pyrrolones, 1- ethyl -2- pyrrolones, 1- propyl group -3- dodecyls pyrrolones, 1-dodecyclazacycloheptan-2-one, ethylene thiourea, hydantoins, oxalylurea, imidazoline urea, N- octadecyls morpholine, dococylpyridinium, N- dodecyl pyrrolidines, N- dodecyls piperidines, the piperidines of N- dodecyls two and its compound.

Aromatic nitrogenated heterocyclic usually contains one 5 yuan-or 6 yuan-substituent, or the ring or the 5 yuan or 6 yuan rings connected that two rings are merged, such as imidazole radicals, indyl, pyridine radicals, pyrimidine radicals, pyrrole radicals, quinoline base, tetrazole radical, 1,2,4- triazolyls etc..

The aromatic nitrogenated heterocyclic described here for being adapted as organic base includes, for example, 2- amino-pyridines, benzimidazole, 2,5- di-amino-pyrimidines, 2,4- methylimidazoles, 2,3- lutidines, 2,4- lutidines, 3,5- lutidines, imidazoles, pyridinyl methoxy, γ-picoline, 2,4,6- trimethylpyridines and its compound.

Non-aromatic nitrogenated heterocyclic, 4 yuan are usually contained to 6 yuan of rings, such as acetylimino, morphine base, lactams and acid imide (such as butyrolactam, epsilon-caprolactams, N- phenyl-azetidinone), benzene two (first) acylimino, piperidyl, piperazine base, piperazinyl, pyrrole radicals, succinimido.

Non-aromatic nitrogenated heterocyclic includes, such as 1,2- lupetidines, 2,5- lupetazins, 1,2- dimethyl pyrroles, 1- ethyl piperidines, n- methylpyrroles, morphine, piperazine, piperidines, pyrroles, 2,2,6,6- tetramethyl piperidines and its compound.

For all pharmacological activity alkali described here, the optimum amount of any particular agent depends on other any acidic species in the power of alkalescence, the molecular weight of alkali, and other factorses, such as the number ethyl preparation or paster in the given secondary ionizable site of medicine.Those of skill in the art can determine the optimum amount of any specially orchard worker reagent easily, it is ensured that preparation reaches effective pH in skin surface, and preparation is once used, and pH scopes are 7.5 to 13.0 or so, it is intended to 8.0 to 11.5 or so, and 8.5 to 10.5 or so scope is more preferably.So ensure to reach the treatment level of maximum successively, while excluding to the damage possibility of body surface or it at least being greatly lowered.

In the composition of topical preparation, active agent can be dispersed in emulsifiable paste, ointment, wax or other liquid or semi-liquid-like solution, so, the topical preparation of emulsifiable paste or ointment or washing lotion or wax or liquid can take active agent on patient's biological surface or internal.Term as used herein " biological surface " contemplates any surface of organ outside or inside.For example, " biological surface " of the topical preparation of the present invention is possibly used for including any epithelial surface such as skin exhaling, and suction road, intestines and stomach, including oral mucosa and genitourinary tract.Term " local application " includes, and is administered in the testis of biological surface, and fissure or crack administration.

One " topical preparation " usually contains the pharmaceutically acceptable carrier of a local treatment, including but not limited to neutral sterile emulsifiable paste, emulsifiable paste, washing lotion, wax, glue, jelly, ointment, paste, smoke agent, paster, powder, and/or its compound.Preferred pharmaceutically acceptable carrier has emulsifiable paste, such as Cetaphil Moisturising Cream (Galderma Laboratories, L.P.), QV cream (Lision Hong), Sorbolene etc..In another embodiment, pharmaceutically acceptable carrier has washing lotion, such as Alpha Keri Moisturising Lotion (Mentholatum), DermaVeen Moisturing Lotion (DermaTechLaboratories), QV Skin Lotion (Lision Hong), Cetaphil MoisturingLotion (Galderma Laboratories, L.P.) etc..In another embodiment, pharmaceutically acceptable carrier includes oil, such as emu oil.

Emulsifiable paste is viscous fluid or semisolid emulsions, oil-in-water or Water-In-Oil.Cream base is that water is washable, by an oil phase, an emulsifying agent and an aqueous phase composition.Oil phase, also referred to as " inside " phase, are generally made up of vaseline and aliphatic alcohols such as a lauryl alcohol or octadecyl alcolol.The volume of aqueous phase is usually more than oil phase, but is not essential, and generally comprises a diluent.Emulsifying agent in cream preparation is typically nonionic, anion, cation or amphoteric surfactant.

Preferred emulsifying agent includes, but it is not limited to, AEO (Peregal A-20), stearic acid such as polyethylene glycol stearic acid (softening agent SG), any polyalcohols form such as FEG-5 tristerins of tristerin and tristerin, hexadecanol, Dithranol or its compound.

Preferred oil-phase component includes, but are not limited to dimethicone, dimethiconol, cyclomethicone, diisopropyl adipic acid, hexadecanol, octadecyl alcolol, paraffin, vaseline, apricot kernel oil and stearic acid.

In special aspects, water-phase component includes but is not limited to pure water, glycerine (glycerine), propane diols, ethyl-para-hydroxybenzoate and any diluent.

In some concrete schemes, emulsifiable paste is further made up of one or more film forming agents, but is not limited to polybutene acid glyceride, acrylate/C10-30 alkyl acrylic heterozygosis polymers；Antioxidant includes but is not limited to tocopherol acetate；Preservative includes but is not limited to Phenoxyethanol, phenmethylol；Other additives include but is not limited to octyl ether, EDETATE SODIUM, sodium hydroxide and lactic acid.

In a specific embodiment, emulsifiable paste includes pure water, polybutene acid glyceride, propane diols, vaseline, octyl ether, PEG-5 tristerins, glycerine, dimethicone, dimethiconol, hexadecanol, Sweet Almond Oil, acrylate/C10-30 alkyl acrylic heterozygosis polymers, alpha-tocopherol acetate, Phenoxyethanol, phenmethylol, EDETATE SODIUM, sodium hydroxide, lactic acid.

In another embodiment, emulsifiable paste includes glycerine, liquid paraffin,light, soft white paraffin wax, dimethicone, saualane, methyl hydroxybenzoate, dichlorbenzyl alcohol.

Ointment is semisolid preparation, generally based on vaseline or other petroleum derivatives.Optimal administration can be obtained by the specific ointment bases that those of skill in the art are had a preference for, while other expected features can also be obtained, such as softening etc..It is similar with other carriers or medium, ointment bases should be it is inactive, stably, nonirritant and not sensitization.Ointment bases is segmented into four classes：Oleaginous base；Emulsifiable matrix；Emulsion bases；And water-soluble base.Oleaginous base includes, for example vegetable oil, the fat from animal, the semisolid hydrocarbons from oil.Emulsifiable matrix, is also so-called absorbable ointment bases, containing a small amount of water or anhydrous, including such as hydroxystearin sulfate, lanolin, and hydrophilic pelpolatum.Emulsion bases is Water-In-Oil (W/O) emulsion or oil-in-water (O/W) emulsion, and wherein oil component includes, such as hexadecanol, glycerin monostearate, lanolin, and stearic acid.Preferred water-soluble base is prepared from polyethylene glycol, with different molecular weight.

Glue is transparent, sticky, jelly sample semisolid or solid, and heavy polymer is dispersed in water-based or alcohol matrix and obtained.Alcohol glue is needed to dry, freezed, and is most suitable for acute exudative itching rash；Non-alcohol glue more lubricates, suitable for the dry fish scale shape damage in scalp.

Washing lotion, is the preparation for skin surface without friction, typically liquid or semi-liquid preparations, and solid particle therein includes active ingredient, is present in water or alcohol matrix.Washing lotion is typically the suspension of solid, for existing purpose, it is intended to be made up of the liquid oily emulsifying agent of oil-in-water type.Here, for treating during large area body region, washing lotion is preferred preparation, and this is due to the ease of use of the preparation with more mobility.It is preferably scattered to obtain that washing lotion generally includes suspending agent, and is conducive to the collection when contacting skin to neutralize the compound for storing active agent, such as methylcellulose, sodium carboxymethylcellulose etc..

Paster is semisolid dosage form, and active agent therein is suspended in a suitable matrix.Property based on matrix, paster is divided into lipid paster or the paster obtained from single-phase aqueous gels.Matrix in lipid paster is typically vaseline, hydrophilic pelpolatum etc..The paster obtained from unisexuality gel aqueous phase is routinely incorporated into carboxymethyl cellulose etc. as matrix.

In one embodiment, the pharmaceutical composition of the present invention can be used in combination individually or with other medicines or therapy, the other medicines or therapy employ the mode as protein or its chimeric molecule that non-human cell lines express, such as Escherichia coli, yeast or the protein or chimeric molecule of CHO expression, for individually or with other medicines treating disease together, the disease includes：Abetalipoproteinemia,A-V,Beta-2-Microglobulin amyloidosis,A-T,A1AD,A1AT,Aagenaes,Aarskog's syndrome,Aarskog-Scott syndrome,Aase-Smith syndrome,Aase syndrome,AAT,Abderhalden-Kaufmann-Lignac syndrome,Abdominal muscle deletion syndrome,Abdominal-wall defect,Abdominal epilepsy,Abdominal migraine,Abductor dysphonia spastica,Abductor spastic dysphonia,Abercrombie's syndrome,Eyelid meloschisis syndrome,ABS,HPRT lacks,Corpus callosum Schinzel Typ lack,Four limbs scalp and cranium defect,Amenorrhea Primar,HGPRT lacks,Absorptive Hyperoxaluriaor Enteric,Abt-Letterer-Siwe diseases,ACADL,ACADM lacks,ACADM,ACADS,Acanthocytosis neuropathy,Acanthocytosis,Epidermolysis bullosa,Acanthosis nigricans (disease),Acanthosis bullosa,Acanthosis nigricans (disease) with A type insulin resistances,Acanthosis nigricans (disease) with Type B insulin resistance,Acanthotic nevus,Acatalasemia,Acatalasia,ACC,Accessory atrioventricular pathways,Acephalia,With heart defect ACF,Relaxation can not,A Chade-ladder syndrome,A Chade (all mutation of horse),A Shaer Cotards,Acholuric jaundice,Achondrogenesis,The achondrogenesis of type four,3rd class achondrogenesis,Achondroplasia,Achondroplasia is slow,Achondroplastic dwarf,Sneeze syndrome,Colour blindness,Monochromat,Monochromasia,Monochromasia,Colour killing mole,Acid ceramidase deficiency,Acid maltase deficiency,Acid β-glucosidase deficiency disease,Acidaemia,Acidaemia,The sporadic incoordination of malonic acid mass formed by blood stasis and weakness,Acid poisoning,Tarsoepiphyseal aclasis,ACM,Acoustic nerve neurilemmoma,Acoustic neurinoma,With referring to (toe) more the undergrown tip of lower limb and refer to (toe) deformity,Refer to (toe) more tip and refer to (toe) deformity II,Refer to (toe) more tip and refer to (toe) deformity IV,Refer to (toe) more tip and refer to (toe) deformity III,The day after tomorrow aphasia convulsions disease,The day after tomorrow brown's syndrome,Acquired epileptic aphasia,Factor XIII deficiency,The ACC that the day after tomorrow is formed is (caused by infection,And be still in intrauterine),Acquired hyperoxaluria,Acquired hypogammaglobulinemia,Immune Deficiency Syndrome (AIDS),Acquired iron overload,Acquired fat nutritional disorders,Acquired partial fat dystrophia,Acquired splenectopia,ACR,Acrodysostosis is with face and genital malformation,Acro Renal,Acrocallosal Syndrome Schinzel Type,Acrocephalosyndactylism (toe) (deformity),I types acrocephalosyndactylism (toe) (deformity),I types acrocephalosyndactylism (toe) (deformity) hypotype I,II types acrocephalosyndactylism (toe) (deformity),Type III acrocephalosyndactylism (toe) (deformity),IV types acrocephalosyndactylism (toe) (deformity),V-type acrocephalosyndactylism (toe) (deformity) (ACS5 or ACS V) hypotype I,Tip skull is asymmetric and slight and refers to,Tip,acrochondrohyperplasia,Acrodermatitis enteropathica,Acrodysostosis,Acrodystrophic neuropathy,Acrofacial dysostosis Nager types,Acrofacial dysostosis Postaxial types,Acrofacial dysostosis Genee-Wiedep types,Acrogeria family,Acromegalia,Acromelalgia,Acromesomelic dysplasia,Acromesomelic dwarfism,Akromikrie dysostosis,Akromikrie depauperation,Acroosteolysis and skull and mandibular change with osteoporosis,Acroosteolysis,Acroparesthesia,ACSI,ACS II types,ACS type IIIs,ACS,ACS3,Corticotropin deficiency disease,Action myoclonia,Acute brachial plexus neuritis syndrome,Acute brachial plexus nerve radicular syndrome,Acute brain Gaucher disease,Acute cholangitis,It is acute to send out encephalomyeloradiculopathy,Acute disseminated histiocytosis X,Acute hemorrhagic polioencephalitis,Extra urgent property polyneuritis,Acute immune reconciles polyneuritis,Children's acute familial centrolobar sclerosis,Accute porphyrin,Acute porphyria,Acute sarcoidosis,Acute shoulder neuritis,Acute toxic epidermolysis bullosa,Acyl-coenzyme dehydrogenase deficiency long-chain,Acyl-coenzyme dehydrogenase deficiency short chain,Acyl coenzyme A dihydroxyacetone (DHA) acyltransferase,Acetylation of coenzyme A oxidase lacks,ADA,ADA lacks,AdamComplex,Adamantiades-Behcet syndrome,Adamantinoma,Adams Oliver syndromes,Adaptive colitis,ADD combined type,ADD,Ai Di diseases carry cerebrosclerosis,Ai Di disease anaemias,Addison's disease,A Disen (family name) anaemia,Adrenoleukodystrophy,A Disen (family name) pernicious anaemia,Interior receipts thumb-mental retardation,Adductor spastic dysphonia,Adductor spastic dysphonia,Manlike elderly woman adenoma,Colon and rectal gland epithelial hyperplasia,Colonic adenoma polyposis,Family's adenomatous polyp,Adenosine deaminase deficiency,Adenylosuccinase deficiency,Hyperactivity mainly moves impulsive style,Absent-minded type based on hyperactivity,Hyperactivity,Arachnoid adhesion,Adie's syndrome,Adie's syndrome,Ai Di myotonic pupils,Adie's pupil,Referring to adipogenital (drawing) retinitis pigmentosa more,Adipogenital (drawing) retinitis pigmentosa syndrome,Adiposa Dolorosa,Adiposis dolorosa,Adiposogenital dystrophy,Adolescent cystinosis,Polycystic kindey,Adrenal cortical adenoma,Adrenal gland diseases,Adrenal cortex function is hyperfunction,Cause pituitary adrenocorticotropic hormone superfluous,Adrenal aplasia,Adrenal insufficiency,Adrenal tumor,Adrenal gland is manlike,Adrenal gland-retinitis pigmentosa-many toes syndrome,Adrenal insufficiency,Hypoadrenocorticism,Corticotropin lacks single,(congenital) adrenogenital syndrome,Adrenoleukodystrophy,Adrenomyeloneuropathy,Adrenal gland-retinitis pigmentosa-many toes syndrome,Adult's cystinosis,Adult dermatomyositis,Adult hypophosphatasia,Into macular degeneration in humans,Adult Onset ALD,Adult onset ceroidosis,Adult Onset's medullary substance cystic disease,Adult Onset's pernicious anaemia,Adult Onset's Schindler Disease,Adult onset subacute necrotizing cerebrospinal cord disease,Adult polycystic kidney disease,Adult Onset's medullary substance cystic disease,Adynlosuccinate lyases are lacked,AE,AEC syndrome,AFD,Fibrinogenemia,African siderosis,AGA,Aganglionic megacolon,Age-related macular degeneration,Agenesis of corpus callus,Agenesis of corpus callus,Agenesis of corpus callus-infantile spasm eye is abnormal,Agenesis of corpus callus and chorioretinal are abnormal,Agenesis of corpus callus-choroidoretinitis,Mastocytosis,Agnosis Primary,AGR Triad,AGU,Agyria (deformity),Agyria-pachygyria frequency range,AHC,AHD,AHDS,AHF lacks,AHG lacks,AHO,Ahumada Del Castillo,Ai Kaerdi syndromes,AIED,AIMP,AIP,AIS,Akinetic seizure,ALA-D porphyrias,Alactasia (disease),Alagille syndrome,Oran island illness in eye (x is chain),alaninuria,Ai Erbai-Arnold Schoenberg disease,Albefaction,Albinism,Albinoidism,Albright constitutional bone disease,Alkaptonuria,The related inborn defect of alcohol,Alcohol embryopathy,Alcoholic cirrhosis,Ald,ALD,ALD,Aldosterone,With normotensive aldosteronism,Congenital cataract,Alexander disease,Alexandria disease (infantile leukodystrophy),Algodystrophy,Algoneurodystrophy,Melanuria,Alkaptonuric ochronosis,Alkyl DHAP synthase is lacked,Allan-Herndon-Dudley syndromes,A Lan-henry syndrome,A Lan-henry-Dudley baryencephalia,Allergic granulomatous angiitis,The Canadian allergic granulomatous angiitis of Cronkhite-,Alobar holoprosencephaly,Alopecia areata,Alopecia circumscripta,Alopecia circumscripta,Alopecia circumscripta,Alopecia-white hair (disease-dermatoglyph uveitis-leucoderma-deafness skin uvea-O,Alopecia seminuniversalis,Whole alopecia,General alopecia,Gray-matter degeneration,The ectocinerea and hepatic sclerosis of alpers diffusivities denaturation,Children's poliodystrophia (brain) of alpers progress,Alpha-1-antitrypsin deficiency,The glucosides enzyme deficiency disease of Alpha -14,Alpha-galactoside enzyme defect,Alpha-galactoside boron deficiency,Alpha's HDL lacks,Sick 3 type of α-L- mycoside azymia mycosides (storing up),α-GalNAc defect Schindler types,Alpha lipoprotein,Mannosidosis,Alpha-N-Acetylgalactosaminidase defect Schindler types,α-NAGA lack Schindler types,α-nerve ammonia (sugar) neuraminidase defect,α-thalassemia/mental retardation syndrome non-deletion type,Alpha lipoprotein,Alport syndrome,ALS,Alstroem Cotards,alstroem,Alstrom syndromes,Alternating hemiplegia syndrome,Children's alternating hemiplegia,Alzheimer's,Family black horny idiots,Family black horny idiots are grown up,Family black horny idiots children,Vulva sex is failed to understand,AMC,AMD,Ameloblastoma,Amelogenesis imperfecta,Amenorrhea-galactorrhea nonpuerperal,Amenorrhea-galactorrhea-FSH reduces syndrome,Amenorrhoea,Amino acid is not normal,Amino acid-osteomalacia-hyperphospheremia syndrome,AMN,Amniocentesis,Amniotic sheets,Amniotic band syndrome,Amnion desmorrhexis is complicated,Amniotic sheets sequence,Amniorrhexis sequence,Congenital amputation,AMS,The short and small syndrome de Lange in Amsterdam,Amylo-1:4,1:6-transglucosidase,6- glucosides enzyme deficiency diseases,Long term hemodialysis starch joint,Amylaceous corneal dystrophy,Amyloid polyneuropathy,Amyloidosis,Starch familial Mediterranean fever,Amylopectinosis,Amyoplasia congenita,ALS,ALS,ALS-glucan,AN,AN1,AN2,Hedratresia,Anal plate,Anorectal malformation,The stricture of anus,The amyloidosis of Analine 60,Blood (interior) alpha-lipoprotein lacks,analrectal,analrectal,Glioblastoma multiforme,Andersen's disease,Anderson -- Fabry disease,Andersen's glycogenosis,Anderson-Hua Bao syndromes,An Delie syndromes,The type of An Delie syndromes two,Androgen insensitivity,Androgen-insensitivity syndrome is local,Androgen-insensitivity syndrome is local,Androgenic Steroids,Anaemia autoimmune hemolytic,Anemia Blackfan Diamond,Anaemia,It is geneogenous,Triphalangeal thumb syndrome,The cold antibody of hemolytic anemia,Hemolytic anemia is lacked with PGK,Pernicious anaemia,Anencephalia,Angelman syndromes,Angio-osteohypertrophy syndrome,Blood vessel folliculus lymph node hyperplasia,Angiohemophilia,Angioceratoma body,Unrestrained property body angioceratoma,Diffusivity angioceratoma,Retinal angiomatosis,Lymphangiohemangioma,Angioneurotic edema heredity,Anhidrotic ectodermal dysplasia,Anhidrotic x is chain ectodermal dysplasia,Irideremia,Irideremia-unclear the mental retardation of genitals sex,Iris and mental retardation,Irideremia-cerebellar ataxia intelligence is residual,Iris part cerebellar ataxia mental retardation,Iris part cerebellar ataxia oligrophrenia,Irideremia type I,Irideremia type II,Aniridia-Wilms tumor association,Aniridia-Wilms tumor-gonadoblastoma,Ankyloblepharon-ectoderm developmental defect harelip/cleft palate,Ankylosing spondylitis,Annular groves,Anodontia,Anodontia vera,Anomalous trichromatism,Anormogenesis exception dentine,Coronal dentin dysplasia,Anomic aphasia,Anophthalmia,Anal intestine,Anorectal malformation,Anosmia,Front arcuate leg is with nanism,Tooth film corneal dystrophy,Anticonvulsion disease,Anti- Epstein-Barr Virus Nuclear Antigens (EBNA) antibody deficiency,Antibody deficiency,Antibody deficiency is carried close to normal immunoglobulin,Antihemophilic factor deficiency disease,Antihemophilic globulin deficiency,Antiphospholipid syndrome,Antiphospholipid antibody syndrome,Antithrombin III lacks,Typical Antithrombin III lacks (I types),Antitrypsin is lacked,Antley-Bixler syndromes,Antoni is benumbed,Shin is uneasy,Aortic arch syndrome,Sustainer and mitral atresia merge left heart syndrome,Aortic stenosis,aparoschisis,APC,Apeced syndromes,Apert syndrome,aperts,Aphasia,Congenital cranium outer shaft sexual dyspenesis,Congenital skin defect,The horizontal physical impairment of congenital skin defect and end,Alpastic anemia,Alpastic anemia birth defect,APLS,Apnea,Appalachian type amyloidosis,Apple skin syndrome,Appraxia (disease),Buccal surface appraxia (disease),Structure appraxia (disease),Ideational appraxias (disease),Ideokinetic appraxias (disease),Ideomotor movement appraxia (disease),Motor appraxia (disease),Eye movement appraxia (disease),APS,Archnoiditis,Arachnodactyly contracture Beals types,Arachnodactyly,Arachnoid cyst,Spider web periosteal ossification,Archnoiditis,Aran-Duchenne,Aran-Duchenne muscular atrophy,Alpastic anemia,Arginine depletion,Argininemia (disease),Arginino succinase are lacked,Argininosuccinase deficiency,Argininosuccinase Defect,Argininosuccinic acid-ASL,Argininosuccinate synthetase lacks,Smart ammonia (base) amber uraturia,Argonz-Del Castillo syndromes,Arhinencephalia [deformity],Armenia's syndrome,Arnold-Chiari malformation,Arnold-plus syndrome,ARPKD,Myoclonic arrhythmia cordis,Right ventricle depauperation,Arteriohepatic dysplasia,Arteriovenous malformation,Venous malformation brain,Arteritis giant cell,Arthritis,Reiter syndromes,Arthro-dento-osteo dysplasia,Arthro-ophthalmopathy,Arthrochalasis multiplex congenita,Distally,IIA types,ARVD,Fragrant (perfume) base sulfatase B deficiency diseases,AS,ASA lacks,Ascending paralysis,ASD,Atrial septal defect,ASH,Asherman syndrome,Ashkenazi type amyloidosis,ASL deficiency,Aspartylglucosaminuria,Eisberg syndrome,Eisberg type self-closing disease,Asphyxiating thoracic dysplasia,Splenic syndrome,ASS deficiency,Asthma,I grades of astrocytoma (benign),II grades of astrocytoma (benign),Asymmetric phase heart defect of crying,Asymmetric septal hypertrophy,Asymptomatic callosity,AT,AT III defects,AT III variants IA,AT III variants Ib,AT 3,Incoordination,Ataxia telangiectasia,Incoordination and lactic acidosis II types,Incoordination brain paralysis,Ataxiadynamia,Ataxiophemia,ATD,Brothers' brain paralysis,Dermatitis and eczema,Atretolemia is with or without esophago-tracheal fistula,Atrial septal defect,Original atrial septal defect,Atrial septum and small ventricular septal defect,Auricular flutter,Auricular fibrillation,Atriodigital dysplasia,Atrial septal defect,Atrioventricular block,Atrioventricular anomaly,Atrioventricular septal defect,(family name) disease in promise,Atrophic type athlete's foot,Olive atrophy,Attention deficit disorder,Attention deficit hyperactivity disorder [with how dynamic],It is attenuated colonic adenoma,Atypical amyloidosis,Atypia mass formed by blood stasis,Internal auditory meatus locking,Auriculotemporal syndrome,Autism,Self-closing disease-Eisberg type,Self-closing disease dementia incoordination and purpose hand use defect,Autism children autism,LADA Addison's disease,Autoimmune hemolytic anemia,Oneself immunity hepatitis,LADA-many (kind) endocrine adenopathy-candidiasis,Autoimmune polyglandular disease I types,Autosomal dominant inheritance albinism,Autosomal dominant CompellingHelioophthalmic Outburst syndromes,Autosomal dominant inheritance desmin distal myopathy is simultaneously tardy,Autosomal dominant EDS,Autosomal dominant eme ry-dreifuss type muscular dystrophies,Autosomal dominant keratoconus,Autosomal dominant Pei-plum Er Shi cerebral sclerosises,Autosomal dominant polycystic kidney disease,Autosomal dominant inheritance spinal cerebellar degeneration,Autosomal recessive inheritance agammaglobulin disease,Autosomal recessive inheritance central nucleus myopathy,Autosomal recessive inheritance Kang-Xu Er 's syndrome,Autosomal recessive inheritance EDS,Autosomal recessive inheritance ecuador's syndrome is malnourished,The eye albefaction of autosomal recessive inheritance form,Autosomal recessive inheritance callosity hypoplasia,Autosomal recessive keratoconus,Recessive hereditary polycystic kidney disease,Autosomal recessive inheritance severe combined immunodeficiency,AV,AVM,AVSD,AWTA,Oxter abscess,Axonal neuropathy is huge,Azorean sacred diseases,B-K mole syndrome,Babinski-Froelich syndromes,BADS,Baillarger Cotards,Balkan disease,Baller-Gerold syndrome,Sacculus bicuspid valve,Balo disease concentric sclerosis,Baltic Sea lafora's disease,Bannayan- Zuo Nana syndromes (bzs),Bannayan- FilippoGammarelli-ruvalcaba syndromes,This Di Shi diseases,Bardet-biedl syndromes,Bare lymphocyte symdrome,Concentric sclerosis,Bar [clarke that]-west [illiteracy] Er Shi diseases,Barrett esophagus,Bei Ruite ulcer,Bartter syndrome,Bartter's syndrome,Basal cell naevus syndrome,Basedow disease,A-betalipoproteinemia,Batten disease,Batten-Mayou syndromes,Batten-Spielmeyer-Vogt disease,The honest Turner's synodrome of shellfish,Bei Dun Teners type congenital myopathy,Batten-Vogt syndromes,BBB syndromes,BBB syndromes (opitz),BBBG syndromes,BCKD is lacked,BD,BDLS,BE,Bei Yaersi syndromes,Bei Yaersi syndromes,Bei Yaersi-Hecht syndrome,Bean syndrome,BEB,Bechterew syndromes,Becker disease,Becker muscular dystrophy,Becker's nevus,Beckwith Wiedemann syndromes,Beckwith syndrome,Begnez-Cesar syndromes,Behcet syndromes,Behcet diseases,Behr1,Behr2,Bell's palsy,Benign acanthosis nigricans,Benign astrocytoma,Benign cranial neuroma,Benign cystinosis,Benign essential blepharospasm,Benign essential tremor,Benign familial hematuria,Benign focal amyotrophia,The benign focal amyotrophia of ALS,Benign hydrocephalus,Benign hypermobility syndrome,Angling benign acanthosis nigricans,Benign paroxysmal peritonitis,Benign recurrent hematuria,Benign recurrent intrahepatic cholestasis,Benign spinal muscular atrophy and calf plumpness,Benign symmetric lipomatosis,Benign central nerve neuroma,Berardinelli-Seip syndrome,Primary Jie Shi diseases,Tinea pedis,Shellfish Mann syndrome,The Nellie syndrome of Claude Bernard-suddenly,Bernard Soulier syndrome,Bass Nie's pruigo,Vitelliform macular degeneration,Beta-alanine-Pyruvic Transaminase,Beta-Galactosidase deficiency Morquio syndrome,Beta-Glucuronidase deficiency,Beta-oxidation defect,β major thalaseemias,β minor thalassemias,Beta lipoprotein is lacked,Bei Telun myopathies,Beuren syndromes,BH4 deficiency diseases,Biber-Haab-Dimmer corneal dystrophies,Bicuspid aortic valve,Biedl-Bardet,Bifid brainpan,Bifunctional enzyme defect,Bilateral listens multiple neurofibromatosis,Bilateral Acoustic Neroumas,Bilateral right-sidedness sequence,Potter syndrome,Bilateral temporal lobe obstacle,Bile breaks out,Glucosiduronate (base) shifts enzyme defect I types,Binder syndromes,Binswanger diseases,Binswanger encephalopathics,Biotinidase deficiency,Bird-headed dwarf of Seckel Seckel types,Inborn defect,Birthmark,Double temporos clamp trace syndrome,Myocardial fibrosis,Bjornstad syndromes,B-K mole syndrome,Black lock-albinism-deafness sensoneural types (BADS),Blackfan-Diamond anemia,Pyorrhea primary arthritis,Blepharophimosis,Ptosis,Epicanthus inversus syndrome,Blepharospasm,Blepharospasm is benign primary,Oral cavity blepharospasm,Blessig's cysts,BLFS,Blindness,Bloch-Siemens incontinentia pigmenti melanoblastosis skins Linearis,Bloch-Siemens-Sulzberger syndromes,Bloch-Sulzberger syndrome,Blood group,Blood group A,Blood group B,Blood group AB,Blood group O,Facial telangiectasis of dwarfs syndrome,Bloom-Torre-Mackacek syndromes,Blue rubber bleb nevus,Blue baby,Blue diaper,BMD,BOD,BOFS,Bone tumour epidermoid cyst polyp,Bonnet-Dechaume-Blanc syndrome,Bonnevie-Ulrich syndromes,Book syndrome,BOR syndrome,BORJ,Borjeson syndromes,Borjeson's syndrome,Cerebrohepatorenal syndrome,Bowen-Conradi syndromes,Bowen-Conradi Hutterite,Bowen-Conradi type Hutterite syndromes,Bowman’s Layer,BPEI,BPES,Brachial plexus neuritis,Brachial plexus neuritis syndrome,Brachial plexus neuritis,Brachial plexus neuropathy,Arm ischemic,Brachmann-de Lange syndrome,Brachycephaly (deformity),Short shape type is congenital,Bradycardia,Craniocerebral injury is because of perinatal asphyxia,Brain tumor,Brain tumor is benign,Brain tumor is pernicious,Branched-chain alpha-keto acid dehydrogenase deficiency disease,Side chain beta-oxybutyria I,Brancher deficiency,Branchio-Oculo-Facial syndromes,Branchio-oto renal aplasias,Branchio-oto kidney syndromes,Branchiooculofacial syndromes,Branchiooculofacial syndromes,Brandt syndrome,Brandywine type hypoplasias of dentin,Brandywine type hypoplasia of dentin,Breast cancer,Benign recurrent intrahepatic cholestasis syndrome,Brittle bone disease,Broad-beta disease,Wide thumb syndrome,The facial MR of wide thumb and big toe feature,Wide thumb,Broca aphasias,Brocq-Duhring diseases,Bronze diabetes,Bronze diffusivity sclerosis,Brown albinism,Brown glaze heredity,Blang-fork clip that (family name) syndrome,Brown's syndrome,BRRS,Bo Lugeer syndromes,Bruton agammaglobulinemias (disease),BS,BSS,Buchanan syndromes,Budd syndromes,Budd-Chiari syndrome,Buerger-Gruetz syndromes,Bulbospinal muscular atrophies x is chain,Overwork syndrome,Heredity bleb,Bleb CIE,Epidermolysis congenital ichthyosis sample erythroderma,Epidermolysis ichthyosis,Bullous pemphigoid,Burkitt lymthomas,Burkitt lymthomas Africa type,Burkitt lymthomas,African type,BWS,Byler diseases,C syndromes,C1 esterase inhibitors exception II type angioedemas,C1-INH,C1 esterase inhibitors lack I type angioedemas,C1NH,Cacchi-Ricci disease,CAD,CADASIL,CAH,Valgus calcaneus,calcaneovalgus,Calcium pyrophosphate dihydrate is calm,Agenesis of corpus callus and eye are abnormal,The plump myeloid muscular dystrophy of calf,Campomelic dysplasia,Trunk nanism,Trunk syndrome,Count on one's fingers-cleft palate -- clubfoot,Limited jaw of counting on one's fingers is offset,Camptomelic dwarfism,Camptomelic syndrome,The long limb type of camptomelic syndrome,Camurati-Engelmann disease,Canada-Cronkhite diseases,Canavan's disease,What canavan's disease included,Ka Nawan leukodystrophies,Cancer,Cancer family syndrome Lynch types,Cantrell syndromes,Cantrell-Haller-Rayich syndromes,Cantrell pentalogys,Carbamyl phosphate synthetase deficiency,Carbohydrate defect glycoprotein syndrome,Carbohydrate defect glycoprotein syndrome Ia types,The hyperlipidemia that carbohydrate induces,The carbohydrate of glucose lactose is not tolerated,Carbon dioxide acid poisoning,Multiple carboxylation enzyme defect,Heart limb syndrome,Heart comprehensive hearing is levied,Jervell and and Lange-Nielsen cardioauditory syndromes,Cardiocutaneous syndrome,The heart-face-skin syndrome,Cardiofacial syndrome Cayler types,II type glycogen storage diseases,Cardiomyopathies lentiginosis,Cardiomyopathy,Cardiomyopathy and desmin storage myopathy,Cardiomyopathy is due to desmin defect,Cardiomyopathy-neutrophilic granulocytopenia,The lethal myocardium in children disease of cardiomyopathy-neutrophilic granulocytopenia,Heart disease amyloidosis,Cardiospasm,Cardocardiac syndromes,Carnitine-acylcarnitines transposition azymia,Carnitine lacks and disorderly,Carntine deficiency is primary,Carntine deficiency is secondary,Carntine deficiency is secondary to be lacked to MCAD,Meat [poison] base deficit disease,Carnitine palmitoyltransferase I＆II (cPTI＆II),Carnitine palmitoyltransferase lacks,Carnitine palmitoyltransferase lacks I types,Carnitine palmitoyltransferase lacks I types includes infant's severe form including benign typical muscle form,Carnitine transport defect (primary carnitine deficiency disease),Carnosinase deficiency,Carnosinemia,Caroli disease,Carpenter's syndrome,Ka Pengte's,Cartilage-hair hypoplasia,Castleman diseases,Castleman disease hyaline-vascular types,Castleman disease thick liquid cell types,Castleman tumours,Cat's eye syndrome,Cat is cried syndrome,Catalayse is lacked,Cataract- dental composites are levied,The chain syphilitic teeths of Cataractx,Catecholamine hormones,Catel-manzke syndromes,Catel-manzke type palatodigital syndromes,Tail dysplasia,Caudal dysplasia sequence,Caudal dysplasia syndrome,Cusalgia syndrome is grown up,Cvernous hemangioma,Cvernous hemangioma,Cvernous hemangioma,Spongy lymphatic vessel,Spongy deformity,Cayler syndromes,Cazenave leucoderma,CBGD,CBPS,CCA,CCD,CCHS,CCM syndromes,CCMS,CCO,CD,CDGla,CDGlA,CDGS Ia types,CDGS,CDI,CdLS,Celiaca,Sprue,Abdominal cavity sprue-dermatitis,Cellular immunity deficiency and purine nucleoside phosphorylase deficiency,Celsus leucoderma,Central choking,Central core disease,Central diabetes insipidus,Center forms multiple neurofibromatosis,Maincenter hypoventilation,Centric sleep apnea,Telecentricity fat nutritional disorders,Centronuclear myopathy,CEP,Naoning tablet,Head (with) thorax fat nutritional disorders,Ceramide trihexosidase deficiency,Cerebellar hypoplasia,Cerebellar hypoplasia,Cerebellum hemiageusia,Cerebellar hypoplasia,Vermis of cerebellum hypoplasia,The dynamic incoordination of vermis of cerebellum hypoplasia-hypernea-curtain formula eye is slow,Cerebellar syndrome,Cerebellarparenchymal diseases IV,Celand-Arnold-Chiari syndrome syndrome,Cerebellum Oculocutaneous telangiectasis,Cerebellarparenchymal disease IV familials,Cerebellopontine angle tumours,Cerebral arachnoiditis,Brain autosomal inheritance cerebrovascular disease is with infarct and leukodystrophy under cortex,Wernicke-Korsakoff syndromes,Cerebral diplegia,Cerebral gigantism,Cerebral ischemia,Cerebrovascular malformation,Cerebral paralysis,Brain oculorenal is malnutritive,Brain eye facial skeleton syndrome,Cerebro-costo-mandibular syndrome,Cerebrohepatorenal syndrome,Cerebromacular degeneration,Cerebromuscular malnutrition Fukuyama types,Brain (with) agenesis of eye,Brain (with) agenesis of eye leyden-Mobius myodystrophia,Cerebro-oculo-facio-skeletal syndrome,Cerebroretinal arteriovenous aneurysm,Cerebroside lipidosis,Brain glucoside (deposition) disease,Brain-tendon xanthomatosis,Cerebrovascular Ferrocalcinosis,Ceroid lipofuscinosis adult form,Cervical dystonia,Cervical dystonia,Neck eye listens distress syndrome,Cervical spinal stenosis,Cervical vertebral body is merged,CES,CF,CFC syndromes,CFIDS,CFND,CGD,CGF,General hair Chalasodermia,Chanarin Dorfman diseases,Chanarin Dorfman syndromes,Chanarin Dorfman fish scale ringworms,Chandler syndromes,Charcot diseases,Charcot-Marie-Tooth atrophy,Figure thinks (family name) disease,Charcot-Marie-Tooth disease,Figure thinks (family name) disease,Charcot-Marie-Tooth disease mutation,Charcot-Marie-Tooth-Roussy-Levy diseases,CHARGE association,Charge syndromes,CHARGE syndromes,Chaund is ectodermal dysplasia,Chédiak-Higashi syndrome,CSH syndromes,Granulomatous cheilitis,Harelip,Chemke syndromes,Cheney syndromes,Cherry red spot and myoclonic syndrome,CHF,CHH,Chiari diseases,Chiari deformities I,Chiari deformities,ChiariI types (chiari deformities I),ChiariII types (chiari deformities II),ChiariI pattern synthesis is levied,Chiari-Budd syndromes,Chiari-Arnold syndrome,Chiari deformities II,Child syndrome,Children's fish scale ringworm,Child syndrome ichthyosis,Children's adrenoleukodystrophy,Children with dermatomyositis,Children's hair style myodystony,Children's cyclic vomiting,Children's giant axonal neuropathy,Childhood hypophosphatasia,Childhood muscular dystrophy,CHN,Cholestasia,Cholestasia genotype Norway type,Intrahepatic cholestasis,Neonate's cholestasia,Oral contraceptive user's cholestasia,Cholestasia and peripheral pulmonary artery stenosis,Cholestasis of pregnancy,Cholesterol desmolase deficiency,Chondrodysplasia punctata,Heng Naman (family name) syndrome,Fetal cartilage nutritional disorders,Chondrodystrophic myotonia,Chondrodystrophy,Chondrodystrophy and clubfoot,Epiphyseal cartilage dystrophia,Hyperplasia form chondrodystrophy,Chondroectodermal dysplasia,Achondroplasia,chondrohystrophia,Chondro-osteodystrophy,Choreoacanthocytosis,Chorionic villi sampling,Choroidal abnormalities,Choroidal abnormalities and ACC,Chorireninal defect-Joubert syndromes,Choroidal sclerosis,Choroideremia,Tip-many and zygodactyly syndrome,Christ-Siement-Touraine syndrome,Christ-Siement-Touraine syndrome,Christmas Day disease,Christmas disease,No. 3 chromosome deficiency distal end 3p,No. 3 distal chromosome 3p monosomy,No. 3 distal chromosome 3q2 overlap,No. 3 bodies of distal chromosome 3q2 tri-,No. 3 chromosome monosomy 3p2,Chromosome 3q partly overlaps syndrome,Chromosome 3q,Partial trisomy syndrome,No. 3 chromosome-trisomy 3q2,No. 4 chromosome 4q31-qter deletion syndromes,No. 4 chromosome 4q32-qter deletion syndromes,No. 4 chromosome 4q33-qter deletion syndromes,No. 4 chromosome long arm missings,No. 4 chromosome long arm missings,No. 4 chromosome monosomy 4q,No. 4 chromosome monosomy 4q,No. 4 chromosome monosomy distal side 4q,No. 4 partial deletion of chromosome 4p,No. 4 chromosomes,Local deletion of short arm,No. 4 chromosomal section monomer distal side 4q,No. 4 chromosomal section monomer 4p,No. 4 bodies 4 (q25-qter) of chromosomal section three,No. 4 bodies 4 (q26 or q27-qter) of chromosomal section three,No. 4 bodies 4 (q31 or 32-qter) of chromosomal section three,No. 4 body 4p of chromosomal section three,No. 4 chromosomal sections three body 4q2 and 4q3,No. 4 body of chromosomal section three distal ends 4,No. 4 rings,No. 4 chromosome 4q terminal deletion syndromes,Chromosome 4q syndromes,Chromosome 4q syndromes,No. 4 chromosome trisomies 4,No. 4 chromosome trisomy 4p,No. 4 chromosome x Y/47 XXY (Mosiac),No. 5 chromosome monosomy 5p,No. 5 chromosomes,Local deletion of short arm syndrome,No. 5 chromosome trisomy 5p,No. 5 chromosome trisomy 5p are all (5p11-pter),The 5p parts (5p13 or 14-pter) of No. 5 chromosome trisomies,Chromosome 5p syndromes,No. 6 body 6q of chromosomal section three,No. 6 rings,No. 6 chromosome trisomy 6q2,Monomer 7p2 on No. 7 chromosomes,No. 7 chromosomal section deletion of short arm (7p2-),No. 7 chromosome terminal 7p lack [del (7) (p21-p22)],No. 8 chromosome monosomy 8p2,No. 8 chromosome monosomy 8p21-pter,No. 8 partial deletion of chromosome (galianconism),No. 8 chromosomal section monomer 8p2,The complete three bodies 9P of No. 9 chromosomes,No. 9 partial deletion of chromosome galianconism,No. 9 chromosomal section monomer 9p,No. 9 chromosomal section monomer 9p22,No. 9 chromosomal section monomer 9p22-pter,No. 9 body 9P of chromosomal section three include,No. 9 rings,No. 9 body 9p of chromosome four,No. 9 body 9p of chromosome four inlay,No. 9 chromosome trisomy 9p (multiple mutation),No. 9 chromosome trisomies 9 (pter-p21 to q32) include,No. 9 chromosome trisomies are inlayed,No. 9 chromosome trisomies are inlayed,No. 10 body 10q of distal chromosome three,No. 10 chromosome monosomies,No. 10 chromosome monosomy 10p,No. 10 chromosomes,Excalation (galianconism),No. 10 chromosomes,10p- is local,No. 10 chromosomal section chromosome 10q24-qter,No. 10 chromosome trisomy 10q2,No. 11 chromosome long arm partial monosomies,No. 11 chromosomal section monomer 11q,No. 11 bodies of chromosomal section three,No. 11 body 11q13-qter of chromosomal section three,No. 11 body 11q21-qter of chromosomal section three,No. 11 body 11q23-qter of chromosomal section three,Chromosome 11q,Partial trisomy,No. 12 chromosome isochromosome 12p inlay,No. 13 chromosomal section monomer 13q,No. 13 chromosomes,Arm portion monomer,No. 14 rings,No. 14 chromosome trisomies,No. 15 body 15q of distal chromosome three,Chromosome r15,No. 15 rings,No. 15 chromosome trisomy 15q2,Chromosome 15q,Partly overlap syndrome,No. 17 chromosome deletion 17p,Grouchy-Royer-Salmon-Lumy syndrome,No. 18 chromosome monosomy 18p,No. 18 chromosome monosomy 18Q,No. 18 rings,No. 18 body 18p of chromosome four,Chromosome 18q syndromes,No. 21 syndromes of chromosomal mosaic 21,No. 21 rings,No. 21 syndromes of chromosome translocation 21,No. 22 chromosomes are overlapping (22pter-22q11),No. 22 bodies of chromosomal section three (22pter-22q11),No. 22 rings,No. 22 chromosome trisomies are inlayed,Chromosome 48xxyy,Chromosome 48xxxy,Chromosome r15,Chromosome trisomy,Chromosome trisomy,Trisome syndrome,X chromosomes,Chromosome x XY,Chronic acholuric jaundice,Chronic arachnoid adhesion,Addison disease,Carotid cavernous body is scorching,Congenital chronic aplastic anemia,Chronic dysphagocytosis,Familial chronic granuloma,Chronic familial jaundice,Chronic fatigue immune dysfunction syndrome (CFIDS),Chronic granulo matosis,Chronic guillain-Barre syndrome,Chronic idiopathic jaundice,Chronic special hair polyneuritis (CIP),Chronic inflammation demyelinating polyneuropathy,Chronic inflammation demyelinating neuropathy,Chronic motor tic,Chronic mucocutaneous candidiasis,Chronic multiple is twitched,Chronic nonspecific ulcerative colitis,Chmnic. obstructive's cholangitis,Chronic peptic ulcer and esophagitis syndrome,Chronic progressive chorea,Chronic progressive ballet's disease syndrome,Chronic progressive ballet's disease and myopathy,Chronic progressive ballet's disease and ragged red fiber,Chronic recurrent neuropathy,Chronic sarcoid,Chronic spasm,Children chronic is vomitted,CHS,Churg-Strauss syndrome,Cicatricial pemphigoid,CIP,The congenital pigment of hepatic sclerosis,Hepatic sclerosis,cistinuria,Citrullinemia,CJD,Typical Schindler diseases,The general syndrome of classic form,Classic form Pfeiffer syndromes,Classical haemophilia,Canonical form Cockayne syndrome I types (A types),Typical Leigh disease,Typical PKU,The typical chain Pei of x-plum Er Shi cerebrosclerosis,CLE,Harelip/cleft palate mucinous cyst lower lip PP finger-like and genitals are abnormal,Harelip-cleft palate blepharophimosis lagophthalmos and broadening,Harelip/cleft palate thumb deformity and microcephaly's deformity,The cleft palate contracture of joint-stretcher walking aid deformity,Cleft palate and harelip,Clavicular skull hypoplasia w/ micrognathias (disease),Lack thumb ＆ distal ends aphalangia (toe) deformity,Agenesis of clavicle,Clavicular skull hypoplasia,Click murmur syndrome,CLN1,Palmospasmus,Cloustons syndromes,Clubfoot,CMDI,CMM,CMT,CMTC,CMTX,COA syndromes,Aortic coaractation,Coats disease,Cobblestone dysplasia,Cochin Jewish diseases,Cockayne syndrome,COD-MD syndromes,COD,Coffin Lowry syndromes,Coffin syndromes,Coffin Siris syndromes,COFS syndromes,Cogan corneal dystrophies,Cogan reese's syndromes,Cohen syndrome,Cold coagulation disease,Antibody disease,Cold antibody hemolytic anemia,Ulcerative colitis,Ulcerative colitis,Ulcerative colitis chronic nonspecific ulcerative colitis,Collodion baby,Defect heart defect locking nostril retarding of growing development urogenital system exception and abnormal ear,Defect,Colon neurosis,Colour blindness,Colour blindness,Colpocephaly,Columnar samples oesophagus,With reference to cone rod cell denaturation,Combined immunodeficiency and immunoglobulin,Joint mesoectodermal defect,Common variable hypogammaglobulinemia,Common variable immunodeficiency,Common ventricle,Communicating hydrocephalus,Complete lack of hypoxanthine-guanine phosphoribosyl transferase,Treatment of Complete Atrioventricular Defect,The inhibitor of complement component 1 lacks,Complement component c1 modifying ingredients is lacked,Complete cardiac conduction block,Complex carbohydrate is not tolerated,Complicated regional pain syndrome,Complicated VATP synthase missing,Composite I,Composite I nadh dehydrogenase defect,Complex II,Complex II succinate dehydrogenase,Succinate dehydrogenase deficiency disease,Complex II I,Complex II I Co-Q10 cytochrome c oxidoreductase defects,Complex IV,Complex IV cytochrome C oxidase defect,Complex IV is lacked,Compound V,Cerebral concussion,Bore rod cell denaturation,Bore rod cell denaturation progress,Cone dystrophy,Bore rod cell malnutritive,Converge reticulated papillomatosis,Congenital low pk dynamics,It is congenital without abdominal muscles,Congenital athymia and parathyroid gland,Albinism,Congenital Addison's disease,Adrenal,congenital hyperplasia,Adrenal,congenital hyperplasia,Congenital afibrinogenemia,Congenital alveolar hypoventilation,Neonatal Congenital anaemia,Congenital bilateral persylvian syndromes,Congenital brown's syndrome,Congenital heart defect,The low hypopnea syndrome of congenital central,Congenital cerebral palsy,Congenital cervical vertebra bony union,The tight simultaneously MR of Congenital Thumb,CCA (toe),Congenital multiple contracture and arachnodactyly,Congenital cyanosis,Congenital craniofacial and scalp defect,Stones in intrahepatic bile duct congenital dilatation,Congenital dysmyelination,Congenital dysphagocytosis,Congenital sexual abnormality blood vessel dilatation,CEP,Congenital factor XIII deficiency diseases,It is congenital can not autonomous control breathing,Nonhemolytic jaundice,congenital familial I types,Congenital family's delayed diarrhea,Congenital form Cockayne syndrome II types (Type B),Congenital generalized fibromatosis,Congenital German measles,Congenital giant axonal neuropathy,CHB,Congenital heart defect,Congenital Hemidysplasia and fish scale sample erythroderma and physical impairment,Acholuric familial jaundice,Congenital hemolytic anemia,Congenital hepatic fibrosis,Congenital hereditary corneal degeneration,Congenital hereditary lymphedema,Congenital hyperchondroplasia,Congenital hypomyelinating DPNs,Congenital hypomyelination neuropathy,Congenital hypomyelination,Congenital hypomyelination (Onion Bulb) DPN,Congenital ichthyosis sample erythroderma,Congenital keratoconus,Congenital hyperlactacidemia,Congenital lactose intolerance,Congenital lipodystrophia,Congenital cirrhosis,CLE,Congenital localized emphysema,Congenital macroglossia (disease),Congenital marrow is narrow,Congenital megacolon,Congenital mole,Congenital mesoderm dysmorphodystrophy,Congenital mesoderm is malnutritive,Congenital microvillus atrophy,Congenital multiple joint,Congenital muscular dystrophy,Congenital neuropathy caused by hypomyelination,Congenital whole blood trace elements,Congenital pernicious anemia,Congenital pernicious anemia is due to lacking intrinsic factor,Congenital pernicious anemia is due to lacking intrinsic factor,Congenital pigmentation,Congenital porphyria,Congenital peri position myopathy and desmin storage myopathy,Congenital emphysema,Congenital pure red cell anaemia,Congenital dyserythropoietic anemia,Congenital retinal is blinded,Congenital cyst of retina,Congenital retinitis pigmentosa,Congenital retinal is cleaved,Congenital Rod diseases,Congenital rubella syndrome (CRS),Congenital scalp defects and distal limb defect,Congenital sensory neuropathy,Congenital SMA and arthrogryposis,Congenital congenital hemolytic jaundice,Pediatric congenital myogenic torticollis,Congenital bolt neck marrow syndrome,Congenital tyrosinosis,Congenital varicella syndrome,Congenital cavernous malformations,Congenital vascular covers retina,Congenital word blindness,Congenital splenectopia (paediatrics),Congestive cardiomyopathy,Keratoconus,Conjugated hyperbilirubinemia,Conjunctivitis,Conjunctivitis Ligneous,Conjunctivo-urethro-synovial syndrome,Conn syndromes,CTD,Conradi disease,Conradihunermann syndromes,Systemic aplastic anemia,Constitutional red blood cell development is not complete,Constitutional eczema,Constitutional dyshepatia,Constitutional thrombopathy blood platelet,Restrict congenital rank,Constrictive pericarditis and nanism,Continuous muscle fibre activity syndrome,Contracted arachodactylia (toe) syndrome,The double-legged muscular atrophy contracture and utilization of dynamic eye (motion) can not,Chi Zong,Cooley anaemias,Copper transports disease,Fecal porphyria,Cor triatriatum,Cor triatriatum Sinistrum,Cor triloculare biatriatum,Cor biloculare,Cori disease,Corneal dystrophy,Cornea amyloidosis,The opacity of the cornea-skin Laxa- MRs,Corneal dystrophy,Cornelia de Lange syndromes,Corona hypoplasia of dentin,Coronary heart disease,Coronary heart disease,Agenesis of corpus callus,Corticobasal ganglionic is denatured,Crust deformity,Corticobasal ganglionic is denatured (CBGD),Corticobasal is denatured,Cortex methloxidase lacks I types,Cortex methyloxidase lacks II types,Cortisol,Costello syndromes,SIDS sudden infant death syndrome,COVESDEM syndromes,COX,COX is lacked,The Canadian type of COX defects France,COX defect babies mitochondrial myopathy includes deToni-Fanconi-Debre,The benign infant's mitochondrial myopathy of COX deficiencies,CP,CPEO,CPEO and myopathy,CPEO and ragged red fiber,CPPD familial forms,CPT defects,CPTD,Cranial arteritis,The meningoencephalocele of cranium,Cranio-Oro-Digital syndromes,Cranio-carpo-tarsal dystrophy,Naoning tablet,Cranium syndrome MR Scott types,Craniofacial dysostosis,Craniofacial dysostosis-PD arteries-hirsutism-atelocheilia,Craniofrontonasal dysplasia,Craniometaphyseal dysplasia,Cranioorodigital syndromes,Cranioorodigital syndrome i I types,Craniostenosis crouzon types,Craniostenosis,Craniosynostosis nasal atresia radius humerus bony union,Craniosynostosis hirsutism face and other exceptions,Craniosynostosis mid facial hypoplasia and foot deformity,Craniosynostosis is primary,Craniosynostosis-radial aplasia syndrome,Craniosynostosis and radial segmental defect,Cranioschisis,CREST syndromes,Creutzfeldt-Jakob disease,Cat's cry syndrome,Cot death,Crigler-Najjar syndrome I types,Clone disease,Cronkhite-Canada syndrome,Cross syndromes,Cross syndromes,Cross-McKusick-Breen syndrome,Crouzon,Crouzon syndromes,Crouzon craniofacial dysostosis,Cryoglobulinemia primary is mixed,Cryptophthalmus-syndactyly syndrome,Cryptorchidism-dwarf-feeblemindedness,Shi Naide (family name) crystalloid corneal dystrophy,CS,CSD,CSID,CSO,CST syndromes,Hair crimping ankyloblepharon nail dysplasia,Curschmann-Battern-Steinert,Curth Macklin type fish scales hystric,CurthMacklin types,Cushing’s,Cushing's syndrome,Cushing’s III,The heredity of malignant melanoma of skin,Cutaneous porphyria,Cutis laxa,Cutis laxa-growth deletion syndrome,Cutis marmorata telangiectatica congenita,CVI,CVID,CVS,Cyclical vomiting syndrome,Kidney medulla disease,Cystic hygroma,Cystic fibrosis,Cystic lymphangioma,Cystine-Lys-Arg ornithinuria,Cystinosis,Abderhalden-Kaufmann-Lignac syndrome,Cystinuria,Cystinuria and dibasic aminoaciduria,Cystinuria I types,Cystinuria II types,Cystinuria type III,Congenital kidney medulla tumour,Cytochrome c oxidase defect,D.C.,dacryosialoadenopathy,dacryosialoadenopathia,dalpro,Dalton,Colour blindness,Danbolt-Cross syndromes,Twitching of the eyelid is stamped one's foot syndrome,Dandy-Walker syndrome,Dan Di-Wo Ke tumours,Dan Di-Wo Ke deformities,Dan Di-Wo Ke deformities,Danish heart-type amyloidosis (type III),Darier disease,Davidson diseases,Davies diseases,DBA,DBS,DC,DD,De Barsy syndromes,De Barsy-Moens-Diercks syndromes,De Lange syndrome,De Morsier syndromes,De Santis Cacchione syndromes,De Toni Fanconi syndrome,Congenital deafness and functional cardiac disease,Deafness-dwarf-neurodeatrophia,Deafness-functional cardiac disease,Deafness refers to (toe) Onychodystrophy osteodystrophy and baryencephalia,Deaf and curly hair Bjornstad types,Nerve deafness and hedratresia and thumb hypoplasia,Debrancher deficiency,Deciduous skin,Enterocyte intrinsic factor receptor lacks,Constant killer cell lacks,Carnitine kidney reabsorption defect,Glycoprotein neuraminidase lacks,Mitochondrial respiratory chain complex IV defect,Lack platelet membrane glycoprotein ib,Lack von Willebrand factor acceptor,Short chain acyl coenzyme dehydrogenase deficiency (ACADS),Mesomelic dwarfism deformity,Degeneration chorea,Treatment of Degenerative Lumbar Spinal Canal Stenosis,Degos' disease,Degos-Kohlmeier diseases,Netted pigmentation disease,DEH,Dejerine-Roussay syndrome,Dejerine-Sottas disease,9p minus syndrome is local,11q deletion syndromes are local,13q minus syndrome is local,Delleman-oorthuys syndromes,Delleman syndromes,Dull-witted and lobe of the lung atrophy and Neuronal cytoplasmic inclusions,Demyelinating disease,DeMyer syndromes,Hypoplasia of dentin corona,Hypoplasia of dentin root of the tooth,Hypoplasia of dentin I types,Hypoplasia of dentin II types,Hypoplasia of dentin brandywine types,Hypoplasia of dentin shielded type,Hypoplasia of dentin type III,Tooth-eye-dysosteogenesis,Dentooculocutaneous syndromes,Denys-Drash syndromes,Depakene,DepakeneTM exposes,Divalproex sodium,Depakote Sprinkle,Discoloration-gingival fibroma-microphthalmia,Dercum disease,Atopic dermatitis,Exfoliative dermatitis,Dermatitis herpetiformis,Multiformity dermatitis,General hair property Dermatochalasia,General hair property Dermatochalasia,Dermectasia,Dermatomyositis sine myositiss,Dermatomyositis,Dermatosparaxis,Dermatostomatitis StevensJohnson types,Desbuquois overstates syndrome,Desmin stores myopathy,Neonate peels,Deuteranomalia,Developmental reading dyslexia,Develop Gerstmann syndrome,De Veulle Ji disease,Devic's disease,Neuromyelitis optica,Dextrocardia-bronchiectasis and nasosinusitis,Dextrocardia and visceral reversal,DGS,DGSX Golabi-Rosen syndromes include,DH,DHAP alkyl-transferases are lacked,DHBS is lacked,DHOF,DHPR is lacked,Diabetes insipidus,Diabetes insipidus diabetes optic atrophy and deafness,Diabetes insipidus is basophilic adenoma of pituitary,Diabetes insulin dependent form,Diabetes,Diabetes Addison's disease oedema,Diabetic ketoacidosis,Diabetic bearded woman syndrome,Diabetic neuropathy,Diamond-Blackfan anemia,Diaphram apnea,Diaphysial aclasis,Diastrophic dwarfism,Diastrophic dysplasia,Diastrophic nanism syndrome,Dicarboxylic aminoaciduria,The dicarboxylic aciduria that aliphatic acid beta oxidation defect is caused,The dicarboxylic aciduria that aliphatic acid beta oxidation defect is caused,The dicarboxylic aciduria that MCADH defects are caused,Dichromasia,Dicker-Opitz,DIDMOAD,Diencephalic syndrome,Children's diencephalic syndrome,Become thin diencephalic syndrome,Dienoyl- CoA reductase defects,Baby diffusivity cerebral degeneration,Diffusivity brain degenerative disease,Diffusivity idiopathic osteoproliferation,Diffusum-Glycopeptiduria,DiGeorge syndromes,Digital-Oro-Cranio syndromes,Digito-Oto-Palatal syndromes,Digito-Oto-Palatal syndrome I types,Digito-Oto-Palatal syndrome II types,Dihydrobiopterin synthetase deficiency,Dihydropteridine reductase deficiency,Dihydroxyacetone phosphate synthase,Expanding (hyperemia) cardiomyopathy,Dimitri disease,The diplegia of cerebral paralysis,Diplo-Y syndromes,Disaccharidase is lacked,Disaccharide intolerance I,Lupus erythematosus discoides,Lupus erythematosus discoides,DISH,Keratosis,Keratosis I types,Keratosis 4,Keratosis 6,Keratosis 8,Keratosis 9Netherton types,The phytane acid type of keratosis 11,Keratosis 12 (neutral fats memory type),Keratosis 13,Keratosis 14,The hair sulphur dystrophia type of keratosis 14,Keratosis 15 (keratitis deafness type),Keratosis 16,The form variation crythrokeratodermia type of keratosis 18,Keratosis 19,Keratosis 20,Keratosis 24,Spleen is shifted,Lupus erythematosus disseminatus,Disseminated neurodermatitis,Multiple sclerosis,Distal end 11q monomers,Distal end 11q syndromes,Distal end AMC IIA types,Distal end AMC IIA types,Far end arthrosis bend IIA types,Far end arthrosis bend 2A types,Distal end repeats 6q,Distally overlapping 10q,Repeat (10q) syndrome,Distally overlapping 15q,Distal end monomer 9p,Distally three body 6q,Distally three body 10q syndromes,Distally three body 11q,Divalproex sodium,DJS,DKC,DLE,DLPIII,DM,DMC syndromes,DMC diseases,DMD,Hereditary DNS,DOCI,DOC2,DOC4,DOC 6 (Harlequin types),DOC 8Curth-Macklin types,The phytane acid types of DOC 11,DOC 12 (neutral fats memory type),DOC 13,DOC 14,The hair sulphur dystrophia types of DOC 14,DOC 15 (keratitis deafness type),DOC 16,The UnilateralHemidysplasia types of DOC 16,DOC 18,DOC 19,DOC 20,DOC 24,Sinus Le Shi bodies-myelopathy,Dolichospondylic dysplasia,Dolichostenomelia (toe),Dolichostenomelia (toe) syndrome,Phenotype Kenny-Caffe syndromes,Phenotype myotonia congenita,Donahue syndromes,Donath-Landsteiner hemolytic anemias,Donath-Landsteiner syndrome,It is deaf-to refer to (toe) first hypoplasia-dysosteogenesis-MR syndrome,It is deaf-to refer to (toe) first hypoplasia-dysosteogenesis-MR syndrome,DRD,DorfmanChanarin syndromes,Dowling-Meara syndromes,Down's syndrome,DR syndromes,Drash syndromes,DRD,Dreifuss-Emery types muscular dystrophy and contracture,Postmyocardial infarction syndrome,Drift about spleen,Drug-induced acanthosis nigricans,Drug-induced lupus erythematosus,Medicine correlation adrenal insufficiency,Drummond syndromes,Dry type athlete's foot,Xerophthalmia,DTD,Duane retraction syndromes,Duane's syndrome,Duane's syndrome IA 1B and 1C types,Duane's syndrome 2A 2B and 2C types,Duane's syndrome 3A 3B and 3C types,1867 johnsen syndromes,Dubin-Johnson syndrome,Dubowitz syndrome,Progressive Erb's atrophy,Duchenne paralyses,Duhring diseases,Duncan diseases,Duncan diseases,Duodenal atresia,Duodenal stenosis,Duodenitis,Overlapping 4p syndromes,Repeat 6q local,Dupuy syndromes,Dupuytren contractures,Dutch-Kennedy syndromes,Nanism,Nanism campomelic,Nanism tubular bone cortex is thickened and instantaneous low calcium,Nanism Levi types,Tropism between nanism,Nanism-onychodysplasia,Nanism-pericarditis,Nanism and nephrarctia and deafness,Nanism and rickets,DWM,Dyggve MelchiorClausen syndromes,Dysautonomia,Familial dysbetalipoproteinemia,Dyschondrodysplasia and hemangioma,Dyschondrosteosis,Dyschromatosis universalis hereditaria,Dysencephalia splanchnocystica,Congenital dyskeratosis,Congenital dyskeratosis autosomal recessive inheritance,Congenital dyskeratosis Scoggins type,Congenital dyskeratosis syndrome,Follicular dyskeratosis Vegetans,Dislexia,Dysmyelogenic leukodystrophy,Dysmyelogenic leukodystrophy-megalobare,Dysphonia spastica,Heng Naman (family name) syndrome,Epiphysis osteodysplasty Hemimelica,Nail dysplasia and metodontiasis,Clavicle atelocephaly,Fibre Development is abnormal,Dysplasia giant's syndrome X is chain,Osteodentin dysplasia,Dysplastic nevi syndrome,Dysplastic nevus type,Dyssynergia cerebellaris myoclonica,Oesophagus dyssynergia,Myodystony,Dystopia canthi,Adiposogenital dystrophy,Malnutritive endothelitis cornea,Malnutritive mesodermalis,Malnutrition bullous epidermolysis,It is malnutritive,Asphyxiating thorax,Myotonia dystrophy,E-D syndromes,Eagle-Barrett syndromes,Retina Eales,Periphlebitis of retina,Ear exception-contracture-osteodysplasty and scoliokyphosis,The short and small syndrome of ear kneecap,Early stage about harness defects,Early stage hypercalcemia syndrome and Elfin Facie,Early onset myodystony,Eaton Lambert syndromes,EB,Ebstein is abnormal,EBV neurological susceptibilities (EBVS),EBVS,ECD,ECPSG,It is ectodermal dysplasia,Ectodermal dysplasia and cleft lip and cleft palate,Ectodermal dysplasia-exocrine pancreas insufficiency,Ectodermal dysplasia Rapp-Hodgkin types,Ectoderm and mesoderm depauperation are congenital,Ectoderm and mesoderm depauperation are participated in bone,Ectodermosis erosiva pluriorificialis,Ectopia lentis,Ectopia vesicae,Ectopic ACTH syndrome,Ectopic adrenocorticotropic hormone syndrome,Hedratresia,Hand ectrodactyly,Adactylism (deformity),Adactylism (toe)-ectodermal dysplasia-cleft lip and palate syndrome,Adactylism (toe)-ectodermal dysplasia-cleft lip and palate syndrome,Adactylism (toe)-ectodermal dysplasia-harelip,Eczema,Eczema-decrease of platelet acquired immunodeficiency syndrome,EDA,EDMD,EDS,EDS artery ecchymosis types,EDS arthrochalasis,EDS typical case's severe forms,EDSdysfibronectinemic,EDS is heavy,EDS hyperkinesias,EDS scoliokyphosis,EDS scoliokyphosis,EDS mitigation types,EDS eyeball scoliosis,EDS Progeroid,EDS periodontosis,EDS blood vessels,EEC syndromes,EFE,EHBA,EHK,Hlers-Danlos syndrome,Hlers-Danlos syndrome,Ai Lesi-as Lip river IX,Eisenmenger complex,Eisenmenger complex,Ai Senmangeer diseases,Ai Senmangeer reacts,Eisenmenger's syndrome,Eisenmenger's syndrome,Ekman-lobstein diseases,Hand ektrodactyly,EKV,Elastosis,Extensive elastic fibrous tissue (fiber) rupture,Elastosis dystrophica's syndrome,Elective mutism (obsolete),Elective mutism,Electrocardiogram (ECG or EKG),Electron transfer flavoprotein (ETF) dehydrogenase deficiency：(GAII & MADD),Electrophysiologic study (EPS),Birth is as nail,Elephantiasis congenital angiomatosis,Distensibility of blood vessel is loose,Elfin facies and hypercalcinemia,Ellis-van Creveld syndrome,Ellis-van Creveld syndrome,Embryoma kidney,Embryonal adenomyosarcoma kidney,Embryo's renal cancer kidney,Embryo's mixed rumour kidney,EMC,Emery Dreyfus muscular dystrophies,Ecuador's syndrome is malnourished,Diamond dust-dreifuss pattern synthesis is levied,EMF,EMG syndromes,Empty sella syndrome,Encephalitis periaxialis,Encephalitis periaxialis concentrica,Naoning tablet,Cranium face angiomatosis,Encephalopathic,Encephalotrigeminal angiomatosis,The multiple cvernous hemangioma of chondrodysplasia,Endemic multiple neuritis,Endocardial cushions defect,Endocardial cushions defect,Endometrial hyperplasia,Endocardial fibroelastosis (EFE),Endogenous hypertriglyceridemia,Endolymphatic hydrops,Endometrial growth,Endometriosis,Myocardium internal membrane of heart fibrosis,The congenital malnutrition of corneal endothelium,Esoderma cornea epithelium is malnutritive,Endothelium,Engelmann disease,Macroglossia,Enterocolitis,Enterocyte vitamin B12 malabsorption,Eosinophia syndromes,Acidophilia cellulitis,Eosinophilic fasciitis,Eosinophilic granuloma,Eosinophil syndrome,Epidermal nevus syndrome,Epidermolysis bullosa,Acquired bleb epidermidolysis,Epidermolysis bullosa hereditaria,Epidermolysis bullosa is lethal,Epidermidolysis Hereditaria Tarda,Epidermolytic hyperkeratosis (disease),Epidermolytic hyperkeratosis (disease) (bleb CIE),Cursive epilepsy,Epilepsy,Adrenaline,Epiphysis changes and high myopia,Epiphyseal cartilage knurl is benign,Epiphysealis Hemimelica dysplasia,Accidental abnormal eye movement,Basement membrane of epithelium corneal dystrophy,Meesmann teenager's dystrophia epithelialis corneae,Multiple epitheliomatosis is with mole,Epithelium,Epival,EPS,Male's Epstein-Barr virus induction lymphoproliferative diseases,Eaton-Lambert myasthenic syndrome,Erdheim Chesters disease,Erythema multiforme oozes out,Multiform (property) erythema Stevens Johnson types,erythroblastophthisis,Fetal red blood cells,Neonate's erythremia,Children's erythroblastosis anaemia,Red blood cell phosphoglycerate kinase deficiency,Infull property red blood cell occurs,Progressive erythrokeratodermia is symmetrical,The symmetrical ichthyosis of progressive erythrokeratodermia,Erythrokeratodermia variabilis,Erythrokeratodermia variabilis type,Erythrokeratolysis hiemalis,Erythropoietic porphyria,Erythropoietic porphyria,Ai Sikewaer syndromes,Atresia oesophageal,Esophageal peristalsis stops,Esophagitis peptic ulcer,Atresia oesophageal and/or tracheoesophageal fistula,Familial hyperlipemia,essential,Essential fructosuria,Essential hematuria,Essential hemorrhagic thrombocythemia,Essential mixed cryoglobulinemia,Primary moschowitz diseases,Essential thrombocythemia,Essential thrombocytopenia is reduced,Piastrenemia,Essential tremor,Esterase inhibitor defect,Fanconi anemia Estren-Dameshek mutation,The cholestasia relevant with estrogen,ET,ETF,ethylmalonic adipicaciduria,Eulenburg disease,pc,EVCS,Exaggerate startle reaction,Exencephalia (deformity),Exogenous hypertriglyceridemia,Umbilical hernia-macroglossia-giant's syndrome,Anemic goiter,Expanded rubella syndrome,Ectopia vesicae,EXT,External osteochondromatosis syndrome,Extrahepatic biliary atresia,Extramedullary plasmacytoma,Exudative retinitis,Eyes retreat syndrome,FA1,FAA,Fabry disease,FAC,FACB,FACD,FACE,FACF,FACG,FACH,Facioplegia,Facioplegia,Face is ectodermal dysplasia,Face is ectodermal dysplasia,Facio-scapulo-humeral dystrophy,Facio-auriculo-vertebral spectrum,Face-the heart-skin syndrome,Face-volume-nose hypoplasia,Faciocutaneoskeletal syndromes,Face-refer to (toe)-genital disease,Faciogenital dysplasia,Faciogenitopopliteal syndromes,Faciopalatoosseous syndromes,Faciopalatoosseous syndrome i I types,Facioscapulohumeral muscular dystrophy,Artificial hypoglycemia,Factor VIII deficiency,Factors IX deficiency disease,Factor XI deficiency disease,Factor XII deficiency,FXIII defect,Fahr disease,Fahr disease,Stomachial secretion gastric anti-pernicious anemia factor exhaustion,Fairbank diseases,Fallot tetra logys,Family is acrogeria,Family's akromikrie,Familial Adenomatous polyp of colon,Familial adenomatosis polyposis and parenteral performance,Familial alobar holoprosencephaly,Familial alpha-lipoprotein deficiency disease,Familial amyotrophic chorea and acanthocytosis,Familial myoclonia arrhythmia cordis,Familial articular cartilage calcification,Familial atypical mole-malignant mela noma syndrome,Familial broad beta disease,Family's calcium gout,Family's calcium pyrophosphate arthropathy,Familial chronic obstructive pulmonary disease,Family persistently peels,Familial amyloidosis cutis,Familial paraproteinemia,Familial emphysema,Familial enteropathy microvillus,Familial central fixation retinoschisis,Family's hibernation syndrome,Familial high cholesterol,Familial hemochromatosis,Familial hypercholesterolemia,Familial high-density lipoprotein deficiency,Familial high anteserum cholesterol,Family's hyperlipidemia,Family's hypoproteinemia and angioleucitis enteropathy,Icterus gpavis familiaris,The juvenile eye with nephronophtisis of familial is abnormal,Familial lichen amyloidosis (IX types),Familial lumbar spinal stenosis,Familial primary lymphedema,Familial Mediterranean fever,Multiple familial polyposis,Familial neck blister,Familial paroxysmal polyserositis,Familial colon polyp,Familial primary pulmonary hypertension,Familial renal glucosuria,Familial splenic anemia,The astonished disease of familial,Familial visceral amyloidosis (VIII types),FAMMM,FANCA,FANCB,FANCC,FANCD,FANCE,Fanconi panmyelopathys,Fanconi pancytopenias,FanconIII,Fanconi anaemias,Fanconi anaemia I types,Fanconi anaemia complementation groups,Fanconi anaemia complementation groups A,Fanconi anaemia complementation groups B,Fanconi anaemia complementation groups C,Fanconi anaemia complementation groups D,Fanconi anaemia complementation groups E,Fanconi anaemia complementation groups G,Fanconi anaemia complementation groups H,Fanconi anaemia Estren-Dameshek mutation,FANF,FANG,FANH,FAP,FAPG,Farber diseases,Farber lipogranulomas,FAS,Fasting hypoglycemia,Hyperlipidemia caused by fatty,The fatal granulomatosis of children,Fat oxidation obstacle,Fatty liver and encephalopathic,FAV,FCH,FCMD,FCS syndromes,FD,FDH,Febrile mucocutaneous mucous membrane syndrome Stevens Johnson types,Heat generation neutrality acute dermatological,Febrile seizure,Feinberg syndromes,Model [Singh]-Le [Roy] two Cotard,Women puppet Turner's synodrome,The bilateral Robin of femoral hypoplasia is abnormal,Femoral hypoplasia is bilateral,Distal femoral surface syndrome,Femoral hypoplasia-unusual facies syndrome,Fetal alcohol syndrome,Fetus anticonvulsant disease,Fetus cystic hygroma,Alcohol fetus effect,Varicella fetus effect,Reaction stops fetus effect,Varicella virus fetus effect,Myocardium internal membrane of heart fibrosis,Fetus face syndrome,Fetal iritis syndrome,Placental transfusion syndrome,Fetal valproate syndrome,Fetus valproic acid exposes syndrome,Fetal infection varicella,Fetus varicella zoster syndrome,FFDD II types,FG syndromes,FGDY,FHS,Fibrin stabilizing factor deficiency,Fibrinase is lacked,Star (colloid) cell cellulose is denatured,Class (blood) fibrin leukodystrophy,Fibrinoligase is lacked,Desmocytoma,(ductus pancreaticus) mucoviscidosis,(drawing) progressive fibrodysplasia ossificans,Fibrous elasticity tissue endocarditis,Fibromyalgia,Fibromyalgia-fibromyositis,Fibromyositis,Fibrosis cholangitis,Fibrositis,Multi-joint fibrous ankylosis,Fibrous cavernitis,Fibrous dysplasia,Penis fibrous plaque,Penis fibrosclerosis,Fickler-Winkler types,Fiedler's disease,Fifth Digit syndromes,Filippi syndromes,Finnish type amyloidosis (V-type),First order CHB,First and second branchial arch syndromes,Fischer syndromes,Fishiness syndrome,Crack tongue,Adenoma syndrome,Flatau-Schilder disease,Flavin containing monooxygenase 2,Floating beta disease,Floating-Harbor syndromes,Splenectopia,Floppy infant syndrome,Floppy valve syndrome,Fluent aphasia,FMD,FMF,FMO Adult LiverForm,FMO2,FND,Focal cerebral ischemia,Focal dermal hypoplasia syndrome,Focal dermal hypoplasia,Focal skin phalanges hypoplasia,Focal dystonia,Focal seizure,The infull II types of focal dermal facial development,Focal neuromyotonia,FODH,Folling syndrome,Fong diseases,FOP,Glycogen storage disease type III,Forbes-Albright syndrome,Forestier disease,Aland's disease (X is chain),Fothergill disease,Fountain syndromes,Progressivity macular dystrophy,FPO syndrome i I types,FPO,Fraccaro types achondrogenesis (IB types),Fragile X syndrome,Franceschetti-Zwalen-Klein syndromes,Francois dyscephaly syndromes,Francois-Neetens spots are malnutritive,Mottled corneal nutrition is not,Fraser syndrome,FRAXA,FRDA,Fredrickson type I hyperlipoprotememias,Cranio-carpo-tarsal dysplasia,Freire-Maia syndromes,Not thunder Cotard,Friedreich incoordination,Friedreich diseases,Friedreich is weak,FRNS,Froelich syndromes,Frommel-Chiari syndrome,Frommel-Chiari syndrome lactation atrophy of uterus,Volume refers to (toe) syndrome,Frontofacionasal hypoplasia,Frontofacionasal dysplasia,Volume nose dysplasia,Volume nose dysplasia and coronal craniosynostosis,Fructose-1-phosphate aldolase deficiency,Fructosemia,Fructosuria,Fryns syndrome,FSH,FSHD,FSS,Fuchs' dystrophy,Sick 1 type of mycoside (storing up),Sick 2 type of mycoside (storing up),Sick 3 type of mycoside (storing up),Fukuhara syndrome,Fukuyama diseases,Fukuyama type muscular dystrophy,Fumarylacetoacetase is lacked,Ditch tongue,G syndromes,G6PD defects,G6PD,GA I,GA IIB,GA IIA,GA II,GAII & MADD,Overflow breast-amenorrhoea syndrome nonpuerperal,Breast-amenorrhoea overflow without pregnancy,Amine-galactose 6-sulfatase is lacked,Galactose-1-phosphate uridyl transferase is lacked,Galactosemia (disease),GALB is lacked,Galloway-Mowat syndromes,Galloway syndromes,GALT is lacked,Agammaglobulinaemia,GAN,Gangliosides nerve ammonia (sugar) neuraminidase defect,Ganglioside sialidase deficiency,The types of gangliosidosis GM1 1,The types of gangliosidosis GM1 2,Gangliosidosis β hexosaminidases B lacks,Gardner syndromes,Gargoylism,Garies-Mason syndromes,Gasser syndrome,Gastric anti-pernicious anemia factor fails secretion,Enterocyte vitamin B12,Gastrinoma,Gastritis,Stomach oesophagus lacerated wound bleeding,Pipe intestinal polyp and ectoderm change,Gastroduodenal ulcer,Abdomen splits (deformity),Gaucher disease,Gaucher-Schlagenhaufer,Gayet-Wernicke syndromes,GBS,GCA,GCM syndromes,GCPS,Gee-Herter diseases,Gee-Thaysen disease,Gehrig diseases,Southern net Cotard,Genee-Wiedemann syndromes,Systemic myodystony,General race's neuromyotonia of building up a family fortune,General hair fibromatosis,Generalized flexion epilepsy,Generalized glycogenosis,Generalized hyperhidrosis,General hair lipofuscinosis,General hair myasthenia gravis,General hair myotonia,It is systemic to distribute neuromyxoma,Genetic disease,Genitals defect,Reproductive system and urinary system defect,Gerstmann syndrome,Gerstmann Tetrad,GHBP,GHD,GHR,Giant axon disease,Giant axonal neuropathy,Huge benign lymphoma,Giant cell glioblastoma,Giant cell arteritis,Giant cell lesion liver,Giant cell hepatitis,Giant cell neonate's hepatic sclerosis,Retina giant cyst,Castleman disease,Bernard-Soulier syndrome heredity,Macroglossia,Gic macular dystrophies,Gilbert diseases,Gilbert syndrome,Gilbert-Dreyfus syndrome,Gilbert-Lereboullet syndromes,Gilford syndromes,Gilles de la Tourette syndromes,Gillespie syndromes,Gingival fibromatosis-exception finger nail nose ear splenomegaly,GLA lacks,GLA,GLB1,Glaucoma,Retinal glioma,Total aphasia,Globoid leukodystrophy,Glossoptosis micrognathia (disease) and cleft palate,Glucocerebrosidase is lacked,Glucocerebrosidosis,Glucose 6 phosphate dehydrogenase deficiency,G6P trafficking defect,G-6-P salt transfer missing,Glucose G phosphoric acid enzyme deficiency diseases,Glucose-galactose malabsorption,Ceramide lipidosis [disease],Glutaric aciduria I,Glutaric acidemia I,Glutaric acidemia II,Glutaric aciduria II,Glutaric aciduria type II,Glutaric aciduria type III,Glutaric acidemia I,Glutaric acidemia II,Glutaric aciduria I,Glutaric aciduria II,Glutaric aciduria IIA types,Glutaric aciduria IIB types,Glutaryl-CoA dehydrogenase deficiency disease,Glutaurate- aspartic acid trafficking defects,Glutelin sensitive enteropathy,Muscle glycogen disease type VII,Glycogenic thesaurismosis I,Glycogenic thesaurismosis III,Glycogenic thesaurismosis IV,Glycogenic thesaurismosis V-shaped,Glycogenic thesaurismosis VI,Glycogenic thesaurismosis VII,Glycogenic thesaurismosis VIII,Glycogenic thesaurismosis II types,Glycogenic thesaurismosis type II,Glycogenosis,Glycogenosis I types,Glycogenosis IA types,Glycogenosis IB types,Glycogenosis II types,Glycogenosis II types,Glycogenosis type III,Glycogenosis type IV,Glycogenosis V-shaped,Glycogenosis VI types,Glycogenosis type VII,Glycogenosis type VIII,Glycolic aciduria,Glycolipid lipidosis,The type of GM2 gangliosidosis 1,GNPTA,Goitrous autoimmune thyroiditises,Ge Erdengha (family name) syndrome,Goldenhar-Gorlin syndromes,Goldscheider diseases,Ge Erci syndromes,Goltz-Gorlin syndromes,Gonadal agenesis 45X,Gonadal agenesis X0,Goniodysgenesis-metodontiasis,Goodman syndromes,Goodman,Goodpasture's syndrome,Gordon's protein-losing enteropathy,Gorlin syndromes,Gorlin-Chaudhry-Moss syndrome,The congenital erythrokeratodermia of gottron progressive symmetry,Gottron Cotards,Gougerot-Carteaud syndrome,Grand mal,Granular pattern corneal dystrophy,Granulomatous arteritis,Granulomatous colitis,Granulomatous dermatitis and eosinophilia,Granulomatous ileitis,Graves disease,Robert Graves hyperthyroidism,Graves disease,Greig short rib-polydactyly syndromes,Groenouw I type corneal dystrophies,Groenouw II type corneal dystrophies,Graefe-Sjogren syndrome,Grotton syndromes,Growth hormone receptor lacks,Growth hormone binding protein is lacked,Growth hormone deficiency,Myhre Growth-Mental deletion syndromes,Hypoevolutism-Rieger is abnormal,GRS,Gruber syndrome,GS,GSD6,GSD8,GTS,GTP cyclohydrolase is lacked,GTP cyclohydrolase is lacked,Guenther porphyrias,Guerin-Stern syndrome,Guillain-Barré,Guillain-Barre syndrome,Congenital erythropoeitic porphyria,H diseases,H.Gottron syndromes,It is accustomed to spasm,HAE,Congenital factor XII deficiency diseases,Hageman factor (HF),Haim-Munk syndromes,Hajdu-Cheney syndrome syndrome,Ha jduCheney,HAL is lacked,Hall-Pallister syndrome,Hallermann-Streiff-Francois syndrome,Hallerman-Streiff syndrome,Hallervorden Spatz disease,Hallerman-Streiff syndrome,Hallopeau-Siemens diseases,Thumb repeats postaxial polydactyly and lacks callosity,Halushi-Behcet syndromes,Lymph hamartoma,Hand-Schueller-Christian syndromes,HANE,Hanhart syndromes,Angelman's syndrome,Harada's syndrome,HardE syndromes,HARD syndromes,Harelip,Harlequin fetus,Harlequin types DOC6,Harlequin type ichthyosis,Harley syndromes,Harrington syndromes,Hart syndromes,How Hart flutters disease,How Hart flutters illness,Hartnup syndrome/disease,Hashimoto diseases,Hashimoto-Pritzker syndromes,Hashimoto syndromes,Hashimoto thyroiditis,Hashimoto-Pritzker syndromes,Hay Well syndromes,Hay-Wells syndrome of ectodermal dysplasia,HCMM,HCP,HCTD,HD,Heart-hand syndrome (Holt-Oram types),Heart disease,Hecht syndrome,HED,Heerferdt-Waldenstrom and Lofgren syndromes,Hegglin diseases,Heinrichsbauer syndromes,Hemangioma,Familial hemangioma,Hemangioma-thrombocytopenia syndrome,Chondrodystrophic dwarf,Hemangioma gill slit lip pseudocleft syndromes,Demifacet (stature) is short and small,Hemimegalencephaly,Cerebral paralysis hemiparesis,Cerebral paralysis hemiplegia,Spinal cord half is cut off,Hematochromatosis (disease),Hemochromatosis syndrome,Dialysis associated amyloidosis,Hemoglobin touch-control syndrome,Hemolytic anemia neonate,Cold cut hemolytic hemolytic anemia antibody,Neonatal hemolytic disease,Hemolytic uremic syndrome,Hemophilia,Hemophilia A,Hemophilia B,Hemophilia B factors IX,Congenital XI factor deficiency,Hemorrhagic decrease of platelet is malnutritive,Hemorrhagica Aleukia,Iron xanthematin thesaurismosis,Hepatic fructokinase is lacked,Liver phosphokinase is lacked,Hepatic porphyria,Hepatic porphyria,HVOD,Hepatitis C,Hepatorenal syndrome,Hepatolenticular degeneration,Hepatophosphorylase is lacked,Hepatorenal glycogenosis,Hepatorenal syndrome,Liver kidney tyrosinemia,Hereditary acromelalgia,Hereditary Melanuria,Hereditary amyloidosis,Hereditary angioedema,Heredity Areflexic dysstasias,Heredopathia atactic polyneuritifor,Ataxia hereditaria,Ataxia hereditaria spinocebellar ataxia type,Hereditary benign acanthosis nigricans,Hereditary cerebellar ataxia,Huntington's chorea,Genetic chronic progressive chorea,Heredity CTD,Hereditary coproporphyrin,Hereditary coproporphyrin,Heredity skin malignant melanoma,Hereditary hearing impairment-retinal pigment degeneration,Heredity zinc-deficiency disease,Heredity DNS,Hereditary dystopic lipidosis,Hereditary emphysema,Hereditary fructose intolerance,Hereditary hemorrhagic telangiectasia,Hereditary hemorrhagic telangiectasia type I,Hereditary hemorrhagic telangiectasia type II,Hereditary hemorrhagic telangiectasia type III,Heredity hyperuricemia and choreoathetosis,Hereditary leptocytosis Major,Hereditary leptocytosis Minor,Hereditary lymphedema,Heredity lymphedema tarda,Heredity lymphedema tarda I types,Heredity lymphedema tarda II types,Hereditary motor and sensory neuropathy,Hereditary motor and sensory neuropathy I,Hereditary motor and sensory neuropathy type III,Hereditary nephritis,Hereditary nephritis and nerve deafness,Hereditary amyloidosis nephrosis,Heredity nephrosis and deafness,Hereditary nonpolyposis colorectal cancer,Hereditary nonpolyposis colorectal cancer,Hereditary nonspherocytic hemolytic anemia,Heredity onycho-osteodysplasia,Hereditary optic retinopathy,Hereditary polyposis coli,Hereditary sensory nerve and autonomic neuropathy I types,Hereditary sensory nerve and autonomic neuropathy II types,Hereditary sensory nerve and autonomic neuropathy type III,Hereditary sensorimotor neuropathy,Hereditary sensory neuropathy I types,Hereditary sensory neuropathy I types,Hereditary sensory neuropathy II types,Hereditary sensory neuropathy group iii,Hereditary sensory radicular neuropathy I types,Hereditary sensory radicular neuropathy I types,Hereditary sensory radicular neuropathy II types,Heredity locus specificity cancer,Heredity spherocyte hemolytic anemia,Hereditary spherocytosis,The type of hereditary tyrosinemia 1,Heredity CTD,Herlitz syndrome,Hermans-Herzberg phakomatosses,Hai-general two Cotard,Hermaphroditic,Herpes zoster,Herpes iris Stevens-Johnson types,Glycogenosis type VI,Heterozygous β-thalassemia,Hexoaminidase alpha subunits missing (variation B),Hexoaminidase alpha subunits missing (variation B),HFA,HFM,HGPS,HH,HHHO,HHRH,HHT,Ceasma hernia-microcephaly's deformity-nephrosis Galloway types,Suppurative hidradenitis,Hidrosadenitis axillaris,Hidrosadenitis suppurates,Perspiration is ectodermal dysplasia,HIE syndromes,High hedratresia,High potassium,High scapula,HIM,Congenital megacolon,Acquired congenital megacolon,Referring to ulnar side ＆ hallux toes and VSD congenital megacolon more,Congenital megacolon and D type brachydactylias,Hirsutism,HIS lacks,Histadine ammoniacal (HAL) is lacked,Histidase is lacked,Histidinemia,Histocytosis,Histiocytosis X,HLHS,HLP II types,HMG,HMI,HMSN I,HNHA,HOCM,Hodgkin's disease,Hodgkin's disease,He Jiejin lymphomas,Hollaender-Simons diseases,Holmes-Adie syndrome,Holocarboxylase synthetase deficiency,Holoprosencephaly,Holoprosencephaly is complicated,Holoprosencephaly sequence,Holt-Oram syndrome,Holt-Oram type heart-hand syndromes,Blood (interior) homocystine is excessive,Homocystinuria,Homogentisic acid oxidase deficiency,homogentisicacidura,Homozygosis alpha-1-antitrypsin deficiency,HOOD,Nellie syndrome suddenly,Horton diseases,HOS,HOS1,Houston-Harris types Achrondrogenesis (IA types),HPS,HRS,HS,HSAN I types,HSAN II types,HSAN-III,HSMN,HSMN type IIIs,HSNI,HSN-III,Huebner-Herter diseases,Hunner’s Patch,Hunner ulcer,Hunter syndrome,Hunter-Thompson type acromesomelic dysplasias,Huntington's chorea,Huntington's chorea,I type mucopolysaccharidosis,Hurley syndrome,Hurler-Scheie compound,HUS,Hao-Ji Er Shi early ageing syndromes,Gilford's syndrome,Hutchinson-Weber-Peutz syndrome,Hutterite syndrome Bowen-Conradi types,Hyaline Panneuropathy,Hydranencephaly (deformity,Hydrocephalus,Hydrocephalus agyria (deformity) and retinal development are abnormal,Dandy-Walker types inside hydrocephalus,Hydrocephalus hydrocephalus noncommunicating hydrocephalus Dandy-Walker types,Hydrocephalus,Uronephrosis and peculiar facial expression,Hydroxylase defect,Hygroma colli,Hyper-IgE syndromes,Hyper-IgM syndromes,Hyperaldosteronism,Hyperaldosteronism and hypopotassemia Alkatosis,Hyperaldosteronism is not accompanied by hypertension,Hyperammonemia,Hyperammonemia caused by ammonia [base] formyl phosphate synthase lacks,Hyperammonemia caused by ornithine carbamyltransferase lacks,Hyperammonemia II types,Super β carnosinemias,Physiologic liver dysfunction,Hyperbilirubinemia II,Familial hypercalcinemia and nephrocalcinosis and urocyanosis,Hypercalcinemia-supravalvar aortostenosises,Hypercalciuric rickets,Hypercapnic acidosis,Hypercatabolism protein losing enteropathy,Hypercholesterolemic acidosis,Hypercholesterolemia,Hypercholesterolemia type IV,The excessive disease of blood (interior) chylomicron,Homocystinemia,hyperekplexia,Can hyper-extended joint,Hyperglobulinemic purpura,Hyperglycinemia and DKA and lactic acidosis,The excessive non-ketosis of glycine in blood,Hypergonadotropic hypogonadism,Hyperimmunoglobulin E syndrome,Hyperimmune globulin E-recurrent infection syndrome,Blood (in clear) excessive disease E- staphylococcics of immunoglobulin,Hyperkalemia,Hyperkinetic syndrome,The Hyperlipemic retinitiss,Hyperlipidemia I,Hyperlipidemia IV,Hyperlipoprotememia type I,Hyperlipoprotememia type III,Hyperlipoprotememia type IV,Hyperoxaluria,Hyperphalangy- forefingers refer to bending and micrognathia-glossoptosis syndrome,Hyperphenylalaninemia,Hyperplastic epidermolysis bullosa,Hyperpnea,Potassemia,Hyperprebetalipoproteinemia,Hyperprolinemia (disease) I types,Hyperprolinemia (disease) II types,Hypersplenism,Away from broadening and esophagus malformation and hypospadia,Hypertelorism-hypospadias syndrome,Hypertrophic cardiomyopathy,Hypertrophic interstitial neuropathy,Hypertrophic interstitial neuritis,Stromal hyperplasia nerve root neuropathy,The hypertrophica neuropathy of refsum,Hypertrophic obstructive cardiomyopathy,Hyperuricemia choreoathetosis autotomy syndrome,Hyperuricemia-oligrophrenia,Hypervalinemia,Hypocalcification (hypomineralization) type,Hypochondrogenesis,hypochrondroplasia,Blood (interior) gamma globulin is very few,The temporary blood of baby (interior) gamma globulin is very few,The malnutritive simultaneously potential diabeteses of hypogenital,Lack tongue-adactylism (toe) syndrome,Hypoglycemia,Exogenous hypoglycemia,Hypoglycemia and macroglossia (disease),Hypoglycosylation syndrome 1a types,Hypoglycosylation syndrome 1a types,Hypogonadism and anosmia,Low gonadotropic hormone hypofunction and anosmia,Ectodermal dysplasia,hypohidrotic,Sweat ectodermal dysplasia disease autosomal dominant inheritance type,Sweat ectodermal dysplasia disease autosomal dominant inheritance type autosomal dominant type,Hypokalemia,Hypokalemic alkalosis and hypercalciuria,Hypokalemic syndrome,Lactase lacks,Hypomaturation types (milky white teeth),Ito hypomelanosises,Hypomelia-hypotrichosis-facial hemangioma syndrome,Hypomyelination neuropathy,Hypoparathyroidism,Hypophosphatasia,Phosphate hypophosphatemic rickets and hypercalcinemia,Hypopigmentation,Pigment reduces ARM,Fallen scalp hypoplasia and heart defect,Hypoplastic anemia,Congenital aplasia anaemia,Chondrodystrophia hypoplastica,Hypoplasia enamel-onycholysis-hypohidrosis,Hypoplasia (hypoplasia-explastic) type,Hypoplastic left heart syndrome,Hypoplasia-(thumb) triphalangia,Hypokalemia,Hypospadia-dysphagia syndrome,Hyposphresia,Referring to hypothalamus hamartoblastoma hypopituitarism hedratresia more,Thalamus-childhood obesity,Hypothyroidism,H3O syndrome,Hypoxanthine-guanine phosphoribosyl transferase defect (missing completely),I- cytopathies,Iatrogenic hypoglycemosis,IBGC,IBIDS syndromes,IBM,IBS,IC,I- cytopathies,ICD,ICE syndrome Cogan-Reese types,Iceland type amyloidosis venereal disease (type VI),I- cytopathies,Ichthyosiform erythroderma cornea is participated in and deaf,The red skin trichosis of ichthyosiform and male,Ichthyosiform erythroderma and leucocyte vacuolization,Ichthyosis,Ichthyosis congenita,Congenital fish scale and trichothiodystrophy,Ichthyosis hystrix,Ichthyosis hystrix,Ichthyosis linearis circumflexa,Ichthyosis simplex,Ichthyosis Tay syndromes,Ichthyosis vulgaris,Ichthyotic neutral lipid storage disease,Leptospira icterogenes disease,Leptospira icterogenes disease,Jaundice (chronic familial),Icterus gravis neonatorum,Jaundice intermittens juvenalis,Primary alveolar hypoventilation,Primary amyloidosis,Primary Takayasu arteritis,Primary basal ganglion calcification (IBGC),Primary brachial plexus neuropathy,Primary cervical dystonia,Primary pulmonary arterial dilatation,Primary peripheral facial paralysis,Essential familial hypelipemia,The hypertrophica aortic stenosis of primary,Primary hypoproteinemia,Primary immunoglobulin deficiency,Primary neonatal hepatitis,Primary nonspecific ulcerative colitis,It is scorching around primary PeV,Idiopathic pulmonary fibrosis,Intractable primary iron granule erythrocyte anemia,Primary renal hematuria,Idiopathic steatorrhea,Idiopathic thrombocythemia,Primary thrombocytopenic purpura,Primary thrombocytopenic purpura (ITP),IDPA,Iga nephrosis,IHSS,Ileitis,Ileocolitis,Illinois type amyloidosis,ILS,IM,IMD2,IMD5,Due to lacking the immunodeficiency of thymus gland,Immune hemolytic anemia paroxysmal is cold,Immune deficiency and ataxia telangiectasia,Immune deficiency cell and abnormal immunoglobulin synthesis,Immune deficiency is often variable not to open classification,Immune deficiency and Hyper-IgM,Immune deficiency and Neuroleptic Leukocytopenia,Immune deficiency -2,Immune deficiency -5 (IMD5),Immunoglobulin deficiency,Hedratresia,Hedratresia and brothers and ear are abnormal,Locking nasolacrimal duct and early ageing syndrome,Impotent neutrophil syndrome,It can not dehisce completely and short flexor digitorum muscle of hand,INAD,Birth defects urea synthesizing arginine-type,Birth defects UreaSynthesis Arginino Succinic types,Congenital urea synthesizing carbamyl phosphate type,Birth defects urea synthesizing citrullinemia type,Congenital urea synthesizing glutamic acid synthesis type,INCL,Inclusion body myositis,Treatment of Complete Atrioventricular Defect,Infull testicular feminization,Incontinentia pigmenti,Incontinenti Pigmenti Achromians,The abnormal simultaneously micrognathia-glossoptosis syndrome of forefinger,Amyloidosis,Indiana type (Equations of The Second Kind),Indolent systemic mastocytosis,The children's aphasia day after tomorrow,Children's recessive hereditary polycystic kidney disease,Infantile beriberi,Children's cerebroganglion glycosides fat,Children's cerebral palsy,Children's abderhalden-Kaufmann-Lignac syndrome,Children epilepsy,Children's Fanconi syndrome and abderhalden-Kaufmann-Lignac syndrome,Baby Finland type neuronal waxy lipophilic disease,Children's Gaucher disease,Pediatric hypoglycemia,Children hypophasphatasia,Children's lobe of the lung pulmonary emphysema,Children's myoclonic encephalopathic,Children's myoclonic encephalopathic and polymyoclonus,Infantilism Myofibromatosis,Infant's necrotizing encephalopathy,Infantile neuronal ceroid lipofuscinosis,Children's neural axis is malnutritive,Infant's morbidity Schindler diseases,Children's phytanic acid storage disease,Children's Refsum disease (IRD),Children sipoidosis GM-2gangliosideosis (s types),Baby sleeping apnea,Infantile spasms,The myeloid muscular dystrophy of children (all types),Children's spinal muscular atrophy ALS,Children's Duchenne-Arandisease type I types,Infantilism neuronal waxy lipophilic disease,Catarrhal jaundice,IBD,Inflammatory breast cancer,Inflammatory linear sebaceous nevus syndrome,Iniencephaly,The acanthosis nigricans of insulin resistance,Insulin lipodystrophy,Insulin-dependent diabetes mellitus,Purpose myoclonia,Intermediate cystinosis,Intermediate maple syrup urine disease,Episodic ataxia and pyruvic dehydrogenase deficiency,Intermittent maple syrup urine disease,Internal hydrocephalus,Interstitial cystitis,Interstitial deletion includes 4q,Intestinal lipodystrophy,Intestines lipophagia granuloma,Intestinal lymphatic dilatation,Intestinal polyp I,Intestinal polyp II,Intestinal polyp III,Intestinal polyposis-cutaneous pigmentation syndrome,Intestinal pseudo-obstruction and ballet's disease,Intracranial neoplasm,Intracranial tumors,Intracranial vascular malformation,Intrauterine nanism,Intrauterine adhesion,Stand upside down and smile and invisible nervous bladder,Iowa types amyloidosis (IV types),IP,IPA,Iridocorneal endothelial syndrome,Iris corneal endothelium (ICE) syndrome Cogan-Resse types,Iridogoniodysgenesis and body exception,Atrophy of iris and corneal edema and glaucoma,Cogan-Reese syndrome,Iron overload anaemia,Iron overload disease,Irritable bowel syndrome,Irritable colon syndrome,Isaacs syndromes,Isaacs-Merten syndromes,Ischemic myocardium disease,Isolate agyria sequence,The amyloidosis of isoleucine 33,Isovaleric acid CoA dehydrogenase deficiency,Isovaleric acidemia,Isovaleric acidemia,Isovaleryl coenzyme A carboxylation enzyme defect,ITO black (color) plain very few disease,ITO,ITP,IVA,Ivemark syndromes,Iwanoff tumours,Jackknife twitches,Jackson-Weiss craniosynostosises,Jackson-Weiss syndromes,Jacksonian epilepsy,Jacobson syndrome,Jadassohn-Lewandowsky syndrome,Jaffe-Lichenstein diseases,Jakob diseases,Jakob-Creutzfeldt disease,Janeway I,Janeway dysgammaglobulinemias,Jansen metaphyseal dysostosises,Jansen type metaphysial chondrodysplasias,Jarcho-Levin syndrome,Jaw winking (reflection),JBS,JDMS,Jegher syndromes,Imperforate jejunum,Jejunitis,Jejunoileitis,Jervell and Lange-Nielsen syndrome,Jeune syndrome,JMS,Job's syndrome,Job-Buckley syndromes,Johanson-Blizzard syndrome,John Dalton,About [Han Xun]-this [Di Wen] Er Shi diseases,Jonston alopecias,Joseph diseases,Joseph disease I types,Joseph disease II types,Joseph disease type IIIs,Joubert syndromes,Joubert-Bolthauser syndromes,JRA,Juberg Hayward syndromes,Juberg-Marsidi syndromes,Juberg-Marsigi baryencephalia syndromes,France ramaninjana,Maine France ramaninjana,Juvenile arthritis,Childhood recessive hereditary polycystic kidney disease,Juvenile cystinosis,Childhood (children) dermatomyositis (JDMS),Childhood diabetes,Childhood Gaucher disease,Juvenile gout choreoathetosis and baryencephalia syndrome,Childhood vitamin b12 intestinal malabsorption,Childhood vitamin b12 intestinal malabsorption,Juvenile macular degeneration,Pernicious anemia,juvenile,Juvenile retinoschisis,Juvenile rheumatoid arthritis,Adolescent spinal muscular atrophy includes,Adolescent spinal muscular atrophy includes ALS,Adolescent spinal muscular atrophy type III,Jaxta-articular adiposis,Hyperplasia by glomerulus,Kabuki's cosmetic syndrome,Kahler's disease,Kallmann syndromes,Kanner syndromes,Kanzaki diseases,Card ripple disease (non-Kaposi sarcoma),κ light chains are lacked,Karsch-Neugebauer syndromes,Kartagener syndromes-chronic sinobronchial diseases and dextrocardia,Kartagener triad,Hemangioma-thrombocytopenia syndrome,Kast's syndrome,Kawasaki disease,Kawasaki syndrome,KBG syndromes,KD,Kearns-Sayre diseases,Kearns-Sayre syndromes,Kennedy diseases,Kennedy syndromes,Kennedy type spinal and bulbar muscular atrophies,Kennedy-Stefanis diseases,Kenny diseases,Kenny syndromes,Kenny types tubular bone (pulp cavity) is narrow (disease),Kenny-Caffe syndromes,Kera.Palmoplant.Con.Pes Planus Ony.Periodon.Arach.,Keratitis ichthyosis and deafness syndrome,Keratoconus,Keratoconus posticus circumscriptus,Cuticula is separated,Keratolysis exfoliativa congenita,Winter exfoliating erythema,Keratomalacia,Darier's disease,Keratosis follicularis spinulosa decalvans,Keratosis follicularis spinulosa decalvans ichthyosis,Angling acanthosis nigricans,Keratosis palmoplantaris and periodontosis and onychogryp(h)osis,Keratosis palmoplantaris congenital flat foot's onychogryp(h)osis periodontosis arachnodactylies,CDK palmoplantaris,Flat foot,Hook first,Periodontosis,Arachnodactyly,Acroosteolysis,Keratosis rubra figurata,Seborrheic keratosis,Keto-acid decarboxylase enzyme defect,Ketone acid is urinated,Ketosis glycinemia,KFS,KID syndromes,Kidney development is not complete,The dirty retinal aplasia Joubert syndromes of cystic kidney,Killian syndromes,Killan/Teschler-Nicola syndromes,Kiloh-Nevin syndrome is II,Hair nodules,Dancing eyes syndrome,Kleeblattschadel deformities,Kleine-Levin syndrome,Kleine-Levin hibernation syndromes,Klinefelter,Cervical vertebrae synostosis,Cervical vertebrae synostosis I types,Cervical vertebrae synostosis II types,Cervical vertebrae synostosis type III,Klippel Trenaunay syndromes,Io-osteohypertrophy's syndrome,G-Buji 's syndrome,KMS,Kniest depauperations,Kniest syndrome,Ke Nisike diseases,Koebberling-Dunnigan syndromes,Kohlmeier-Degos disease,Kok diseases,Korsakoff mental diseases,Korsakoff syndromes,Krabbe diseases include,Krabbe leukodystrophies,Kramer syndromes,KSS,KTS,KTW syndromes,Kufs disease,Ku-Wei disease,Kugelberg-Welander syndrome,Kussmaul-Landry paralysis,KWS,L-3- hydroxy-acyls-coenzyme a dehydrogenases (LCHAD) defect,Laband syndromes,Labhart-Willi syndromes,Labyrinthine syndrome,Hydrolabyrinth,Tear stains-ear-tooth-refers to (toe) syndrome,Lactase is not tolerated alone,Alactasia,Lactation atrophy of uterus,Lactic acidosis leber hereditary optic neuropathy,Lactic acid and Pyruvic acid acidaemia and the sensitive sense of carbohydrate,Lactic acid and Pyruvic acid acidaemia and sporadic incoordination and weakness,Lactic acid and Pyruvic acid salt,Hyperlactacidemia,Adult is intolerant to lactose,Lactose intolerance,Children are intolerant to lactose,LADD syndromes,LADD,Lafora disease includes,Lzfora corpusculums disease,Fibrinase,fibrin stabilizing factor-Factor XⅢ deficiency disease,LAM,Lambert type ichthyosis,Lambert-Eaton syndrome,Lambert-Eton myasthenic syndrome,Stratiform recessiveness ichthyosis,Lamellar ichthyosis,Lancereaux-Mathieu-Weil spirochetosises,Landau-kleffner syndromes,Landouzy Dejerine muscular dystrophies,Landry ascending paralysises,Langer-Salidino types achondroplasia (Equations of The Second Kind),Langer Giedion syndromes,Histiocytosis X,Langerhans cell histiocytosis (LCH),Big atrium and ventricular loss,Laron dwarfism,Laron type pituitary dwarfisms,Draw Ademilson (family name) syndrome,Laryngeal dystonia,Lata (in Malaysia's discovery),Late period children's neural axis is malnutritive,Late period children's neural axis is malnutritive,The tardy type of Cockayne syndrome three (c types),Tardive dystonia,Late immunoglobulin deficiency disease,Tardy Pei-plum Er Shi cerebrosclerosis,Lattice corneal dystrophy,Lattice dystrophy,Launois-Bensaude,Launois-Cleret syndrome,Laurence syndromes,Laurence-Moon syndromes,Laurence-Moon-Bardet-Biedl syndrome,Lawrence-Seip syndrome,LCA,LCAD is lacked,LCAD,LCAD,LCADH lacks,LCH,LCHAD,LCPD,Jeune syndrome,Leband syndromes,Leber amaurosises,Leber congenital amaurosises,Amaurosis,Congenital post cone missing,Leber ' s congenital retinals blanket layer is denatured,The congenital blanket retinal developments of Leber ' s are abnormal,Leber ' s diseases,Leber ' s optic atrophies,Leber ' s optic neuropathies,Left ventricle fibrosis,Ulcus cruris,Legg-Calve- Perthes diseases,Leigh ' s diseases,Leigh ' s syndromes,Leigh ' s syndromes (subacute necrotizing cerebrospinal cord disease),Leigh brain necrosis,Lennox-Gastaut syndromes,Freckle-hyperposia-digestion syndrome (Lentigio-Polypose-Digestive Syndrome),Lenz DysmorphogeneticSyndrome,Lenz dysplasia,Lenz nanophthalmus syndromes,Lenz syndromes,Leopard syndrome,Donohue's syndrome,Pia-arachnoid hemangioma,Leptospiral jaundice,Leri-Weill Disease,Leri-Weill dyschondrosteosis,Leri-WeilSyndrome,Lermoyez syndrome,Leroy Disease,Lesch Nyhan Syndrome,Lethal baby's caradrin myopathy,Lethal neonate's nanism,Lethal osteochondrodysplasia,Letterer-Siwe disease (letterer-Siwe disease),Leukocyte disorder albinism,Leucocyte embedding is with blood platelet disorders,Leukodystrophy,Leukodystrophy is with Rosenthal fiber,Leukoencephalitis (leukoencephalitis) PeriaxialisConcentric (centrality),Levine-Critchley syndrome,Fructosuria,Levy-Hollister syndromes,Limbus girdle muscular dystrophy,Lactoglobulin,Lerbe hereditary neuropathy,Left internal carotid,Lichen ruber acuminatus,Lichen acuminatus,Lichen amyloidosis,Lichen planus,Lichen psoriasis,Lignac-Debre-Fanconi Syndrome,Lignac-Fanconi syndrome,Ligneous conjunctivitis,Limb girdle type muscular dystrophy,LimbMalformations-Dento-Digital Syndrome (Deformities of Limbs ...),Limit dextrinosis,Linear mole hypermelanosis,Naevus linearis Sebacous syndromes,Linear scleroderma,Linear naevus sebaceus sequence,Linear sebaceous nevus syndrome,Bifid tongue,Fissured tongue,Fissured tongue,Lingual surface dyskinesia,Lip Pseudocleft-hemangiomatous Branchial CystSyndrome (- hemangioma branchial cleft cyst syndrome is split in lip vacation),Lipid granuloma disease,Lipid histocytosis,Angle glucoside epoxy-type,Lipid storage disease,Lipid storage myopathy is not enough (Lipid-Storage myopathy Associated with SCAD Deficiency) with SCAD,Baby's ganglioside lipidosis,Lipoatrophic diabetes (Lipoatrophic DiabetesMellitus),Fat nutritional disorders,Lipid corneal dystrophy,Lipid hyperplasia pseudo (Lipoid Hyperplasia-Male Pseudohermaphrotitism),Congenital lipomatosis of pancreas,Lipomucopolysaccharidosis,Lipomyelomeningocele,The Deletional schilder's disease,Familial of lipoprotein lipase,Atomicvapor laser isotope separation,LIS1,Agyria 1,Agyria I types,Agyria abnormity is with corpus callosum cerebellar hypoplasia or other exceptions,Infantile spasm Diplegia,Liver phoshorylase,Liver,Kidney,Spleen,Lactose malabsorption syndrome,Circumscribed atrophy of brain,Intelligence circumscribed atrophy of brain,Lobar holoprosencephaly,Baby's lobe of the lung anxiety pulmonary emphysema,Lobstein disease (I types),Lobster claw deformity,Local epidermolysis bullosa,Local fat dystrophia,Local cingulum membri superioris neuritis,Lv Fule bacterium disease,Lv Fule bacterium cardiac muscle internal membrane of heart fibrosis is with eosinophilia,Lv Fule bacterium mural endocarditises,Lip river Kern's syndrome,Senior Lip river Kern syndrome,Long-chain 3-hydroxyacyl-CoA dehydrogenase,Long-chain fatty acyl-CoA dehydrogenase defect,Long-chain fatty acyl-CoA dehydrogenase,Long acyl-CoA dehydrogenase deficiencies,Without deafness QT syndromes,Hypoglycemia,Low-density beta lipoprotein is lacked,Low level hedratresia,Low potassium syndrome,Oculo cerebro renal syndrome Lowe ' s Syndrome,Lowe-BickelSyndrome,Lowe-Terry-MacLachlan Syndrome,Low back pain,Milk of sulfur,Leukotrienes d,Lubs syndromes,Luft disease,Waist spinal canal stenosis,Lumbar spinal stenosis,Waist sacrum spinal canal stenosis,Lundborg-Unverricht Disease,Lundborg-UnverrichtDisease Included,Lupus,Lupus,Lupus erythematosus,Luschka-MagendieForamina Atresia,Lyell's syndrome,Lyell syndrome,Lymphadenoid goitre,The protein losing enteropathy of lymphangiectasis,Lymphangioleiomyoma,Lymphangioleiomyoma,Lymphangioma,Lymphatic malformation,Lynch Syndromes,Lynch SyndromeI,Lynch Syndrome II,Schindler type lysosomes alpha-N-Acetylgalactosaminidase lacks (Lysosomal Alpha-N-Acetylgalactosaminidase DeficiencySchindler Type),Lysosomal Glycoaminoacid StorageDisease-Angiokeratoma Corporis Diffusum,Lysosomal glycosidase is lacked,Single joint arthritis,Machado Disease,Ma-about sick,Macrencephaly,Megacephaly,Megacephaly hemihypertrophy,Megacephaly is with lipomatosis and Hemangiomata,Megacephaly is with pseudopapilloedema and Multiple (multiple) Hemangiomata,Macroglobulinemia,Macroglossia (disease),Macroglossia-omphlexoche-visceral obesity syndrome,Macrostomia (meloschisis) Ablepheron Syndrome,Bernard-Soulier Type familials Macrothrombocytopenia (Macrothrombocytopenia Familial Bernard-Soulier Type),Macula retinae sexual involution,Macular amyloidosis,Macular degeneration,Macular disciform degeneration,Macular disciform degeneration,Macular disciform degeneration,Mottled corneal nutrition obstacle,Maximum allowable amount,Madelung’s Disease,Maffucci syndrome (multiple chondroma is with internal organ cvernous hemangioma),Grand mal,Malabsorption,Malabsorption-ectodermal dysplasia-wing of nose hypoplasia,Maladie de Roger,Maladie de Tics,Malaria,Male four limbs and deformity of kidney,Male gonad maldevelopment disease,Pernicious acanthosis,Malignant acanthosis nigricans,Malignant astrocytoma,Malignant atrophic papulosis,Malignant malaria,Malignant hyperphenylalaninemia,Malignant fever,Pernicious hyperpyrexia,Malignant mela noma,Central nervous system malignant tumour,Mallory-Weiss tear,Mallory-Weiss tear,Ma-Wei syndrome (orifice of the stomach mucous membrane lacerated wound syndrome),Mammary Paget’s Disease,Mandibular ameloblastoma,Faciomandibular dysostosis,Mannosidosis,Surface-point-finger pattern corneal dystrophy (Map-Dot-Fingerprint Type Corneal Dystrophy),Maple syrup urine disease,Marble bone,Paroxysmal nocturnal hemoglobinuria (marchiafava-Micheli syndrome),Ma Kasigeen jaw winking syndromes,Ma Kasigeen (family name) phenomenon,Ma Kasigeen ptosis is with gunn syndrome,Ridge grace syndrome is (during unilateral ptosis,Ipsilateral upper eyelid is moved with mandibular symphysis),Ma Kasigeen (gunn syndrome) syndrome,Ma Kasigeen sagging (with gunn syndrome),Marden-Walker syndrome (recessive hereditary multiple arthrogryposis deformity),Ma-fertile Er Shi CTDs,Malaysia side nutrition exhaustion,Malaysia side-Achard syndromes,Malaysia side syndrome,Malaysia side syndrome I types,Malaysia side variant,Malaysia side type hyperkinesia syndrome,Marginal corneal dystrophy,Mary's incoordination,Mary's disease,Marie-Strumpell disease,MarieStrumpell Disease,Marie-Strumpell spondylitis (ankylosing spondylitis),Marinesco-Sjogren syndrome (ataxia hereditaria,Cataract,Dwarf and amentia syndrome),Ma-Si-lattice syndrome,Marker X syndrome,Ma Shaoer syndromes,Maroteaux LamySyndrome,Maroteaux Type Acromesomelic Dysplasia,Ma Shaoer is ectodermal dysplasia with vision auditorily handicapped,Ma Shaoer-Smith's syndrome,Ma Shaoer syndromes,Ma Shaoer types are deaf-myopia-flood-saddle nose (Marshall TypeDeafness-Myopia-Cataract-Saddle Nose),Ma-Austria's syndrome (false accessory thyroid glands function is low to subtract disease),Ma-shellfish syndrome,Martorell syndromes,MASA Syndrome,Large area myoclonia,Mast cell leukemia,Mastocytosis,Mastocytosis is with haematological disorders,Maumenee Corneal Dystrophy (corneal dystrophy),Maxilla ameloblastoma,Maxillary bone dysbolism,Maxillonasal dysplasia,Maxillonasal dysplasia adhesion type,Maxillary sinus-eyelid synkinesia,Abnormal (the may-Hegglin anomalies of Mei-sea Er Shi (blood platelet),Heredity nucleic acid is abnormal),Medium-chain acyl-coenzyme A dehydrogenase deficiency,McArdle disease (glycogen storage disease IV types),McCune-Albright,(kidney) medullary cystic disease,McKusick type metaphysial chondrodysplasias,Mitral valve closure speed,Mixed connective tissue disease,U.S. syndrome,Meckel-Gruber syndrome (internal organ tumour-head depauperation syndrome),Median cleft face syndrome,Thalassemia,Medium chain acyl coa dehydrogenase,Medium chain acyl coa dehydrogenase is lacked,Medium chain acyl coa dehydrogenase is lacked,(kidney) medullary cystic disease,Medullary sponge kidney,Maximum expiratory flow volume,Mega-esophagus,Macrencephaly,Macrencephaly is with hyaloid inclusion body,Megalencephaly with Hyaline Panneuropathy,Megaloblastic anemia,Juvenile erythrocyte anemia during pregnancy,Macrocornea-mental retardation's syndrome,Basal cell naevus syndrome,MeigeShi lymphedemas,Meige syndromes (Meige ' s Syndrome),Macrotooth (macroteeth) leukodystrophy,Intestinal mucosa melanoplakia polyposis,Intestinal mucosa melanoplakia polyposis,MELAS Syndrome,MELAS,Melkersson Syndrome,BOR syndrome,Osteodysplasty of Melnick and Needles,Melnick-Needles syndrome,Mucous membrane fat nutritional disorders,Da Costa's syndrome (cardiac neurosis),Meniere disease (auditory vertigo),Meniere disease,Meninx capillary blood tuberculation,Menkes disease,Menkes disease I,Aphasia body disorders and hallux valgus type baryencephalia,Mental retardation-deafness-skeletal abnormality-coarse face and thick lip (Mental Retardation-Deafness-Skeletal Abnormalities-CoarseFace with Full Lips),5th thumb and toe hypoplasia type mental retardation,Osteochondrodysplasia type mental retardation,The short and small type mental retardation of growthing lag-deafness-genitals (Mental Retradation-X-linked with Growth Delay-Deafness-Microgeni-talism),Menzel type olvopontocerebellar atrophies,Mermaid syndrome,Myoclonus epilepsy and ragged red fibrers syndrome,Myoclonus epilepsy and ragged red fibrers syndrome,Merten-Singleton Syndrome,Limb injury syndrome,Glomerular mesangium immunoglobulin-a type nephrosis,Mesenteric fat dystrophia,Mesiodens-Cataract Syndrome,Mesodermal Dysmorphodystrophy,Mesomelic Dwarfism-MadelungDeformity,Metabolic acidosis,Metachromatic leukodystrophy,Metatarsus varus,Metatropic dwarfism syndrome,Metatropic dysplasia,Metatropic dysplasia I,Metatropic dysplasia II,Methylmalonic acidemia,Methylmalonyl CoA carboxyls mutase lacks disease,Meulengracht’s Disease,Faciomandibular dysostosis I,Macroglobulinemia,Malignant histiocytosis,Microangiopathic hemolytic anemia,Micrencephaly,Primary nanocephalic dwarf I,Microcephaly's deformity,Microcephaly-ceasma hernia-nephropathy Galloway types (Microcephaly-Hiatal Hernia-Nephrosis Galloway Type),Microcephaly-ceasma hernia-nephropathy syndrome,Folliculus cornea (epithelium) is malnutritive,Microcytosis,Microlissencephaly,Nanophthalmus,Anophthalmia or nanophthalmus are with relevant abnormalities,Gyrus is small with muscular dystrophy,Knee cap micrognathia syndrome without microtia,Microvillus embedding disease,Mesangial immune deposits,Midsystolic click late systolic murmur syndrome,Miescher’s Type I Syndrome,Mikulicz syndrome,Mikulicz-RadeckiSyndrome,Mikulicz-Sjogren Syndrome,Slight autosomal recessive inheritance,Light moderate maple syrup urine disease,Mild maple syrup urine disease,Miller syndrome,Miller-Dieker syndrome,The syndrome of meter -not,Meige disease,Minkowski-Chauffard syndrome (familial spherocytosis,Congenital hemolytic anemia,Acholuri familial jaundice),Lapse,Von Willebrand's disease (constitutional blood platelet disorders disease),Mirror image dextrocardia,Mitochondria beta oxidation is disorderly,Mitochondria and cytoplasmic,Mitochondrial cytopathies,Mitochondrial cytopathies,Kearn-Sayre Type,Mitochondrial encephalopathy,Mitochondrial encephalomyopathy lactic acidosis and the breaking-out of apoplexy sample,Mitochondrial myopathy,Mitochondrial encephalomyopathy lactic acidosis and the breaking-out of apoplexy sample,Mitochondria phosphoenolpyruvate carboxykinasedeficiency,Mitral valve prolapse,Mixed apnea,Mixed connective tissue disease,Mixed hepatic porphyria,Nonfluent aphasia,Mixed sleep apnea,Tonic-clonic Combination torticollis,MJD,Mounier-Kuhn syndrome (congenital,Eso-ethmoiditis combines presence with bronchiectasis),Malignant lymphoma I (ML I),Malignant lymphoma II,Malignant lymphoma III,Malignant lymphoma IV,Mediterranean lymphoma disorder I (ML Disorder Type I),ML Disorder TypeII,ML Disorder Type III,ML Disorder Type IV,Membranous lupus nephritis,Moderate nerve growth retardation syndrome,Motor neuron disease,MNGIE,MNS,Mobitz I,Mobitz II,Mo Biesi (family name) syndrome (congenital facial diplegia),Moebius syndrome (pain motion can not),Moersch-Woltmann syndromes,Mohr's syndrome (OFD syndrome i I types,Bifid tongue,Conduction deafness,Thumb portion is overlapping),Monomodal typhloexias (Monomodal Visual Amnesia),Mononeuritis multiplex,Mononeuritis,Single nerve endings lesion,Monosomy 3p2 (Monosomy 3p2),Monosomy 9p Partial,Monosomy 11q Partial,Monosomy 13q Partial,Monosomy 18qSyndrome,Monosomy X,Sub-thread fibrous dysplasia,Morgagni-Turner-Albright Syndrome,Scleroderma circumscriptum,Eccentro-osteochondrodysplasia,Morquio syndrome,Morquio syndrome A,Morquio syndrome B,Morquio syndrome syndrome,Syringomyelia,The body 9p of piebald four (MosaicTetrasomy 9p),Motor neuron disease,Motor neuron syndrome,Motor neuron disease,Motor neuron disease,Motor neuron disease,Dynamical system disease (is assembled and slow),Moyamoya disease,Moyamoya disease (due to brain bottom abnormal vascular net rupture caused by block and small bleeding caused by cerebrum ischemia cause progressive nervous function defect,Moyamoya disease),Mucopolysaccharide (storing up) disease,Mucopolysaccharide (storing up) disease I,Mucopolysaccharide (storing up) disease I H,Mucopolysaccharide (storing up) disease 1H/S Hurler/Scheie Syndrome,MPS IS Scheie Syndrome,MPSII,MPS II A,MPS IIB,MPS II-AR,Autosomal recessive inheritance Hunter syndrome,MPS II-XR,The serious autosomal recessive inheritances of MPS II-XR,Mucopolysaccharide (storing up) disease V,Mucopolysaccharide (storing up) disease VI,Mucopolysaccharide (storing up) disease VI Severe Intermediate MildMaroteaux-Lamy (severe ... in light),Mucopolysaccharide (storing up) disease V II,Mucopolysaccharide (storing up) disease VIIsly syndrome (mucopolysaccharidosis VII types：The disease caused by beta-glucuronidase lacks),Mucopolysaccharide (storing up) disease VIII,Mucopolysaccharide (storing up) is disorderly,MPS Disorder I,MPS Disorder H,MPS Disorder IH,MPS Disorder VI,MPS DisorderType VII,(classical symptom is the facial paralysis of recurrent exerbation to the Cotard of Mai-sieve two,Non-inflammation edema of the face and folding property harelip),Malignant stricture,Multi-system atrophy,Multiple sulfuric acid lps deficiency,Symptoms of excess fat,Muscular subaortic stenosis,Maple syrup urine disease,Maple syrup urine disease Ib types,Maple syrup urine disease II types,Viscous liposteatosis III,Mucolipidosis IV,Mucopolysaccharidosis,Mucopolysaccharidosis I-H,Mucopolysaccharidosis I-S,Mucopolysaccharidosis II,Mucopolysaccharidosis III,Mucopolysaccharidosis IV,Mucopolysaccharidosis VI,Mucopolysaccharidosis VII,Mucopolysaccharidosis I types,Mucopolysaccharidosis II types,Mucopolysaccharidosis type III,Mucopolysaccharidosis IV types,Stickiness,Mucosulfatidosis,Mucous colitis,(ductus pancreaticus) mucoviscidosis,Mulibrey Dwarfism,Mulibrey Nanism Syndrome,Gyneduct hypoplasia-renal aplasia-neck chest body segment hypoplasia,Gyneduct-kidney-neck chest-upper limbs volume defect,Gyneduct and renal aplasia are with upper limbs and rib deformity,Gyneduct-kidney-neck chest segment monster,Binswanger type multi infarct dementias,MulticentricCastleman’s Disease,Many focus eosinocyte granulation knurls,Multiple acyl-CoA dehydrogenase missing/glutaric aciduria II types,Multiple Angiomas (complicated hemangioma) andEndochondromas,Multiple carboxylase deficiency,Multiple chondrophyte,Multiple cancellous exostoses,Multiple enchondromatosis,Multiple endocrine deficiency syndrome,Multiple epiphyseal dysplasia,Multiple exostosis,Multiple exostoses syndrome,Multiple familial polyposis,Leopard's syndrome,Huppert's disease,Shoulder belt multiple neuritis,Osteochondromatosis,Multiple peripheral neuritis,Multiple sulfatase deficiency,Symptoms of excess fat,Multi-system atrophy,Multisynostotic osteodysgenesis,Multisynostotic osteodysgenesis is with long osteomiosis,Mulvihill-Smith syndromes,MURGS association (Miller tubes,Kidney,Cervical vertebra defect),Murk Jansen type metaphyseal chondrodysplasias,Muscle carnitine deficiency,Muscle axis of centres disease,Muscle phosphofructokinase deficiency,Muscle central core disease disease,Muscular dystrophy,Muscular dystrophy x linked recessives (heredity),Congenital muscular dystrophy involves with central nervous system,Mental retardation causes the congenital muscular dystrophy of progressive,Facioscapulohumeral muscular dystrophy,Muscular rheumatism,Myotonia-progressive property breaking-out,Musculoskeletal pain,Four limbs shortage disease,Mutism,,Microvascular pressure,Microvascular pressure,MWS,Myasthenia gravis,Goldflam disease,Lambert-Eaton myasthenic syndromes,Myelinoclasis diffusivity is hardened,Myelomatosis,Myhre syndromes,Myoclonia astatic type lapse,Myoclonia dystonia,Baby's myoclonia encephalopathic,Myoclonic epilepsy,Hartung types myoclonic epilepsy (Myoclonic Epilepsy Hartung Type),Lafora's disease is with ragged red fibers,Myoclonic epilepsy and ragged red fibers disease,The progressive myoclonus epilepsy of familial,Familial myoclonus epilepsy,Myoclonic seizure,Myoclonia,Lafora's disease,Myoencephalopathy Ragged-Red Fiber Disease (... ragged red fibers disease),Myofibromatosis,Congenital Myofibromatosis,Myogenicity face-omoplate-fibula syndrome,Myoneurogastointestinal Disorder andEncephalopathy (encephalopathic),Myopathy AMC,Muscle [poison] base deficit disease,Muscle central area fibrosis,Myopathy Congenital (congenital myopathy ...) Nonprogressive,myopathy Congenital Nonprogressive withCentral Axis,Myopathy is lacked with carnitine palmityl transferase,Myopathy-Marinesco-Sjogren syndromes,Myopathy-metabolism DL-carnitine chloride palmitoyl transferase missing,Myopathy mitochondria-encephalopathic-lactic acidosis-apoplexy,With the myopathy of sarcoplasmic bodies and sarcoplasmic bodies,Myophosphorylase is lacked,Myositis ossificans,This too sick (atrophica of nanotesla (family name),Myotonia atrophica),Myotonia congenita,Myotonia congenita intermittens,Myotonia atrophica,Myotonia myopathy dwarf chondrodystrophy and face are abnormal,Myotubular myopathy,X myotubular myopathy,Myproic Acid,Myriachit (Siberia saying),Myxedema,N- acetylgalactosamine -1- phosphotransferases lack,N- acetylglutamates synthesize enzyme defect,Reduced nicotinamide adenine dinucleotide defect,Naegeli EctodermalDysplasias (ectodermal dysplasia),Nager acrofacial dysostosis syndrome,Nager acrofacial dysostosis syndrome,Nager acrofacial dysostosis syndrome,N- acetyl-β-d- UNAGs lack,(refer to,Toe) Onychodystrophy-deafness syndrome,(refer to,Toe) first hypoplasia and metodontiasis,Nail patella syndrome,Nance-Horan syndrome,Bird-headed dwarf of Seckel,Microcephaly's (deformity),Ommatidium,Hypnolepsy,Hypnolepsy syndrome,Non-aqueous reverse-phase chromatography,Nose-volume-(Nasal-fronto-faciodysplasia),Wing of nose hypoplasia hypothyroidism achylia,Atelognathia on nose,Nose fat nutritional disorders (Nasu Lipodystrophy),NBIA1,Neurotic depression,Nerve defect index,No protein diet,Cause encephalomyelopathy (the Necrotizing Encephalomyelopathy of Leigh ' s of necrosis,),Cause the respiratory apparatus granulomatosis of necrosis,Neill-Dingwall Syndrome,(after bilateral adrenal glands excision hypophysis tumor and mucocutaneous pigmentation occur for Nelson syndrome),Nemaline myopathy,Neonate's adrenoleukodystrophy,Neonate's adrenoleukodystrophy (NALD),Neonate's adrenoleukodystrophy (ALD),Neonate's autosomal recessive inheritance POLYCYSTIC KIDNEY DISEASE,Neonate dwarf,Neonatal hepatitis,Hypoglycemia of newborn,Neonate's lactose intolerance,Exudative enteropathy causes neonate's lymphedema,Neonatal necrotizing enterocolitis,Neonate's Progeroid syndromes,Wiedemann-RautenstrauchShi neonate's pesudohydrocephalus Progeroid syndromes,Neoformation archnoiditis,Nephroblastoma,Kidney primary diabetes insipidus,Familial teenager's nephrophthisis,Nephropathic cystinosis,Kidney-pseudohermaphroditism-nephroblastoma,Nephrosis-microcephaly's deformity syndrome,Nephrosis-neopiran dysmigration syndromes (Nephrosis-Neuronal Dysmigration Syndrome),Nephropathy-diabetes-dwarf-rickets-hypophosphoremia syndrome,Netherton Disease,Netherton syndrome (with the ichthyosis of tubercle hair),Netherton syndrome ichthyosis,Nettleship FallsSyndrome (x linked recessives),Neu-Laxova syndromes,Neuhauser syndromes,NTD,Brachial neuritis,Mucopolysaccharide-n- acetyl nerve ammoniacal liquor solution enzyme defect,Nerve melanosis,Auditory nerve nerve-cell tumor,Nerve-cell tumor,Neuroacanthocytosis,Schindler type neuroaxonal dystrophies,Brain iron aggregation I types neurodegeneration (NBIAI),Auditory nerve neurofibroma,Neural congenital multiple arthrogryposis,Neuromyelitis optica,Neuromyotonia,Local nerve myotonia,Neuromyotonia,Diffusion,Family,Neuromyotonia,Local nerve myotonia,Distribute,Schindler type neopiran aixs cylinder dystrophias,Adult neopiran ceroid lipofuscinosis,Juvenile neuronal waxy lipophilic disease,I type neopiran ceroid lipofuscinosis,Neuronophage acute glucose cerebrosidase deficiency disease,Nerve amyloidosis,Nerve athlete's foot,Neural incoordination and retinitis pigmentosa,Brachialpelxus DPN syndromes,Heredity sensorium DPN I types,Transmissibility sensorium DPN II types,Neuropsychiatry porpharia,Neutral lipid storage disease,Nev II,Nevoid basal cell carcinoma syn drome,Mole,Mole is porous,Acne-like nevus,Naevus anaemicus,Jadassohn sebum type moles,Nezelof syndromes,Nezelof type thymic aplasias,Nezelof type severe combined immunodeficients,Multiple neurofibromatosis,Multiple neurofibromatosis 1,Multiple neurofibromatosis 2,Multiple neurofibromatosis -1,Multiple neurofibromatosis -2,Standard serum,Niemann Pick diseases,Niemann Pick disease A types (acute neuronophage type),Niemann Pick disease Type Bs,Niemann Pick diseases c-type (chronic neuronophage type),Niemann Pick disease D types (variation of Nova spills),Niemann Pick disease E types,Niemann Pick disease F types (sea blue tissue cells disease),Yctalopia (disease),Nigrospinodentatal degenerates (Nigrospinodentatal Degeneration),NII kawakuroki syndromes (N II kawakuroki Syndrome),Neonatal lupus syndrome,Nodular melanoma,Noack syndrome I types,Hereditary primary nocturnal myoclonia,Tubercle corneal degeneration,Non- epidermolysis hyperkeratosis congenitalis sample erythroderma,Non- epidermolysis hyperkeratosis congenitalis sample erythroderma,Obstructive hydrocephalus,Non-deletion type alpha Thalassemia/mental retardation,Syndrome,Non- ketone hyperglycinemia I types (NKHI),Nonketotic hyperglycinemia,Nonlipid reticuloendotheliosis,Non- neuronophage chronic adult Gaucher disease,Non- scarring epidermidolysis bulla generation,Non- arteriosclerotic cerebral calcification,Nonarticular rheumatism,Non- brain type,Teenager's Gaucher disease,Non- diabetic diabetes,Non local ischemic-type caradrin myopathy,Slight coronary artery non-ketosis hypoglycemia and meat [poison] base deficit disease caused by lacking,Non- ketosis hypoglycemia caused by fatty acyl-CoA dehydrogenase missing,Non- ketone glycinemia,Nonne ' s syndromes,Nonne-Milroy-Meige syndromes,Non- opalescence opalescent dentine,Non- postpartum galactorrhoea amenorrhoea,Non-secretory myeloma,Aspherical erythrocyte hemolysis type anaemia,Nontropical sprue,Noonan syndrome (noonan syndrome),Norepinephrine,Standard presses hydrocephalus,Norman-Roberts syndromes,Norrbottnian Gaucher diseases,Norrie diseases,Norway's type heredity cholestasia,Niman-Peek's disease (niemann-Pick disease),NPS,Necrotizing sialometaplasia,Normal serum albumin,Nape dystonia dementia,Nutritional neuropathy,Nyhan syndromes,Ocular spine dysplasia spectrum,Obstructive apnea,Obstructive hydrocephalus,Obstructive sleep apnea,Oat cells cancer syndrome,It is engaged Thromboaortopathy,Oat cells cancer syndrome,Hidden encephalic vascular malformation,Occult spinal dysraphism sequence,Ochoa syndromes,Ochronosus,Ochronotic arthritis,Eye-brain-kidney syndrome,OCRL,Otocephaly,Ocular albinism,Eyes bleb,Eyes myasthenia gravis,Ocular spine dysplasia,Eye ear vertebra (depauperation) syndrome,Behet's disease,Eye brain syndrome is with hypopigmentation,Eye brain skin syndrome,Eye brain kidney,Oculocerebralrenal dystrophy,Eye skin albinism,Eye-brain-kidney syndrome,Oculocraniosomatic syndrome (agensis),Eye skin albinism,Eye skin albinism Chediak-Higashi,Eye,Tooth,Refer to (toe) depauperation,Eye tooth digital synthesis disease,Oculo-dento-osseous dysplasia,Eye intestines and stomach muscular dystrophy,Eye intestines and stomach muscular dystrophy,Oculomandibulodyscephaly is with hypotrichosis,Francois' syndrome,Eye movement contracture and muscle atrophy,Oculosympathetic paralysis,ODD syndromes (eye tooth refers to (toe) syndrome),Odontogenic tumor,Tooth trichodes syndrome,Mouth-face-means (toe),Mouth-face-means (toe) syndrome,Ohio type amyloidosis disease (VII types),Otitis interna,Congenital Inner Ear is scorching,Tardy otitis interna,Old-field syndrome,Oligohydramnios sequence,Oligophrenia,Oligophrenic is malnutritive,Olvopontocerebellar atrophy,Olvopontocerebellar atrophy is with dull-witted and extrapyramidal sign,Olvopontocerebellar atrophy is with retinal degeneration,Olvopontocerebellar atrophy I,Olvopontocerebellar atrophy II,Olvopontocerebellar atrophy III,Olvopontocerebellar atrophy IV,Olvopontocerebellar atrophy V,Osteochondromatosis,Ollier osteochondromatosis,Omphlexoche-visceral obesity-macroglossia (disease) syndrome,Ondine is cursed,Onion-bulbar neurons lesion,Longstamen Onion Bulb DPN,Onycho-osteodysplasia,Onychotrichodysplasia is with neutrophilic granulocytopenia,Olvopontocerebellar atrophy,Olvopontocerebellar atrophy I,Olvopontocerebellar atrophy II,Olvopontocerebellar atrophy III,Olvopontocerebellar atrophy IV,Olvopontocerebellar atrophy V,Ear-palate-refers to (toe) syndrome,Ear-palate-refers to (toe) syndrome i type,Ear-palate-refers to (toe) syndrome i I types,Ear-palate-refers to (toe) I syndromes,Ear-palate-refers to (toe) II syndromes,Ophthalmoplegia disease,Ophthalmoplegia-enteral Pseudo-Obstruction,Ophthalmoplegia,Retina and cadmium myopathy pigment degeneration,Ophthalmoplegia plus syndrome,Ophthalmoplegia syndrome,Opitz bundle-branch block syndromes,OpitzBBB/G compound syndromes,Opitz BBBG syndromes,Opitz-Frias syndromes,Opitz G syndromes (hypertelorism-hypospadia is integrated),Opitz G/BBB syndromes,Opitz g syndromes (hypertelorism-hypospadias syndrome),Opitz-Kaveggia syndromes,Opitz Oculogenitolaryngeal syndromes,Opitz trigonocephalias (deformity) syndrome,(eye distance is wide for opitz syndrome,Hypospadia syndrome),Opsoclonus,Opsoclonus-myoclonia,Opthalmoneuromyelitis,Eyesight atrophy DPN and deafness,Neuromyelitis optica,Optic nerve neuromyelities,Opticomyelitis,Chiasmal arachnoiditis,Mouth facial cleft seam,Mouthful face is done exercises obstacle,Mouth face dystonia,Oral-facial-digital syndrome,Oral-facial-digital syndrome I types,Oral-facial-digital syndrome I,Oral-facial-digital syndrome II,Oral-facial-digital syndrome III,Oral-facial-digital syndrome IV,Socket of the eye (interior) tumour is with brain and Lesional Skin deformity,Ornithine carbamyltransferase,Ornithine transcarbamylase defect,Orocraniodigital syndromes,Oral-facial-digital syndrome,Oromandibular dystonias,Orthostatic hypotension,Hereditary hemorrhagic telangiectasia (hereditary hemorrhagic telangiectasia),Osseous-Oculo-Dento depauperations,Osseous-Oculo-Dento depauperations,Osteitis deformity,Chondro osteodystrophy deformity,Osteochondrosarcoma,Melnick and Needles type osteodysplasties,Osteogenesis imperfecta,Osteogenesis imperfecta,Osteogenesis imperfecta congenita,Delayed osteogenesis imperfecta,Osteohypertrophic capillary hemangiomas,The many types of osteopathy Hyperostotica sclerotitis of baby,The many types of osteopathy Hyperostotica sclerotitis of baby,Osteopathyrosis,Osteopetrosis,Adult type autosomal dominant osteopetrosis,The pernicious autosomal dominant osteopetrosis of infantilism,The intermediate slight autosomal recessive inheritance osteopetrosis of typicalness,Osteosclerosis fragilis generalisata,Osteosclerotic myeloma,Ostium primum defect (including endocardial cushions defect),Ostium secundum defect,Ornithine transcarbamoylase deficiency,Ear nose larynx palate finger-like syndrome,Ear-palate-finger syndrome I types,Ear-palate-finger syndrome II types,Ear-palate-finger syndrome I types,Ear-dysplasia dentalis,Otopalatodigital syndrome,Otopalatodigital syndrome II types,Oudtshoorn skins,Ovary dwarf's syndrome type,Ovarian hypoplasia syndrome type,Ovarian wedge resection,Oxalosis,Oxidizing ferment is not enough,Tip [deformity],Tip [deformity]-tip [deformity],P-V,Shaking plasy,Acrodermatitis papulosa infantum,Onychauxis Ichtyosiforme,Congenital finger (toe) onychauxis is with natal teeth,Congenital finger (toe) onychauxis,Congenital finger (toe) onychauxis spreads seborrheic keratosis (through ketone),Jadassohn-Lewandowsky type is congenital to refer to (toe) onychauxis,Paroxysmal atrial fibrillation is with multi-system atrophy,Paget diseases,Bone Paget diseases,Chest Paget diseases,Nipple Paget diseases,Nipple-areola Paget diseases,Pagon syndrome,Painful ophthalmoplegia,PAIS,Palatal myoclonus,Palate-ear-refers to syndrome II types,List syndrome,Pallister-Hall syndromes,Pallister-Killian piebald syndromes,Li Siteshi piebald aneuploidy,Li Siteshi piebald syndromes,Tetrasomy 12p Pallister chimera syndromes,Pallister-W syndromes,Hyperkeratosis of palms and soles and bald,Paralysis,Pancreatic fibrosis,Aprotinin hyposecretion and thrombophthisis,The ulcerogenic tumour syndrome of aprotinin,Panmyelophthisis,Panmyelopathy,The adjoint neurodegeneration of pantothenate kinase (PKAN),Papillon-Lefevre syndrome (hyperkeratosis of palms and soles-periodontitis complex),Papillotonic Psuedotabes,Paralysis period is tetanic,Atrophic beriberi,Paralytic brachial neuritis,The Paramedian recessed-popliteal pterygium syndrome of lower lip lip,Side center diencephalon syndrome,Paramyloidosis,Complicated myoclonia,Myotonia congenita,Von Eulenburg myotonia congenitas,Parkinson diseases,Paroxysmal anterior chamber's tachycardia,Paroxysmal cold hemoglobinuria,Paroxysmal dystonia,Paroxysmal choreoathetosis,Paroxysmal Kinesigenic dystonias,Paroxysmal nocturnal hemoglobinuria (and having anaemia),Common paroxysmal hemoglobinuria,Hypnolepsy,Parrot syndrome,Parry diseases,Parry-Romberg syndrome,Parsonage-Turner syndromes,Partial androgen insensitivity syndrome,No. 4 the short arm of a chromosome excalations,No. 5 the short arm of a chromosome excalations,No. 9 the short arm of a chromosome excalations,3q replicates syndrome in part,15q replicates syndrome in part,Local facial paralysis is abnormal with uropoiesis,Partial lipodystrophy,The local monosomy of No. 11 chromosome long arms,Local backbone consciousness syndrome,11q partial trisomies,Partington syndromes,Paroxysmal auricular tachycardia,PDA,Pathology myoclonia,Few joint type-starting juvenile arthritis,Paullinia,Pregnancy and childbirth,Atrioventricular beam,PC lacks,PC lacks A clusters,PC lacks B clusters,Myotonia congenita,Eulenburg disease,PCC lacks,The cold property hemoglobinuria of paroxysmal,PCLD,Plasma coagulation time,Diastolic pressure,Patent ductus arteriosus,PDH lacks,Pearson's syndrome pyruvate carboxylase deficiency,Children's obstructive sleep apnea,Cast off a skin syndrome,Pelizaeus Merzbacher disease,Pelizaeus-Merzbacher disease (familial centrolobar sclerosis),Rough skin-cerebellar ataxia-kidney acidaminuria syndrome,Pelvic pain syndrome,Pemphigus vulgaris,Pena ShokeirII pattern synthesis diseases,Pena Shokeir syndrome II types,Penile fibromatosis,Penis fibrosis,Penile induration,Five X syndrome,Cantrell pentalogys,Pentalogy syndrome,Pentasomy X,Phosphoenolpy ruvate carboxy kinase is lacked,Pepper syndromes,Perheentupa syndrome,Periarticular,Muscular rheumatism,Pericardium shrinks not enough with growth,All (enclosing) amyloidosis of collagen,Perinatal period polycystic kidney disease,Perineum anus,Periodicity amyloid syndrome,Periodic sleep morbid hunger,Periodically comprehensive peripheral nerve disorder,Periodicity view membrane vesicle sexual involution,Periodicity dysosteogenesis-nose hypoplasia-mental retardation,Peripheral neuritis,Peripheral neuropathy,Peritonaeum pericardial diaphragmatic hernia,Pernicious anaemia,Peromelia is with micrognathia (disease),Peroneal muscular atrophy,Nervus peronaeus neural paralysis,Peroutka sneezes,Peroxidase acyl coenzyme A is aoxidized,Peroxidase beta oxidation is not normal,Peroxidase bifunctional enzyme,Peroxidase acetyl-CoA acetyltransferase,Peroxidase acetyl-CoA acetyltransferase defect,Truncus arteriosus,persistent,Perthes disease,Lapse,Petit mal variant,Outstanding Gus (family name) syndrome,Peutz-Touraine syndromes,Fibrous cavernositis of penis,Fei Fu,Pfeiffer syndrome I types (acrocephalosyndactylia),Prostaglandin a I,Prostaglandin a II,Prostaglandin a III,Phosphoglycerate kinase,PH I types,PH I types,Pharyngeal pouch syndrome,Pulmonary heart disease short chain acyl coa dehydrogenase is lacked,Phenylalanine hydroxylase deficiency,Phenylalaninemia,PKU,Folling's disease,Phocomelia,Sea dog limb syndrome,Phosphoenolpyruvate carboxykinase deficiency,Phosphofructokinase deficiency,Phosphoglycerate kinase deficiency,Phosphoglycerokinase,The kinases of phosphorylase six is lacked,Phosphorylase deficiency glycogen storage disease,Hepatic phosphorylase kinase is lacked,Light sneezing reflex,Light sneeze,Phototherapeutic keratectomy,PHS,Physicist's John's dalton,Phytanic acid storage disease,Pi Phenotype ZZ,PI,Asphyxia apoptosis,Pick disease,Pik Wei Kan (family name) syndrome (the low obesity syndrome of aloveolar ventilation),Sieve's Pierre spot (family name) robin,Sieve's Pierre spot (family name) compound,Sieve's Pierre spot (family name) sequence,Sieve's Pierre spot (family name) syndrome,Sieve's Pierre spot (family name) syndrome is with Hyperphalangy and refers to (toe) bending,Pierre-Marie diseases,Globus pallidus black substance rubrum pigmentation is degenerated,Pili Torti and nerve deafness,PiliTorti- sensory nerve hearing impairments,Pituitary dwarfism II types,The postoperative hypophysis of epinephroectomy (swollen) knurl,Pityriasis pilaris,Hair (keratosa) pityriasis rubra,Pancreaticojejunostomy,PKAN,POLYCYSTIC KIDNEY DISEASE,POLYCYSTIC KIDNEY DISEASE 1,POLYCYSTIC KIDNEY DISEASE 2,POLYCYSTIC KIDNEY DISEASE 3,Phenyl ketonuria,Phenyl ketonuria 1,Plagiocephaly,Plasma cell myeloma,Plasma cell myeloma,Plasma cell leukemia,Plasma thromboplastin component deficiency,Plasma transglutaminase is lacked,Plastic induration cavernous body,Plastic induration,Poliomyelitis,Fissured tongue,PLS,Pectoralis major muscle defect,Pneumorenal syndromes,Paroxysmal nocturnal hemoglobinuria,,Peripheral nerve myelin,Purine nucleoside phosphorylase) lack,Polycystic ovarian disease,Paroxypropion,Poikiloderma atrophy and racing current,Poikiloderma congenitale,Poland's anomaly,Polish sequence (side chest muscle is lacked and syndactylism),Polish dactylion,Poland's syndrome (pectoralis major is without development and brachydactylia syndrome),Progressive poliodystrophia cerebri,Panarthritis Enterica,PAN,The juvenile arthritis I types for multi-joint-rise,The juvenile arthritis II types for multi-joint-rise,The juvenile arthritis I types and II types for multi-joint-rise,Polychondritis,POLYCYSTIC KIDNEY DISEASE,Marrow type POLYCYSTIC KIDNEY DISEASE,Polycystic liver disease,Polycystic ovarian disease,Polycystic liver nephrosis,Refer to (toe)-Joubert syndromes more,Polydysplastic epidermolysis bullosa,Many dystrophic oligrophrenia,Growth hormone deficiency,Many (endocrine) gland autoimmune syndromes type IIIs,Many (endocrine) gland autoimmune syndromes II types,Many (endocrine) gland autoimmune syndromes I types,Many (endocrine) gland autoimmune syndromes II types,(hormone after excision of endocrines) deletion syndrome II types,Polyglandular syndrome,Polyadenous body macular degeneration,Polymorphic macular degeneration,Platelet glycoprotein Ib polymorphisies,Polymorphous corneal dystrophy,Rheumatic polymyopathy,Polymyositis and dermatomyositis,Primary agammaglobulinemia,Polyneuritis,DPN-deafness-ophthalmatrophy,Peripheral polyneuropathy,DPN and polyradiculoneuropathy,Multiple bone fibrous dysplasia,Polyostotic,Schilder's disease,Familial,Gardner type polyposises,Polyposis paramnesia intestines,Polyp-osteoma-epidermis capsule syndrome,Polyposis cutaneous pigmentation and nail change,Polyp spot syndrome,Recidivity chronic hyperplastic perihepatitis,Y polysomies,Refer to (toe) (deformity) with unusual shape cranium more,Referring to (toe) (deformity)-dysmorphism cranium Greig types more,Pompe disease,Glycogen storage disease-II types,Popliteal pterygium syndrome,Porcupine people,Hole brain,Hole brain,Pancreatin deaminase,Porpharia,Acute intemittent porphyria,ALA-D porpharias,Porphyria cutanea tarda,Porphyria cutanea tarda hereditaria,Porphyria cutanea tarda symptomatica,Hepatic porphyria gonorrhea Variegate,Sweden's type porphyria,Diversity porphyria,Accute ratio coughs up sclererythrin,Porphyrin,Alopecia areata,Port-wine stain,Amyloidosis,Portuguese type,Polyneuritis after infection,Progressive lafora's disease,Progressive osteodysplasty,The pale degeneration syndrome of progressive,Progressive spinobulbar muscular atrophy,Stein-leventhal syndrome,Progressive systemic sclerosis disease,Progressive tapetochoroidal dystrophy,Proline oxidase deficiency,Propionic acidemia,Propionic acidemia I types (PCCA missings),Propionic acidemia II types (PCCB missings),Propionyl CoA carboxylase is lacked,Red color weakness,Protanopia,Congestive heart failure Secondary cases protein losing enteropathy,Proteus syndromes,Base portion including 4q is lost,Primary retinitis pigmentosa,PRS,Prune belly syndrome,Osteitis deformans,Viscous fat stores up disease type III,False malnutrition,False black (color) acanthosis,Pseudoachondroplasia,Pseudocholinesterase deficiency,Pseudogout,Hemogenia,Pseudo,False hermaphroditism-kidney disorder-Wilm tumours,Erb's atrophy,Pseudohypoparathyroidism,Pseudohypophosphatasia,Pseudopolyembryony,Pseudothalidomide syndrome,,Nevus elasticus,Psoriasis,Psorospermosis follicularis,False gestation,Pellegrini-Si Dide (family name) syndrome,Psychomotor shakes,Psychomotor epilepsy,Psychomotor equivalent epilepsy,PTC deficiency diseases,It is pteryium,Pterygium colli,Large area is pteryium,Pterygolymphangiectasia syndrome,Pulmonary atresia,Pulmonary lymphangiomyomatosis,,Pulmonary stenosis,Lung is narrow-interventricular septal defect,Denticle,Marrow depauperation,Aortic arch syndrome,Simple alymphocytosis,Simple epithelial tissue cells increase disease,Purine nucleoside phosphorylase deficiency,Purine nucleoside phosphorylase deficiency,Purtilo syndromes,Nevus elasticus,Nevus elasticus dominant character,Nevus elasticus recessive character,Pycnodysostosis,Pycnodysostosis,Lapse,Pyroglutamic aciduria (disease),Pyroglutamic aciduria (disease),Pyrrolin carboxylate dehydrogenase is lacked,Pyruvate carboxylase deficiency,Pyruvate carboxylase deficiency A clusters,Pyruvate carboxylase deficiency B clusters,Pyruvic dehydrogenase is lacked,Pyruvate kinase deficiency,q25-qter,q26or q27-qter,q 31 or 32-qter,Extend with the QT of extracellular hypocalcipexy,Non- congenital deafness QT extensions,With congenital deafness QT extensions,Cerebral palsy quadriplegia,Cerebral paralysis quadriplegia,Quantum scatters,r4,r6,r14,r18,r21,r22,Rachischisis posterior,Agenesis of radius-megacaryocyte cytopenia,Radial aplasia-thrombocytopenia syn-drome,Radial aplasia-thrombocytopenia syn-drome neural paralysis,Radicular neuropathy consciousness,Radicular neuropathy consciousness is shunk back,Radicular dentin dysplasia,Anxious hair property dystonia Parkinson (family name) syndrome,Rapp-Hodgkin syndromes,Rapp-Hodgkin (hypohidrosis) ectodermal dysplasia's syndrome,Ectodermal dysplasia's syndrome of Rapp-Hodgkin hypohidrosis,Rare ataxia hereditaria changes and deaf with multiple neuritis caused by being hydroxylated enzyme defect by phytanic acid,Rautenstrauch-Wiedemann syndromes,Rautenstrauch-Wiedemann type neonates Ji Fude (family name) syndrome,Raynaud phenomenons,Recombinant dry plasma,Reactive functional hypoglycemia,Congenital stealthy myotonia secondary response hypoglycemia,Neurofibromatosis,Proctoperineoplasty,Recurrent vomiting,Sympathetic reflex neurovascular dystrophia,Reflex sympathetic dystrophy syndrome,It is ametropia,Refractory anemia,Frost paralysis,Refsum disease (heredopathia atactica polyneuritiformis),Refsum disease (heredopathia atactica polyneuritiformis),Regional ileitis,Reid-Barlow syndromes,(sexual gland is insufficient for Reifenstein syndrome,Orchiatrophy Oligospermia,Bifid urethra,Womanlike breast),Reiger exception-growth retardations,Reiger syndromes,Reimann periodic diseases,Reimann syndromes,Reis-Bucklers corneal dystrophies,Reiter syndromes,Recur Guillain-Barre syndromes,Recurrence-remitting type multiple sclerosis,Renal aplasia,Kidney development is bad,Heredity,Bad-Loken-Senior types retinene the hypoplasia of kidney development,Renal glucosuria,Renal glucosuria A types,Renal glucosuria Type B,Renal glucosuria is O-shaped,Renal-oculocerebrodystrophy,Kidney-retinene dysplasia is with (kidney) medullary cystic disease,Intentional myoclonus,Atomic force intentional myoclonus,Miller syndrome,Atomic force intentional myoclonus,Miller syndrome,Postaxial polydactyly,Postencephalitic intentional myoclonus,Corneal dystrophy heredity afterwards,Dejerine-Roussy syndrome,Archnoiditis after myelographin use,Postnatal cerebral palsy,Postoperative cholestasia,Postpartum galactorrhea amenorrhea syndrome,Postpartum hypopituitarism,Postpartum panhypopituitarism syndrome,Postpartum panhypopituitarism,Postpartum pituitary necrosis,Postural hypotension,Potassium-losing nephritis,Lose potassium syndrome,Potter I type infantile polycystic kidney diseases,PotterIII type infantile polycystic kidney diseases,Persistent pulmonary hypertension,PPS,Prader-Willi syndrome,Prader-Labhart-Willi de Toni Fanconi syndromes,77 tyrosine amyloidosis of prealbumin,Pre-excitation syndrome,Pregnenolone is lacked,Premature's atrial contraction,Progeria syndrome,Premature's ventricular contraction,Neonate's ventricular complex,Neonate or childbirth when neuroaxonal dystrophy,Alzheimer's disease,Presenile macular degeneration,Primary adrenal insufficiency,Primary agammaglobulinemia,Primary aldosteronism,Primary alveolar hypoventilation,Primary amyloidosis,Primary anemia,Primary tinea pedis,Primary bile,Primary biliary cirrhosis,Primary brown's syndrome,Primary meat [poison] base deficit disease lacks,Primary maincenter hypoventilation syndrome,Primary ciliary dyskinesia type,Primary cutaneous amyloidosis,Primary dystonia,Initial stage adrenocortical insufficiency,Primary familial maxilla hypoplasia,Primary hemochromatosis,Essential hidrosis (disease),Primary hyperoxaluria [I types],The type of primary hyperoxaluria 1 (PHi),Primary hyperoxaluria I types,Primary hyperoxaluria II types,Primary hyperoxaluria type III,Primary hypogonadism,Primary intestinal lymphangiectasia,Primary lateral sclerosis,Primary extragenetic amyloidosis,Primary inaccessible pulmonary vascular disease,Primary progressive multiple sclerosis,Primary pulmonary hypertension,Primary progressive multiple sclerosis,Primary pulmonary hypertension,Primary Dyslexia,Primary renal glucosuria,Primary sclerosing cholangitis,Hemorrhagic thrombocythemia,Primary tumor of central nervous system,Visual agnosia,Proctocolitis,Proctocolitis,Adult Ji Fude (family name) syndrome,Children Ji Fude (family name) syndrome,Lucky Ford (family name) is short and small,Progeriod short statures are with mole,De Barsy Progeriod syndromes,The autonomous exhaustion of progressive is with multi-system atrophy,Carry out sex bulbus paralysis,Including carrying out sex bulbus paralysis,Progressive cardiomyopathy is denatured lentiginosis,Family's progressive cerebellar ataxia,Progressive poliodystrophia cerebri,Poliodystrophia (brain),Progressive diaphyseal dysplasia,Progressive facial hemiatrophy,Progressive familial myoclonic epilepsy,Progressive hemifacial atrophy,Progressive is insensitive,Progressive infant brain poliodystrophia,Progressive lenticular degeneration,Progressive lipodystrophia,Children's progressive muscular dystrophy,Familial kidney-malnutritive to retina,Kidney-retina syndrome,Rendu-Osler-Weber syndrome,Respiratory acidosis,Respiratory tract dysfunction,Breathe myoclonia restless legs syndrome,RestrictiveCardio myopathy,Retention Hyperlipemia,Rehtore syndromes (unobvious),Reticular dysgenesis,Retinal Aplastic-Cystic Kidneys-JoubertSyndrome,The cone is degenerated,Retinal cone dystrophy,Retinal cone-rod dystrophy,Retinitis pigmentosa,Retinitis pigmentosa and congenital deafness,Retinal neuroblastoma,Retinol defect,Retinoschisis,Teenager's retinoschisis,Retraction syndrome,DPN after eyeball,Dejerine-Roussy syndrome,Rett syndromes,,Reye syndromes,Reye syndromes,RGS,Rh blood factors,Rh diseases,Rh factor incompatibilities,Rh incompatibilities,Rh [blood group] incompatibility,Rheumatic fever,Rheumatic fever,Rheumatic fever,The big cerebral arachnoiditis of nose nasal sinus,Acra chondrodysplasia punctata (RCDP),Acatalasemia,Traditional Refsum is sick (heredopathia atactica polyneuritiformis),Rhythmicity myoclonia,Rib GapDefects with Micrognathia,Ribbing diseases (unobvious),Ribbing diseases (epiphysis end achondroplasia),Richner-Hanhart Syndrome,Rieger syndromes,Rieter syndromes,Right ventricle fibrosis,Riley Day syndrome,Riley-Smith syndrome,Circular chromosome 14,Circular chromosome 18,Ring 4,The chromosome of ring 4,Ring 6,The chromosome of ring 6,Ring 9,The chromosome R9 of ring 9,Ring 14,The chromosome of ring 15 (mosaic pattern),Ring 18,Ring chromosome 18,Ring 21,The chromosome of ring 21,Ring 22,The chromosome of ring 22,Dermatitits exfoliative infantum (exfoliative dermatitis of newborn),Ritter-Lyell syndromes,RLS,RMSS,Roberts SC- manomelia syndromes,Roberts syndrome,Luo Baici tetraphocomelia syndromes,Robertson is ectodermal dysplasia,Robin anomalads,Robin sequences (primary defect-early stage hypoplasia of the mandible),Robin syndromes,Robinow nanisms,Robinow syndromes,Robinow syndrome dominant forms,Robinow syndrome recessive forms,Nemutogen,Roger's disease (congenital ventricular septal defect),Rokitansky diseases,Romano-Ward syndrome,Romberg syndrome (progressive facial hemiatrophy),Without rooted tooth,Rosenberg-Chutorian syndromes,Rosewater syndromes,Rosselli-Gulienatti syndromes,Rothmund-Thomson syndromes (congenital poikiloderma atrophicans vasculare),Roussy-Levy syndrome,RP,X RS,RS (rubinstein syndrome),RSDS,RSH syndromes,RSS (Russian spring and summer encephalitis),RSTS,RTS (Robinstein (family name) syndrome),Congenital rubella,Rubinstein syndrome,Rubinstein's syndrome,Lu-tower Er Shi thumbs and the broadening syndrome of hallux toe,Rufous albinism,Ruhr syndromes,Russell diencephalon cachexia,Russell syndromes,Russell syndromes (diencephalic syndrome of infancy),Russell-Silver nanisms,Russell-Silver syndrome,X russell-Silver syndrome,Ruvalcaba-Myhre syndrome (RMSS),Ruvalcaba syndrome,Ruvalcaba type osseous dysplasias are with mental retardation,Sacrum bone deterioration,Congenital sacrum dysosteogenesis,SAE,Saethre-Chotzen syndrome,Sakati,Sakati syndromes,Sakati-Nyhan syndromes,Spasmus nutans,Salivo-sudoriparous syndrome,Salzman nodular corneal dystrophies,Sandhoff disease,Sanfilippo syndromes,Sanfilippo types A,Sanfilippo types B,Santavuori diseases,Santavuori-Haltia diseases,Boeck sarcoidosises,Sarcoidosis,Sathre-chotzen,Saturday night palsy,SBMA (spinobulbar muscular atrophy),SC amputation syndromes,SC syndromes,SCA3,SCAD defects,The SCAD defects of adult's partial seizures,Congenital whole body SCAD defects,SCAD,SCAD defects,Scalded skin syndrome,Congenital scalp defect,Cymbocephaly,Shoulder blade is raised,Scapuloperoneal myopathies,Scapuloperoneal muscular dystrophies,Scapuloperoneal Syndrome disease types,Cicatrization bulla,SCHAD,Schaumann diseases,Her husky syndrome,SchereshevkII-Turner syndromes,Adrenoleukodystrophy (Schilder),Xi Erde encephalitis,Adrenoleukodystrophy,I types adrenoleukodystrophy (baby's breaking-out),The adrenoleukodystrophy of infancy,Adrenoleukodystrophy,II types adrenoleukodystrophy (adult onset),Schinzel syndromes,Schinzel-Giedon syndromes,Schinzel acras explain syndrome,Schinzel-Giedion Middle faces are shunk back syndrome,Fissure,Schizophrenia,Stupid type metaphysial chondrodysplasia,Stupid type metaphyseal dysostosis,Schmid-Fraccaro syndrome,Schmidt syndrome,Schopf-Schultz-Passarge syndromes,Schueller-Christian diseases,Schut-Haymaker types,Schut-Haymaker-Passarge syndromes,1A types and 1B type schwartz-Jampel syndromes,Schwartz-Jampel syndrome,2 type schwartz-Jampel syndromes,SCID,Scleroderma,The gradual family's sclerosis of whole body,Dispersivity family cerebral sclerosis,Sciatic nerve crush,Scott cranium syndromes are with baryencephalia,Ditch tongue,SCS,SD,SDS,SDYS,Season membranous conjunctivitis,Naevus sebaceus syndrome,Naevus sebaceus,Seborrheic keratosis,Seborrheic wart,Seckel syndrome,Plug Kerr-type dwarf levies,Secondary cases CHB,Secondary amyloidosis,Secondary cases blepharospasm,Secondary cases nontropical sprue,Secondary cases brown's syndrome,Secondary cases athlete's foot,Secondary cases whole body amyloid lesion,Secondary cases dystonia,Secondary cases secretory component deficiency,Secondary cases I gA defects,SED is slow,Congenital SED,SEDC,Intermittent wire is without mole,Intermittent dystonia,Intermittent myoclonia,Seip's syndrome,Seitelberger diseases,Epilepsy,IgG subtype-selective defects,Selective mutism,IgG subtype-selective defects,Selective IgM deficiency,Selective mutism,Selective IgA deficiency,Self healing cytosis disease,Semilobar holoprosencephaly,Seminiferous tubule dysgenesis,Senile retinoschisis,Senile wart,Senior-Loken syndromes,I type hereditary sensory neuropathies,II type hereditary sensory neuropathies,I type hereditary sensory neuropathies,Sensory radicular neuropathy,Degeneration sensory radicular neuropathy,The gradual granulomatosis of septicopyemia,Septo-Optic depauperations,Blood plasma circumscribed meningitis,Serum protease inhibitors defect,Blood plasma carnosine enzyme defect,Setleis syndrome,Severe combined immunodeficiency,Severe combined immunodeficiency is with adenosine deaminase deficiency,Severe combined immunodeficiency (SCID),Property conversion,Sexual infantilism,SGB syndromes,Sheehan syndrome,Shields type dentinogenesis imperfectas,Herpes zoster,Varicella virus,Ship beriberi,SHORT syndromes,The disconnected chromosome deficiency syndrome of arm 18,Short chain fatty acyl-CoA dehydrogenase (SCAD) defect,Of short and small stature and face telangiectasis,Property face/skeletal abnormality-retardance-macrotooth (macroteeth) of short and small stature,Of short and small stature-paratonia-Rieger exception-delayed dentitions,Of short and small stature-onychodysplasia,Of short and small stature and face telangiectasia erythema,SHORT syndromes,Shoshin athlete's foots,Shoulder girdle syndrome,Shwachman-Diamond syndrome,Shwachman syndromes,Shwachman-Diamond-Oski syndromes,Shwachmann syndromes,Shy-Drager syndrome,Shy-Ma gee syndromes,SI defects,Sialidosis type I,II type baby's sialidosises,Sialidosis,Sialolipidosis,Sick sinus syndrome,Sickle-cell anemia,Sickle cell disease,Sickle cell-hemoglobin C disease,Sickle cell-hemoglobin D disease,Microdrepanocytosis,Sickled-shapedcell feature,Sideroblastic anemia,Sideroblastic anemia,Sideroblast stain,SIDS,Siegel-Cattan-Mamou syndromes,The coloured skin disease of Siemens-Bloch types,Siemens syndrome,Silver's syndrome,Sai Erwo-russell's dwarf disease,Silver-Russell syndrome,Simmond diseases,Siemens syndrome,Single epidermolytic bleb,Single malformation syndrome,Simpson-Golabi-Behmel syndromes,Sinding-Larsen-Johansson diseases,Singleton-Merten syndrome,Sinus arrhythmia,Venous sinus,Nodal tachycardia,Sirenomelia sequence,Sirenomelus,Situs inversus viscerum bronchiectasis and nasosinusitis,SJA syndromes,Sjogren Larsson concurrency ichthyosis,Sjogren syndrome,Syndrome,SJS (Stevens-Johnson syndrome),Skeleton development is abnormal,Weismann Netter Stuhl types skeleton development is abnormal,Decortication syndrome,Skin early stage of pyogenic infection of skin,Skull is asymmetric and mild mental retardation,Skull is asymmetric and slight and refers to,SLE (systemic lupus erythematosus),Sleep apnea,SLO,Sly syndromes,SMA,The acute SMA of baby,SMA I,SMA III,I types SMA,II types SMA,Type III SMA,SMA 3,SMAX1,SMCR,Smith Lemli Opitz syndromes,Smith Magenis syndromes,Smith Magenis chromosome regions,Smith-McCort nanisms,Smith diseases,Smoldering Myeloma,SMS,SNE,Light irradiation causes sneeze,Sodium vedproate,Solitary plasmacytoma of bone,Sorsby diseases,Sotos syndromes,Souques-Charcot syndromes,South African genetic porphyria,Dysphonia spastica,Accessory cramp,Accessory cramp,Spastic cerebral palsy,Spastic colon,Dysphonia spastica,Spastic paraplegia,SPD calcinosises,The specific antibody defect of normal immunoglobulin,Specific reading disability,SPH2,Congenital hemolytic jaundice,Spherocytosis,Spherophakia-brachymorphia syndrome,Sphingomyelin lipidosis,Sphingomyelinase deficiency,Spider finger,This Pierre plum Ilyushin-Vogt disease,Spielmeyer-Vogt-Batten syndromes,Spina bifida,Spina bifida aperta,Spina bifida aperta,Backbone arteriovenous malformation,Familial Occurrence spinal ataxia,Spinal ataxia,Spinal cord is crushed,Spinal cord diffusivity idiopathic hyperostosis,Backbone DISH,Duchenne-Arandisease,Various Duchenne-Arandisease,ALS type Duchenne-Arandiseasies,Duchenne-Arandisease-calf hyperplasia,I type Duchenne-Arandiseasies,Type III Duchenne-Arandisease,3 type Duchenne-Arandiseasies,Duchenne-Arandisease-calf hyperplasia,Spinal cord ossify archnoiditis,Spinal stenosis,Spinocebellar ataxia,I type Spinocerebellar Atrophies,I types Spinocerebellar Atrophy (SCA1),II types Spinocerebellar Atrophy (SCA II),Type III Spinocerebellar Atrophy (SCAIII),Type III Spinocerebellar Atrophy (SCA3),IV types Spinocerebellar Atrophy (SCAIV),V-type Spinocerebellar Atrophy (SCAV),VI types Spinocerebellar Atrophy (SCAVI),VII types Spinocerebellar Atrophy (SCAVII),Leptospiral jaundice,Splenic agenesis syndrome,It is splenoptosia,It is splenoptosia,Split hand-faciomandibular dysostosis,Split hand,Arthritis vertebralis,I type backbone dysplasias of rib,Spondyloepiphyseal dysplasia tarda,Spondylothoracic dysplasia,Spondylotic caudal radiculopathy,Sponge kidney,Spongioblastoma multiforme,Spontaneous hypoglycemia,High scapula,Spring ophthalmia,SRS,ST,Corrupt fish syndrome,Staphylococcus scalded skin syndrome,Stargardt diseases,Startle diseases,Status epilepticus,Steele-Richardson-Olszewski syndrome,Bristle disease,Stein-Levensa Er 's syndrome,Steinert disease,Stengel syndromes,Stengel-Batten-Mayou-Spielmeyer-Vogt-Stock diseases,Cholangitis,stenosing,Waist Taper Pipe is narrow,It is narrow,Steroid sulfatase deficiency,Stevanovic ectodermal dysplasias,Stevens Johnson syndromes,STGD,Stickler syndromes,Unyielding man's syndrome,Unyielding man's syndrome,Still diseases,Stilling-Turk-Duane syndrome,Stillis diseases,The myoclonia of stimuli sensitive,Unyielding man's syndrome,Unyielding man,Streeter Anomaly,Autosomal dominant type striatum substantia nigra degeneration,Striopallidodentate Calcinosis,Interstitial,Descemet membrane,Interstitial corneal dystrophy,Struma lymphomatosa,Sturge's syndrome,Sturge-Weber syndrome,Si Teqi-weber phakomatoss,Subacute spongiform encephalopathy,Subacute necrotizing cerebrospinal cord disease,Subacute spongiform encephalopathy,Subacute necrotizing encephalopathy,Subacute sarcoidosis,It is subacute thermophilic neural,Subaortic stenosis,Binswanger's disease,Subendocardial sclerosis,Succinylcholine sensitivity,Congenital invertase-isomaltase defects,Congenital sucrose-isomaltose malabsorption,Congenital sucrose intolerance,Pelizaeus-Merzbacher disease ADL,Pelizaeus-Merzbacher disease's pelizaeus-Merzbacher disease：Familial centrolobar sclerosis's type,Built-in pelizaeus-Merzbacher disease,SIDS,SudeckShi atrophies,Sugio-KajII syndromes,Summerskill syndromes,Acrocephalosyndactylism,SummittShi acrocephalosyndactylisms,Summitt syndromes,Brown's syndrome,Adrenal gland,Supraaortic stenosis,Supraventricular tachycardia,Surdicardiac syndromes,Jervell-Lange Nielsen syndrome,SVT,Sweat gland ulcer,Stink with perspiration syndrome,Sweet's syndrome,Swiss Cheese cartilage syndrome,A Peier (family name) syndrome,I types simultaneously refer to occur together microcephalus and baryencephalia,Syndromatic liver conduit hypoplasia,Syringomyelia,Systemic aleukemic reticuloendotheliosis,SA,Carnitine deficiency,Systemic elastorrhexis,Systemic lupus erythematosus,Systemic mast cell disease,Systemic mastocytosis,Systemic onset juvenile type arthritis,Systemic sclerosis,Systopic spleens,T lymphocyte lacks,Tachyalimentation hypoglycemia,Tachycardia,Acatalasemia,Aortic arch syndrome,Aorto-arteritis,Talipes calcaneus,Equinovarus,Tip-foot,Talipes varus,Talipes valgus,Tandem spinal stenosis,Tangier disease,Tapetoretinal degeneration,Radial aplasia-thrombocytopenia syn-drome,Tardive dystonias,Delayed onset muscular dystrophy,Tardive dyskinesia,Delayed onset mouth movement disorder,Tardive dystonias,Slow ulnar palsy,Target type red cell anaemia,Tarsomegaly,Glycogenosis type VII,Include TAS center lines shortage,TAS center lines lack,Tay Sachs sphingolipidosises,Tay Sachs disease,Tay Ichthyosis syndromes,Tay Sachs sphingolipidosises,Tay Ichthyosis syndromes,I type Taybi syndromes,Taybi syndrome,TCD,TCOF1,Treacher-Collins syndrome,Deformable dystonia,Hair-dental-bone syndrome,I type hair-dental-bone syndromes,II type hair-dental-bone syndromes,Type III hair-dental-bone syndrome,Telangiectasis,Telecanthus and associated malformation,Telecanthus-hypospadia syndrome,Temporal-lobe epilepsy,Temporal arteritis/giant cell arteritis,Temporal arteritis,Toxic epithelial necrosis,Tiltedly adhere on stndon sheath top,Tension force myalgia,Terminal deletion in 4q,Terrian corneal dystrophies,Corneal dystrophy syndrome,Notochord is limited syndrome,Tethered cord malformation sequence,Tethered cord syndrome,Neck notochord is limited syndrome,Tetrahydrobiopterin lacks,Tetrahydrobiopterin lacks,Method pleasure (family name) tetra logy,Tetraphocomelia-thrombocytopenic syndromes,No. 9 the short arm of a chromosome tetrasomies,9p tetrasomies,No. 18 the short arm of a chromosome tetrasomies,Thalamic syndrome,Thalamic pain syndrome,Thalamic hyperesthetic anesthesia,Medium-sized thalassemia,Minor thalassemia,Major thalaseemia,Thiamine deficiency,Thiamine response type maple syrup urine disease,Thin substrate membranous type nephrosis,Acetyl-CoA acetyltransferase deficiency disease,RCDP,Acyl-CoA dihydroxyacetone phosphate acyltransferase,Third and fourth pharyngeal pouch syndrome,Three degree of CHBs,Myotonic cataract,It is malnutritive that chest basin refers to (toe),Dorsal spinal canal,Chest and abdomen syndrome,Chest and abdomen ectopia cordis syndrome,Three M syndromes,The thin bone type nanisms of three M,Glanzmann and Naegeli thromasthenia,Primary thrombocytosis,Thrombocytopenia-absent radius syndrome,Thrombocytopenia hemangioma syndrome,Heredity thrombophilia caused by AT III,Thrombotic thrombocytopenic purpura,Thrombus Ulcerative Colitis,Thymic dysplasia with normal immunoglobulins,Thymic dysgenesis,DiGeorge type thymic dysgenesis,Primary thymic dysgenesis is with agamaglobulinemia,DiGeorge type thymic dysgenesis,Congenital thymic aplasia,Trigeminal neuralgia,Twitch,Tinel ' s syndromes,Tolosa-Hunt syndrome,Tonic spasm torticollis,Stiff pupil' Argyll Robertson pupil syndrome,Tooth and nail syndrome,Toxoplasmosis,Other viruses,Rubella,Cytomegalovirus,Herpes simplex infections,TORCH syndromes,Deformable dystonia,Torticollis,Ophygeneralized lipodystrophy,Completeness pulmonary vein is connected extremely,Touraine ' s aphthosises,Tourette syndrome,Tourette obstacles,Townes-Brocks syndromes,Townes syndrome,Poisoning paralytic anaemia,Toxic epidermal necrolysis,Toxopachyosteose DiaphysaireTibio-Peroniere,Toxopachyosteose,Toxoplasmosis and viral rubella,Cytomegalovirus,Herpes simplex,With or without atretolemia tracheoesophageal fistula,Tracheoesophageal fistula,Transient neonatal myasthenia gravis,Transiens atrioventricular septal defect,Transposition of conducting arteries,Guarded through transtelephonic,The thyroxine-binding globulin of methionine -30 amyloidosis (I types),The multiple bone connection syndromes of Trapezoidocephaly-,Te Leixie Collins (family name) syndrome,Treacher Collins-Franceschetti syndrome,Trevor diseases,Three atrial hearts,Hair-dental-bone syndrome,(hair) hair gray malnutrition,Hair-nose-refers to (toe) syndrome,Tricuspid atresia,Three functional protein deficiency diseases,Trigeminal neuralgia,Triglycerides stores obstacle,Long-chain fat acid oxidase is damaged,Trigonitis,Trigonocephalia,Trigonocephalia syndrome,Trigonocephalia C syndromes,Trimethylamino Aciduria,Three finger joint thumb hypoplasia distal end onychodystrophies,Triphalangeal thumb syndrome,Behcet triplet syndromes,Triple X syndrome,Triplo-Ⅳ syndrome,Triploidy syndrome,Triploidy,Triples syndrome,Trismus pseudocamptodactyly syndrome,Trisomy,Trisomy G syndromes,Trisomy X,6q partial trisomies,Part 6q trisomy syndromes,The trisomy of mosaic type 9,9P patau syndromes,Part 11q trisomys,The trisomy of mosaic type 14,The trisomy syndrome of mosaic type 14,21 (number chromosome) Trisomy syndromes,The trisomy of mosaic type 22,The trisomy syndrome of mosaic type 22,TRPS,TRPS1,TRPS2,TRPS3,True hermaphroditism,Truncus artiriosus,Tryptophan malabsorption,Tryptophan oxygenase syndrome,TS,TTP,TTTS,Nodositas (brain) is hardened,Tubular ectasia,Turcot syndrome,Turner syndrome,Heredity bone-refer to (toe) onychodysplasia syndrome,With the Turner syndrome of normal chromosomal (caryogram),Turner-Varny syndromes,Oxycephaly,Monodidymus transfusion syndrome,Twin-to-twin transfusion syndrome,A types,Type B,AB types,It is O-shaped,Type i diabetes,The incomplete male of I type familials,Incomplete male pseudohermaphroditism,I type Gaucher disease (familial splenic anemias,Gaucher disease),I type histocytosises,II types (PCCB deficiency diseases),II type tyrosinemias,Intermittent ischemic cardiac arrest,Congenital end multi-joint contracture,Type III Gaucher disease (familial splenic anemia,Gaucher disease),Type III tyrosinemia,Type III hereditary opalescent dentin,Typicalness retinoschisis,Tyrosinase feminine gender albinism (I types),Tyrosinase positive albinism (II types),I type tyrosinemia acutes,I type tyrosinemia chronic types,Tyrosinosis,Urea cycle enzyme disease,Ulcerative colitis,Non specific chronic ulcerative colitis,Ulna breast syndrome,Pallister ulna breast syndromes,Ulnar nerve paralysis,Diving Medical Association,Open rate of recovering,Unconjugated Benign Bilirubinemiav,Parathyroid activity is low,Unilateral ichthyosiform erythroderma is accompanied side cacomelia,Unilateral osteochondromatosis,Unilateral chest muscle defect and and refer to,Unilateral half depauperation,Unilateral macrencephaly,Unilateral partial lipodystrophy,Unilateral agenesis of kidney,Colon is irregular,Lafora's disease,Unverricht-Lundborg disease (myoclonia),Unverricht-Lundborg-Laf Disease,Progressive lafora's disease,Upper limbs-angiocarpy syndrome (Holt-Oram),Upper motor neuron disease,Upper respiratory tract apnea,Urea cycle disorder,Arginase type urea cycle disorder,Argininosuccinase type urea cycle disorder,Carbamyl phosphate synthetase type urea cycle disorder,Citrullinemia type urea cycle disorder,Typicalness N- acrylic glutamate synthetase type urea cycle disorders,Ornithine transcarbamylase type urea cycle disorder,Urethral syndrome,Urethra-eye-Articular Syndrome,I type severe uridine diphosphate glucose thuja acids (base) shift enzyme deficiency disease,Urethra defect,Urofacial syndromes,Type III uroporphyrinogen cosynthetase,Urticaria pigmentosa,Usher syndrome (usher syndrome),I type usher syndromes,II type usher syndromes,Type III usher syndrome,IV type usher syndromes,Metrosynizesis,Uoporphyrinogen I synzyme,Uveitis,Uveomeningitis syndrome,The creutzfeldt-Jacob disease of variation,VACTEL associations,VACTERL associations,VACTERL syndromes,Valgus calcaneus,Valine aminotransferase deficiency,Valinemia,Valproic acid,Valproate is contacted,Valproic acid is contacted,Valproic acid,Van Buren ' s diseases,Van der Hoeve-Habertsma-Waardenburg-Gauldi syndromes,Irregular morbidity immunoglobulin deficiency dysgammaglobulinemia,The Creutzfeldt-Jakob disease of variation,Varicella embryopathy,Diversity porphyria,Vascular birthmark,Binswanger ' s types vascular is dull-witted,Vascular angioma cavernosum,Vascular hemophilia,Vascular malformation,Cerebrovascular malformation,Vasculitis,Vasomotor ataxia,Vasopressin-resistant diabetes insipidus,Pitressin sensitiveness diabetes insipidus,Method spy's association,Vcf syndromes,vcf,Velocardiofacial syndromes,The arthritis of venereal disease,Venous malformation,Ventricular fibrillation,Interventricular septal defect,Congenital ventricular defect,Interventricular septal defect,Ventricular Tachycardia,Veinlet deformity,VEOHD,Vermis of cerebellum hypoplasia,Cerebellar hypoplasia,Spring angle (film) conjunctivitis,Verruca,The radius of the tracheoesophageal oesophagus of the anus of vertebra,Vertebral ankylosis hypertrophy,The choreoid early onset thereofs of Huntington ' s,Pole long-chain acyl-CoA dehydrogenase deficiency disease,Vestibular schwannomas,Vestibular schwannomas multiple neurofibromatosis,Vestibulocerebellum,Virchow ' s tips,Internal organ xanthogranulomatosis,Internal organ xanthogranulomatosis,Internal organ myopathy-outside ophthalmoplegia,Visceral obesity-umbilical cord protrusion-beckwith-Wiedemann syndrome,Typhloexia,Vitamine A deficiency,Thiamine deficiency,Macular dystrophy,Leucoderma,Head leucoderma,Degeneratio,hyaloideoretinalis,Spring angle (film) conjunctivitis,Vogt-Koyanagi-Harada syndrome,VLCAD,Vogt syndrome (striatal syndrome),Vogt Cephalosyndactyly,Vogt Koyanagi Harada syndromes,(Feng) spondylitis ankylopoietica,Von Eulenburg myotonia congenitas,Von Frey ' s syndromes,Glycogen storage disease,(Feng) angiomatosis retinae et cerebelli syndrome,Von Mikulicz syndromes,Feng's multiple neurofibroma,Von Willebrandt diseases,Vasopressins,Vrolik disease (I types),Ventricular septal defect,Plain edition keratosis,Plain edition ichthyosis,W syndromes,Wa Erdunbao syndromes,Waardenburg-Klein syndrome,Wa Erdunbao syndromes (I types),Wa Erdunbao syndromes (II types),II A type Wa Erdunbao syndromes,II Type B Wa Erdunbao syndromes,Type III Wa Erdunbao syndromes,IV type Wa Erdunbao syndromes,Waelsch ' s syndromes,WAGR complexs,WAGR syndromes,Waldenstroem ' s macroglobulinemias,Waldenstrom’s Purpura,Waldenstrom ' s syndromes,Waldmann diseases,Walker-Warburg syndromes,Splenectopia,Warburg syndromes,Warm antibody hemolytic anemia,Warm type cross reacting antibody disease,Wartenberg syndrome,Bacterial endocarditis,Brain moisture,Bacterial endocarditis,Watson-Alagille syndromes,Fertile-ergeth syndrome,The disease of wax,Viola-Bertier 's syndrome,Weaver syndrome,Weaver-Smith syndromes,Weber-Cockayne diseases,Wegener ' s granulomatosis,Leptospirosis (leptospiral jaundice),Weil syndromes,Weill-Marchesani,Weill-Marchesani syndrome,Weill-Reyes syndromes,Weismann-Netter-Stuhl syndromes,Weissenbacher-Zweymuller syndromes,Vail syndrome,Wenckebach,Werdnig-Hoffman diseases,Werdnig-Hoffman paralyses,Werlhof ' s diseases,Adult progeria,Wernicke ' s (C) I syndromes,Wernicke ' s aphasias,Wernicke-Korsakoff syndromes,West's syndrome,Beriberi humida,WHCR,Whipple ' s diseases,Intestines lipogranuloma disease,Whistling face syndrome,Whistling face syndrome,White-Darier diseases,Whitnall-Norman syndrome,Verticillate mole sample melanin is excessively sick,WHS,Wieacker syndromes,Wieacker syndromes,Wieacker-Wolff syndromes,Wiedmann-Beckwith syndromes,Wiedemann-Rautenstrauch syndromes,Wildervanck syndrome,Willebrand-Juergens diseases,Hypotonia-hypomentia-hypogonadism-obesity syndrome,Williams syndrome,Williams-Beuren syndromes,The nephroblastoma,The nephroblastoma-irideremia-gonadoblastoma-MR syndrome,The nephroblastoma-irideremia-gonadoblastoma-MR,The nephroblastoma-irideremia-gonadoblastoma-MR syndrome,The nephroblastoma-pseudohermaphroditism-nephrosis,The nephroblastoma and pseudohermaphroditism,The nephroblastoma-pseudohermaphroditism-glomerulopathy,Wilson ' s diseases,Wilson ' s syndromes,Winchester-Grossman syndromes,Victoria-Austrian syndrome,Wiskott-Aldrich type immune deficiencies,Witkop ectodermal dysplasia,Witkop tooth-nail syndrome,Wittmaack-Ekbom syndromes,WM syndromes,Wechsler Memory Scale,WNS,Wohlfart diseases,Wohlfart-Kugelberg-Welander disease (heredity peri position nerve originality muscular atrophy),Wolf syndrome,Wolf-Hirschhorn chromosomal regions (WHCR),Wolf-Hirschhorn syndromes,Wolff-Parkinson-White syndrome,Wolfram syndrome,Primary familial xanthomatosis (lysosomal storage disease caused by acid lipase deficiency),WoodyGuthrie ' s diseases,W-P-Ws,Writer ' s spasm,WS,WSS,WWS,Retinal arterio-venous aneurism,Addison ' the s diseases of X-linkage,The adrenoleukodystrophy of X-linkage,Adult's spinobulbar muscular atrophy of X-linkage,The adult spinal muscular atrophy of X-linkage,X linked agammaglobulinemias are with growth hormone deficiency,X linked agammaglobulinemias,The chain syndromes of lymphadenia type X,Cardiomyopathy and neutrophilic granulocytopenia chain X,Central caryogram myopathy chain X,The chain copper deficiency diseases of X-,Copper uptakie chain X is bad,Dominant Kang-Xu Er 's syndrome chain X,Dominant heredity callosal agenesis chain X,Dystonia-parkinson's syndrome chain X,Ichthyosis chain X,X-linked agammaglobulinemia,Neonate's Nectrotizing encephalopathics chain X,Juvenile retinoschisis chain X,Agyria disease chain X,Lymphoproliferative syndrome chain X,Chain X intelligence development is low-the thumb syndrome held,Intelligence development chain X is low with hypotony,Chain X intelligence development is low and huge testis,Progressive variable immunodeficiency chain X,Recessive Kang-Xu Er 's syndrome chain X,Recessive severe combined immunodeficiency syndrome chain X,Retinoschisis chain X,Spine epiphyseal dysplasia chain X,(lithoxiduria lacks xanthine oxidase deficiency disease,Heredity),The xanthogranulomatosis of whole body,Xanthoma tuberosum,Xeroderma pitmentosum,Dominant xeroderma pitmentosum,AIXPA type xeroderma pitmentosums,Type B II XPB xeroderma pitmentosums,Type E V XPE xeroderma pitmentosums,Type C III XPC xeroderma pitmentosums,Type D IV XPD xeroderma pitmentosums,TypeF VI XPF xeroderma pitmentosums,Type G VII XPG xeroderma pitmentosums,Xeroderma pitmentosum mutation Type XP-V,Xeroderma-clubfoot enamel defect,Xerodermic idiocy,Xerophthalmia,Keratitis sicca,The chain lymphadenias of X,XO syndrome syndrome,Xeroderma pitmentosum,XX male syndrome,Sex is reversed,XXXXX syndromes,XXY syndromes,XYY syndromes,XYY chromosome models,Yellow mutant albinism,Huang refers to (toe) first syndrome,YKL,Young women with arteritis,Yunis-Varon syndromes,YY syndromes,Z-E syndromes,Z and protease inhibitors deficiency disease,Ze Weige syndromes,Zellweger zellweger syndromes,ZE syndrome,Ziehen-Oppenheim disease (deformable dystonia),Zimmermann-Laband syndrome,Congenital zinc deficiency,Zinsser-Cole-Engman syndrome,ZLS,Zollinger-Ellison Syndrome.

In a specific embodiment, can be with other biological products, medicine or treatment use in conjunction or being used alone, including endocrine disrupting effects, human primary gastrointestinal cancers, incidence cancer, kidney and Genito-urinary device tumour, malignant mela noma, the cancer of the esophagus, colorectal cancer, cancer of pancreas, breast cancer, soft tissue sarcoma such as hand and leg, liver cancer, prostate cancer, glioma, astrocytoma, cholangiocarcinoma for treating disease or symptom such as many solid tumors (especially targeting removes tumour) containing the pharmaceutical composition by separation TNF-α or its chimeric molecule；Disease disease such as Kaposi sarcomas, treatment malaria, mycobacterium tuberculosis, mycobacterium avium, listeria monocytogenes, Salmonella typhimurtum, adult's leishmaniasis, schizotrypanum cruzi, toxoplasma gondii, Plasmodium chaubaudi, plasmodium falciparum, hepatitis C, SARS virus infection and the sick bacillus pneumonia of thermophilic lung cenobium of communicable disease such as HIV and correlation；Sleep-disorder such as sleep apnea；Obesity (being used for adipose tissue excision) and a variety of symptom (being used for general cutting tissue).

In another embodiment, pharmaceutical composition containing separation LT- α or its chimeric molecule use in conjunction or can be used alone with other biological products, medicine or treatment, the disease related for treating growth and metastasis of tumours, including adult solid's knurl, endocrine disrupting effects, human primary gastrointestinal cancers, incidence cancer, kidney and Genito-urinary device tumour, malignant mela noma, sarcoma, the cancer of the esophagus, colorectal cancer, cancer of pancreas, glioma, breast cancer and metastatic bone cancer；Cellular damage effect caused by radiation or cell toxicant cancer therapy drug；Disease disease such as Kaposi sarcomas, treatment malaria, mycobacterium tuberculosis, mycobacterium avium, listeria monocytogenes, Salmonella typhimurtum, adult's leishmaniasis, schizotrypanum cruzi, toxoplasma gondii, hepatitis C, SARS, coronavirus infection and the sick bacillus pneumonia of thermophilic lung cenobium of communicable disease such as HIV and correlation；Motion function rehabilitation after nerve regneration such as crushed nerve damage；Septicemia；And cachexia；Autoimmune disease such as rheumatoid arthritis, inflammatory bowel disease, such as Crohn disease；Multiple sclerosis；And diabetes.

In another embodiment, pharmaceutical composition containing separation TNFRI or its chimeric molecule such as TNFRI-Fc use in conjunction or can be used alone with other biological products, medicine or treat, for treating communicable disease such as HIV；Hepatitis C；Tuberculosis related HIV-1；SARS；Coronavirus infection；Severe sepsis；Septic shock, Gram-negative bacteria and gram-positive bacteria；Meat poisoning disposition is suffered a shock；Arthritis includes rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis (JRA), spondyloarthropathy, arthritic psoriasis, severe urarthritis, ankylosing spondylitis, childhood primary arthritis, chronic polyarthritis, systemic lupus erythematosus；As related to swelling, temporomandibular disorders, the chronic back and/or neck joint pain such as the pain after rheumatoid arthritis, operation on oral cavity of pain, severe acute sciatica, pain caused by Bone tumour, sciatica, the type of multizone Pain Syndrome -1 (CRPS 1) caused by nucleus pulposus abjection；Psoriasis；Asthma；The anaphylaxis of respiratory tract and nonallergic inflammatory reaction；Wegener granulomatosis；Dermatomyositis；Polymyositis；Uveitis；Non-infectious sclerotitis；RAEB；Graves illness in eye；The sclerotitis of patients with ankylosing spondylitis；Vasculitis；Polyangitis；Relapsing panniculitis；The related periodic syndrome (TRAPS) of Tumor Necrosis Factor Receptors；Nodular nonsuppruative panniculitis (WCD)；Behcet's disease；Churg-Strauss vasculitis；Qiu-apply two syndromes；PAN；Giant cell artery eye；Boeck's sarcoid；Polymyositis/dermatomyositis；Sjogren syndromes；The hypnosia of sleep apnoea is for example caused in obesity patient by obstructive sleep apnea；Multicentric reticulohistiocytosis；Gangrenous pyaphysia；Aorto-arteritis；Heart mitochondria dysfunction, aorto-arteritis, and apoptosis during heart failure；The aorto-arteritis (AOSD) that adult starts；Crohn diseases；Alcoholic hepatitis；Myositis；Giant cell arteritis；Primary mullerianosis；Chronic Infantile nerve epidermis articular (CINCA) syndrome；Guillain-Barre syndrome；Boeck's sarcoid；Stomacace；The osteolysis of periprosthetic is for example after total hip replacement；Primary amyloidosis；Hyperimmunoglobulinemia and periodic fever syndrome；The sterility of masculinity and femininity；Otitis interna；Langerhans cell histiocytosis；Immunologic thrombocytopenic purpura；Chronic inflammatory demyelinating polyneuropathy；Multicentric reticulohistiocytosis；LADA dacryoadenitis；Peripheral nerve disease such as abdominal disease；Polychondritis；Pneumatosis cystoides intestinalis；Neurosarcoidosis；Pigmented villonodular synovitis；Necrotizing angiitis；Acute children's ulcerative colitis；Inflammatory bowel disease；Kawasaki disease；Myopathy is for example at progressive Erb's atrophy (DMD)；Glasses caused by the syndrome of Ya-shellfish two infect；Hallopeau continuation acrodermatitises；Suppurative hidradenitis；Amyloidosis of kidney；The colitis of unknown cause；Bronchiolitis obliterans after transplanting；Pyostomatitis vegetans；SAPHO syndromes；Necrobiosis lipoidica；Rising star's syndrome；Tumour such as breast cancer includes combined chemotherapy or other biotinylated biomolecules are treated；The related cachexia of tumour；T-cell lymphoma,cutaneous；Graft rejection phenomenon such as graft versus host disease(GVH disease) (GVHD) (such as the subacute pulmonary dysfunction after Acute noninfectious tuberculosis (primary pneumonia syndrome, IPS) and Allogeneic stem cell transplanting)；Lung transplantation ischemia reperfusion injury；The anti-steroidogenic acute GVHD of severe；In HSCT；In organ transplant, such as chronic transplant damage such as kidney transplant.

However, in another embodiment, for treating during rheumatoid arthritis, the pharmaceutical composition of TNFRI molecules or chimeric molecule such as TNFRI-Fc compositions can also be with methopterin administering drug combinations.In another embodiment, of the invention and other bioactive molecule administering drug combinations, such as leflunomide, imuran, cyclosporine A or SASP or other monoclonal antibodies (such as anti-TNF antibody, Mac I or LFA I antibody) or other acceptors related to TNF products include IL-1 or IL-2 acceptors.

In another embodiment, the pharmaceutical composition containing separation TNFRII or its chimeric molecule use in conjunction or can be used alone with other biological products, medicine or treatment, for treating communicable disease such as HIV；Hepatitis C；Tuberculosis related HIV-1；SARS；Coronavirus infection；Severe sepsis；Septic shock, Gram-negative bacteria and gram-positive bacteria；Meat poisoning disposition is suffered a shock；Arthritis includes rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis (JRA), spondyloarthropathy, arthritic psoriasis, severe urarthritis, ankylosing spondylitis, childhood primary arthritis, chronic polyarthritis, systemic lupus erythematosus；Pain such as rheumatoid arthritis, pain and swelling after operation on oral cavity, temporomandibular disorders, chronic back and/or the related pain of neck joint, severe acute sciatica, pain caused by Bone tumour, sciatica, the type of multizone Pain Syndrome -1 (CRPS 1) caused by nucleus pulposus abjection；Psoriasis；Asthma；The anaphylaxis of respiratory tract and nonallergic inflammatory reaction；Wegener granulomatosis；Dermatomyositis；Polymyositis；Uveitis；Non-infectious sclerotitis；RAEB；Graves illness in eye；The sclerotitis of patients with ankylosing spondylitis；Vasculitis；Polyangitis；Relapsing panniculitis；The related periodic syndrome (TRAPS) of Tumor Necrosis Factor Receptors；Nodular nonsuppruative panniculitis (WCD)；Behcet's disease；Churg-Strauss vasculitis；Qiu-apply two syndromes；PAN；Giant cell artery eye；Boeck's sarcoid；Polymyositis/dermatomyositis；Sjogren syndromes；The hypnosia of sleep apnoea is for example caused in obesity patient by obstructive sleep apnea；Multicentric reticulohistiocytosis；Gangrenous pyaphysia；Aorto-arteritis；Heart mitochondria dysfunction, oxidative stress, and apoptosis during heart failure；The Stills of adult onset is sick (AOSD)；Crohn diseases；Alcoholic hepatitis；Myositis；Giant cell arteritis；Primary mullerianosis；Chronic Infantile nerve epidermis articular (CINCA) syndrome；Guillain-Barre syndrome；Boeck's sarcoid；Stomacace；The osteolysis of periprosthetic is for example after total hip replacement；Primary amyloidosis；Hyperimmunoglobulinemia and periodic fever syndrome；The sterility of masculinity and femininity；Otitis interna；Langerhans cell histiocytosis；Immunologic thrombocytopenic purpura；Chronic inflammatory demyelinating polyneuropathy；Multicentric reticulohistiocytosis；LADA dacryoadenitis；Peripheral nerve disease such as abdominal disease；Polychondritis；Pneumatosis cystoides intestinalis；Neurosarcoidosis；Pigmented villonodular synovitis；Necrotizing angiitis；Acute children's ulcerative colitis；Inflammatory bowel disease；Kawasaki disease；Myopathy is for example at progressive Erb's atrophy (DMD)；Glasses caused by the syndrome of Ya-shellfish two infect；Hallopeau continuation acrodermatitises；Suppurative hidradenitis；Amyloidosis of kidney；The colitis of unknown cause；Bronchiolitis obliterans after transplanting；Pyostomatitis vegetans；SAPHO syndromes；Necrobiosis lipoidica；Rising star's syndrome；Tumour such as breast cancer includes combined chemotherapy or other biotinylated biomolecules are treated；The related cachexia of tumour；T-cell lymphoma,cutaneous；Graft rejection phenomenon such as graft versus host disease(GVH disease) (GVHD) (such as the subacute pulmonary dysfunction after Acute noninfectious tuberculosis (primary pneumonia syndrome, IPS) and Allogeneic stem cell transplanting)；Lung transplantation ischemia reperfusion injury；The anti-steroidogenic acute GVHD of severe；In HSCT；In organ transplant, such as chronic transplant damage such as kidney transplant.

When treating rheumatoid arthritis, the pharmaceutical composition containing TNFRII molecules or TNFRII chimeric molecules can also be with methopterin administering drug combinations.But, in another embodiment, of the invention and other bioactive molecule administering drug combinations, such as leflunomide, imuran, cyclosporine A or SASP or other monoclonal antibodies (such as anti-TNF antibody, Maci or LFAI antibody) or other acceptors related to TNF products include IL-1 or il-2 acceptors.

In another embodiment, pharmaceutical composition containing separation OX40 or its chimeric molecule can be with other biological products, medicine or treatment use in conjunction are used alone, for treating the disease such as anaphylaxis for including singly being not limited to T cell mediation, inflammatory and autoimmune disease such as graft rejection, autoimmune disease and inflammation, graft versus host disease(GVH disease) (GVHD), Acute GVHD after allogeneic bone marrow transplantation, rheumatoid arthritis (RA), inflammatory bowel disease, experimental allergic encephalomyelitis (EAE), multiple sclerosis, tumour, lupus nephritis, inflammatory bowel disease, asthma, multiple sclerosis；Crohn diseases；Ulcerative colitis；Ulcerative colitis；Asthma and pneumonia and autoimmune diabetes caused by breast cancer, colorectal cancer, autoimmune encephalitis, inflammatory lung injury such as influenza.

In another embodiment, pharmaceutical composition containing separation BAFF or its chimeric molecule use in conjunction or can be used alone with other biological products, medicine or treatment, for adjust B cell, T cell, dendritic cells, macrophage, Neutrophil-mediated biological process, and BAFFR is activated, for example increase propagation, activation and the survival of bone-marrow-derived lymphocyte；For treat immune deficiency (such as B lymphocyte proliferation, activate or insufficient patient of surviving, or immune deficiency (CVID) patient that typically makes a variation, or IgA defects patient)；For increasing the generation of antibody during vaccine inoculation；For treating B cell malignant tumor such as chronic lymphocytic leukemia (B-CLL), non-Hodgkin lymthomas (NHL), and Huppert's disease (MM).

However, in another embodiment, BAFF is connected to radionuclide, toxin or chemotherapeutics, as a kind for the treatment of method, it can be used for targetting and kill B cell malignant tumor.Suitable radionuclide is for example including iodo- 123, iodine -131, technetium -99 and Yttrium-90.Suitable toxin is for example including vanous toxin and the Pseudomonas exotoxin truncated.

In another embodiment, BAFF (and chimeric molecule containing BAFF) amino acid variant, with BAFF antagonistic activities, can be used for treating the disease that correlation is lowered in the expression of BAFF 3, such as B cell lymphoma and autoimmune disease.

In another embodiment, pharmaceutical composition containing separation NGFR or its chimeric molecule use in conjunction or can be used alone with other biological products, medicine or treatment, for the growth for suppressing breast cancer and other tumours, wherein NGF and other NGFR parts are mitogen；Morbidity for suppressing epidermis caused by neurogenic inflammation and systemic inflammatory disease, such as psoriasis, atopic dermatitis, nettle rash, rheumatoid arthritis, ulcerative colitis and bronchial astehma；In HIV patient, the macrophage of HIV is removed；And block the hyperfunction progress of autonomic dysreflexia such as bladder reflex after spinal cord injury.

In another embodiment, pharmaceutical composition containing separation FasL or its chimeric molecule use in conjunction or can be used alone with other biological products, medicine or treatment, for treating rheumatoid arthritis, osteoarthritis, graft versus host disease(GVH disease), toxic epidermal necrolysis, LADA lymphoproliferative syndrome, immune deficiency, hepatic failure, nerve regneration after Alzheimer diseases, multiple sclerosis, bone marrow injury and apoplexy.

But, in another embodiment, pharmaceutical composition containing separation FasL or its chimeric molecule use in conjunction or can be used alone with other medicines or treatment, survival for promoting allograft, and prevention of autoimmune diseases include the breaking-out of multiple sclerosis and diabetes.

In another embodiment, pharmaceutical composition containing separation FasL or its chimeric molecule use in conjunction or can be used alone with other medicines or treatment, for apoptosis-induced in many different malignant diseases, including aleukemic leukemia, glioma, breast cancer and other solid tumors, they all express Fas.

Moreover, the Pharmaceutical compositions of the present invention have a higher drug effect, stronger heat endurance, longer serum half-life or with expression in the protein of non-human cell lines or its chimera compared with when with higher blood dissolubility.The present invention also shows lower Ia scavenging action or related side effects.Due to the characteristic of these improvement, the pharmaceutical composition of the present invention can be administered with lower frequency compared with expression is in the protein of non-human cell lines or its chimera.Reduction administration frequency can strengthen the adaptability of patient to be conducive to treatment results.The quality of life of patient equally can also increase.

Therefore, in one embodiment, pharmaceutical composition of the invention can be administered with therapeutic dose with expression in the protein of non-human cell lines or its chimera identical administering mode.Therapeutic dose refers to the amount for producing the necessary pharmaceutical composition of activity in vivo.The accurate amount for giving compound is determined by factors such as the other compositions in the exact type such as treatment symptom, the physical qualification and pharmaceutical composition for the treatment of patient.Pharmaceutical composition containing albumen of the present invention or chimeric molecule isoform can be prepared to treat the patient with said one or multiple symptoms in favor of the different formulas of administration.The mean treatment significant degree of pharmaceutical composition may be different.It is expected that effective dose in 0.1ng/kg body weight between 20 μ g/kg body weight；Or the suggestion according to qualified physicians or prescription.

In a specific embodiment, composition and preparation prepared in accordance with the present invention, for topical application, each course for the treatment of uses the active medicine (such as TNFRI and/or TNFRII and/or TNFRI-Fc and/or TNFRII-Fc) between 0.1 μ g to 20g.Can administration per hour, daily, weekly, monthly or every year.

The topical compositions of the present invention can be prepared in the following manner：With pharmaceutically acceptable carrier described here or diluent, mixing TNFRI-Fc or its variant, homologue or the like or TNFRI polypeptides or its variant, homologue or the like and/or TNFRII polypeptides or its variant, homologue or the like and/or TNFRII-Fc or its variant, homologue or the like.In a specific embodiment, pharmaceutically acceptable carrier or diluent are an emulsifiable paste, wherein, the emulsifiable paste is selected from Cetaphil Moisturising Cream (Galderma Laboratories, L.P.), QV Cream (Lision Hong), Sorbolene etc..

In another embodiment, topical preparation's Distaval and pharmaceutically acceptable carrier or diluent mixing mixing TNFRI polypeptides or its variant, homologue or the like or/and TNFRII polypeptides or its variant, homologue or the like and/or TNFRI-Fc or its variant, homologue or the like and/or TNFRII-Fc or its variant, homologue or the like.The ultimate density of TNFRI polypeptides or its variant, homologue or the like or/and TNFRII polypeptides or its variant, homologue or the like and/or TNFRI-Fc or its variant, homologue or the like and/or TNFRII-Fc or its variant, homologue or the like in topical preparation should be equal to or less than 50mg/ml or so.It is as used herein " to include but is not limited to following concentration equal to or less than 50mg/ml "：0.001,0.002,0.003,0.004,0.005,0.006,0.007,0.008,0.009,0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.10,0.11,0.12,0.13,0.14,0.15,0.16,0.17,0.18,0.19,0.20,0.21,0.22,0.23,0.24,0.25,0.26,0.27,0.28,0.29,0.30,0.31,0.32,0.33,0.34,0.35,0.36,0.37,0.38,0.39,0.40,0.41,0.42,0.43,0.44,0.45,0.46,0.47,0.48,0.49,0.50,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5,10.0,10.5,11.0,11.5,12.0,12.5,13.0,13.5,14.0,14.5,15.0,15.5,16.0,16.5,17.0,17.5,18.0,18.5,19.0,19.5,20.0,20.5,21.0,21.5,22.0,22.5,23.0,23.5,24.0,24.5,25.0,25.5,26.0,26.5,27.0,27.5,28.0,28.5,29.0,29.5,30.0,30.5,31.0,31.5,32.0,32.5,33.0,33.5,34.0,34.5,35.0,35.5,36.0,36.5,37.0,37.5,38.0,38.5,39.0,39.5,40.0,40.5,41.0,41.5,42.0,42.5,43.0,43.5,44.0,44.5,45.0,45.5,46.0,46.5,47.0,47.5,48.0,48.5,49.0,49.5 and 50.0mg/ml.

In a specific embodiment, the final concentration of 0.25mg/ml of TNFRI-Fc or its variant, homologue or the like or TNFRII-Fc or its variant, homologue or the like in topical preparation or so.

The topical preparation being made up of TNFRI-Fc and/or TNFRII-Fc, can further include Distaval or its variant, homologue or the like, especially be the analog of non-teratogenesis.The concrete scheme is from two level calculation TNF-αs, i.e., by the biosynthesis pathway that suppresses TNF-α and by neutralizing excessive TNF-α.

Distaval (or α-(N- benzene two (first) acylimino) glutarimide) is a glutamate derivatives.It is a bicyclic ring structures, there is an asymmetric carbon atom in glutarimide ring.It exists in the form of S (-) and R (+) mapping isoform mixed in equal amounts.Distaval is an immune modulatory molecules, with anti-inflammatory and immunosuppressive activity, but its mechanism of action is not completely understood also.Distaval has the ability that the ability for suppressing TNF-α generation and modification TNF-α induced adherence molecule are expressed on epithelial cell and human leukocytes.In the cell type that many LPS are stimulated, including person monocytic cell (Sampaio et al.J Exp Med 173 (3)：699-703,1991) and alveus cell (Tavares et al.Respir Med 91 (1)：31-9,1997) in, the generation of Distaval selective depression TNF-α, this is (the Moreira et al.J Exp Med 177 (6) that the degraded by strengthening TNF-α mRNA reaches：1675-80,1993).

Ultimate density of the Distaval in topical preparation should be equal to or less than 100mg/ml.It is as used herein that " being equal to or less than 100mg/ml " includes following concentration：0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100mg/ml.

In a specific embodiment, ultimate density of the Distaval in topical preparation is 10 to 30mg/ml, and concentration of the Distaval in topical preparation is more preferable when being 20mg/ml.

Invention further provides application of the protein or chimeric molecule and pharmaceutical composition including at least Partial Protein or the separation of its chimeric molecule in different therapy and/or diagnosis.

More specifically, the invention provides the method for treating or preventing subject mammalian diseases, the amount or activity of the albumen in the present invention or chimeric molecule can wherein be increased to alleviate disease, this method includes giving the protein of separation of effective dose, the chimeric molecule comprising the albumen, the chimeric molecule of the extracellular regions segment comprising the protein or protein or the pharmaceutical composition of chimeric molecule comprising the separation to the subject mammal.In a certain particular aspects, the present invention provides treatment method for the illness for treating the inflammatory conditions with the too high feature of TNF-α level or related or TNF-α causes the state of an illness to be aggravated to TNF-α, method of administration includes giving the topical preparation of patient's dose therapeutically effective, and it is made up of TNFRI-Fc described here and/or TNFRII-Fc.The related symptom of TNF-α is normally defined the treatable symptom of TNF-α inhibitor.

TNF-α is present in excess in many autoimmune diseases, including rheumatoid arthritis, the sick and many inflammatory skin conditions described here of Crohn.The TNF-α that TNF-α " excess " can be broadly defined as in blood samples of patients or serum exceedes the number that the solvable TNF-α acceptor of patient's endogenous can be combined.Typically, the excessive result of TNF-α is inflammatory reaction.

In a specific embodiment, " illness " refers to a kind of disease disease, is made up of one or more symptoms, shows skin surface or the inside of patient, method includes giving the topical preparation containing TNFRI-Fc described here and/or TNFRII-Fc, the skin impacted for patient.Illness be selected from list below when more preferably：Psoriasis, behcet's disease, bullous dermatitis, eczema, mycotic infection, leprosy, neutrophilic dermatitis, pityriasis (or pityriasis rosea), tinea nigra (or tinea nigra), pityriasis rubra pilaris, systemic loupus erythematosus, systemic many blood vessels and toxic epidermal necrolysis；Or using illness caused by medication, for example using symptom caused by Aldara emulsifiable pastes include erythema, burn into ulcer, peeling, fish scale, drying, formed pimple, crust, skin diffusate.But, the present invention should not only consider the treatment of these diseases.

Term " patient has the illness of the too high feature of TNF-α level " as used herein should be understood to include the illness with following characteristics：Detect that TNF mRNA in tissue are too high or patients serum in TNF-α it is too high, and no matter whether patient detects that TNF-α is excessive, gives the disease that any medicament that can reduce TNF-α quantity or activity can be treated.

In a specific embodiment, present invention contemplates treat psoriasic method, method includes giving the medicament being made up of TNFRI-Fc described here and/or TNFRII-Fc of patient's dose therapeutically effective.Accordingly, term " psoriasis " as used herein is interpreted as covering all mutation of the disease, including the plague, guttate psoriasis, inverse psoriasis, nail psoriasis, whole body erythrodermic psoriasis, pustular psoriasis, the palm-impetigo of pacing up and downing, Von Zumbusch psoriasis and arthritic psoriasis.

The method of the present invention includes giving patient pharmaceutical formulations.Can administration per hour, daily, weekly, monthly or every year.In addition, medication can include time per unit multiple dosing, for example medication can include give per hour, daily, weekly, monthly or every year medicament 1,2,3,4 or 5 times.Medication can also include per multiple chronomere's single-doses, such as medication can include every 1,2,3,4 or 5 hours, day, week, the moon or give medicament year once.As described above, medication is more suitable for being applied topically to a biological surface or an artificial surfaces and then for biological surface.For example, medicament can be placed on the region that needs are treated with gauze or fragment, then them.

In addition, when being administered every time, per 100cm²The medicament deal that impacted region is given can include from 0.1ml to 10ml.This includes following deal：0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0ml, per 100cm²Need the region for the treatment of.

In a specific embodiment, certain skin area shows the symptom of one or more disease diseases, it is necessary to which treatment daily, uses 0.5ml topical preparation, consisting of TNFRI (0.25mg/ml) and Distaval (20mg/ml), per 100cm²Impacted region, once a day.In some concrete schemes, up to 2ml or so compositions are used for 100cm for the topical preparation of TNFRI (0.25mg/ml) and Distaval (20mg/ml)²The impacted region in left and right.

In another embodiment, areas of inflammation is administered once a day, and gives 0.5ml topical preparations (TNFRI (0.25mg/ml)；Distaval (20mg/ml)), per 100cm²Areas of inflammation, once a day.In some concrete schemes, up to 2ml or so topical preparation (TNFRI (0.25mg/ml)；Distaval (20mg/ml)) it is used for 100cm²The impacted region in left and right, once a day.

In another embodiment, areas of inflammation every two is administered once everyday, gives 0.5ml topical preparations (TNFRI (0.25mg/ml)；Distaval (20mg/ml)), every 100 square centimeters or so of areas of inflammation.In some concrete schemes, up to 2ml or so topical preparation (TNFRII (0.25mg/ml)；Distaval (20mg/ml)) it is used for 100cm²The impacted region in left and right, per once two days.

In a specific embodiment, areas of inflammation is administered once for every two days, gives 0.5ml topical preparations (TNFRII (0.25mg/ml)；Distaval (20mg/ml)), every 100 square centimeters or so of areas of inflammation.In some concrete schemes, up to 2ml or so topical preparation (TNFRII (0.25mg/ml)；Distaval (20mg/ml)) it is used for 100cm²Impacted region, per once two days.

In a specific embodiment, certain skin area shows the symptom of one or more disease diseases, it is necessary to which treatment daily, uses 0.5ml topical preparation, consisting of TNFRI-Fc (0.25mg/ml) and Distaval (20mg/ml), per 100cm²The impacted region in left and right, once a day.In some concrete schemes, up to 2ml or so compositions are used for 100cm for the topical preparation of TNFRI-Fc (0.25mg/ml) and Distaval (20mg/ml)²The impacted region in left and right.

In another embodiment, areas of inflammation is administered once a day, and gives 0.5ml topical preparations (TNFRII-Fc (0.25mg/ml)；Distaval (20mg/ml)), per 100cm²Areas of inflammation, once a day.In some concrete schemes, up to 2ml or so topical preparation (TNFRII-Fc (0.25mg/ml)；Distaval (20mg/ml)) it is used for 100cm²The impacted region in left and right, once a day.

In another embodiment, areas of inflammation is administered once for every two days, gives 0.5ml topical preparations (TNFRI-Fc (0.25mg/ml)；Distaval (20mg/ml)), every 100 square centimeters or so of areas of inflammation.In some concrete schemes, up to 2ml topical preparation (TNFRI-Fc (0.25mg/ml)；Distaval (20mg/ml)) it is used for 100cm²The impacted region in left and right, per once two days.

In a specific embodiment, areas of inflammation is administered once for every two days, gives 0.5ml topical preparations (TNFRII-Fc (0.25mg/ml)；Distaval (20mg/ml)), every 100 square centimeters of areas of inflammation.In some concrete schemes, up to 2ml topical preparation (TNFRII-Fc (0.25mg/ml)；Distaval (20mg/ml)) it is used for 100cm²Impacted region, per once two days.

Present invention further contemplates with the other therapeutic reagents of drug combination by the present invention or the method that constitutes of therapeutic scheme.One or more therapeutic reagents can be with TNFRI and/or TNFRII and/or TNFRI-Fc and/or TNFRII-Fc drug combinations.For " drug combination ", refer to identical preparation or two kinds of different preparations by identical or different by way of being administered simultaneously, or with identical or different by way of successive administration.For " successive " administration, refer to that giving two kinds has a period of time between reagent or therapeutic scheme：Different second, point, hour or day.The reagent or therapeutic scheme being administered in succession can be administered in any order.

When another therapeutic reagent is administering drug combinations, it can be used for whole body or part.

Accordingly, the invention provides a manifold drug packages, consisting of：Part I includes TNFRI and/or TNFRII and/or TNFRI-Fc and/or TNFRII-Fc, it is suitable for the form of local administration, and Part II or successive part include another active agent, it is suitable for part or the form of Systemic administration, Part I also includes the topical vehicle of pharmaceutical acceptable.

Therefore, in a specific embodiment, present invention contemplates treat the method for patient's psoriasis or related dermal illness, method include administering locally to patient's effective dose by TNFRI and/or TNFRII and/or TNFRI-Fc and/or the TNFRII-Fc medicament constituted and another active agent.It is other to be included with medicament of the invention with the active agent of administering drug combinations：

(i) tar：Coal tar is the adjuvant drug in known curing psoriasis, and can be obtained from crude coal tar, tar washing lotion, and is mixed with refined forms in prefabricated emulsifiable paste, washing lotion and shampoo.The chemicals similar to having found material in tar can be used according to material known to it as Dithranol or anthraline.

(ii) UV lines：Typically applied by an artificial light source.

(iii) corticoid：External application corticoid can help to treat psoriasis in a variety of different alkali forms, but it is generally up to about to help 1-2 days.There are some specific regions such as ear and the back of the hand, tar treatment is not highly effective, thus is typically best using corticoid in these regions.Preferably avoid using corticoid tablet using interior, unless in the case that other any treatments are not all helped.The subject matter of these tablets is that they can help to treat, but when they are stopped using, psoriasis can suddenly occur and become than originally more severe, i.e., so-called rebound effect.

(iv)Calcipotriol：It is a kind of synthesized form of vitamin D.Vitamin D has been recognized many years, for improving great exception present on Psoriatic skin, still, even if only the vitamin D more slightly higher than daily recommendation consumption is absorbed, and can result in internal calcium metabolism problem (possible kidney stone and irregular heartbeat).It has been found that Calcipotriol also has a psoriasic ability of improving, and the influence very little to calcium metabolism.If ointment is used in face or neck, there is the risk of facial dermatitis, therefore it is extremely important only to recommend to be used for thoroughly cleaning hand after trunk and four limbs, and application, to avoid being transferred to skin of face accidentally.

(v) PUVA phototherapy：PUVA is the name for treatment, is made up of psoralen (making skin irradiate sensitive to the artificial ultraviolet of A scopes) combined U VA.Both have powerful influence at combination to psoriasic plaques, slow down the quick division of cell, and the quick division of cell is considered as occurring in active psoriasis.The dosage of UVA irradiations should slowly increase, if it is too fast to introduce the treatment, it may occur that skin burn.A kind of a kind of technology of mutation latest developments of PUVA phototherapy, that is, take a shower PUVA.Different from being orally ingested psoralen, take a shower only need to spend 10 minutes in UVA pre-irradiations, contain psoralen chemical substance.On the daytime for the treatment of phase, the PUVA of form of ownership is treated, and sunlight safeguard measure is all critically important.PUVA is not psoriasic first-line treatment scheme.

(vi) methopterin：Methopterin has been used for psoriasis treatment.The active agent is also used to treat some cancers and leukaemia with higher dosage.

(vii) Tigason：Tigason is a kind of " biostearin " (a kind of synthesis of derivatives of vitamin A), can be used for handling very serious psoriasis case, and psoriasic purulence bubble.

(viii) cyclosporin：The inflammation occurred when known cyclosporin can suppress to suffer from psoriasis on skin.

(ix) anthraline：Anthraline is derived from goa, bark of the goa from araroba, and has been used for treatment psoriasis more than 100 years

(x) salicylic acid：Salicylic acid is a kind of chemicals that can help to remove the scales of skin that peel off.

(xi) melatonin：The strong effective antioxidant of a kind of lipophilic, it was demonstrated that inflammatory cutaneous can be relaxed.

Other active agents also include the molecule that other cell factor inhibitors for example suppress IGF-1 or IGF-1R, and suppress igf binding protein such as IGFBP-1,2,3 or 4 molecule.

The suppression for the cell factor being related to includes the expression for suppressing the inhereditary material of the Codocyte factor.This inhibitor includes antisense nucleic acid molecules, just nucleic acid molecule, dsRNA (DNA- derivative or RNA) of synthesis and ribozyme.

The present invention is further illustrated by with non-limiting embodiment below.

Embodiment 1

The preparation of carrier-Fc constructs

(a)pIRESbleo3-Fc

The DNA sequence dna of encoding human IgG1 Fc domains is expanded by PCR (PCR), from EST cDNA storehouses (Clone ID 6277773, Invitrogen), forward primer (the SEQ ID NO for being mixed with restriction enzyme BamH1 and BstX1 sites respectively are used：21) with reverse primer (SEQ ID NO：22).The amplicons cloned enters in pIRESbleo3 (Cat.No.6989-1, BD Biosciences) corresponding restriction enzyme site to prepare construct pIRESbleo3-Fc.PIRESbleo3-Fc discharges the Insert Fragment of 780bp desired sizes with BamH1 and BstX1 digestion, as gel electrophoresis is determined.

(b) preparation of marking protein or albumen-Fc DNA construct

The DNA sequence dna of encoding proteins matter or its ectodomain is expanded by PCR, from ESTcDNA storehouses, uses the forward primer and reverse primer that restriction enzyme site is introduced according to table 8.After amplification, amplicon is through suitable digestion with restriction enzyme and is cloned into expression vector as shown in table 8, to prepare Carrier-protein or carrier-albumen Fc constructs.In the preparation process of encoding proteins-Fc construct, the DNA sequence dna of encoding proteins matter is cloned into the upstream of Fc nucleotide sequences, two sequences is merged with meeting frame, so in protein expression, albumen is merged directly or through connexon with Fc regions.TNFRII-Fc-pCEP-4 preparation include expand TNFRII-Fc sequences and with table 8 to restriction enzyme site be cloned into pCEP-4.Suitable restriction enzyme is used for the carrier for digesting the DNA sequence dna containing encoding proteins matter or albumen-Fc, to discharge the fragment of desired size as shown in table 8.Carrier-protein or carrier-albumen-Fc constructs are sequenced to determine the integrality of cloning procedure described herein.

Table 8

Protein-Fc and related cloning information

Protein	CDNA originates	Forward primer	Reverse primer	It is restricted Restriction enzyme site	Carrier	Size   (bp)
							TNF-α	Clone ID 5216438,   Invitrogen	SEQ ID   NO：25	SEQ ID   NO：26	EcoRV,   EcoRI	pIRESbleo3   (Cat.No. 6989-1, BD   Biosciences)	1048
LT-a	Clone ID 5229942,   Invitrogen	SEQ ID   NO：41	SEQ ID   NO：42	EcoRV,   BamHI	pIRESbleo3   (Cat.No. 6989-1, BD   Bioscience s)	666
							TNFRI	Clone ID 5758757,   Invitrogen	SEQ ID   NO：57	SEQ ID   NO：58	EcoRV,   BamHI	pIRESbleo3-Fc	637
TNFRII	Clone ID 5181070,   Invitrogen	SEQ ID   NO：87	SEQ ID   NO：88	EcoRV,   BamHI	pIRESbleo3-Fc	795
							TNFRII-Fc	TNFRII-Fc   pIRESbleo3	SEQ ID   NO：198	SEQ ID   NO：199	XhoI,   Bam HI	pCEP-4(Cat No. Cat.No.V044-50,   Invitrogen	1059
BAFF	Clone  ID 5173954,   Invitrogen	SEQ ID   NO：145	SEQ ID   NO：146	EcoRV,   BamHI	pIRESbleo3   (Cat.No. 6989-1, BD   Biosciences)	888
							NGFR	Clone ID 5263715, Open   Biosystems	SEQ ID   NO：161	SEQ ID   NO：162	EcoRV,   BamHI	pIRESbleo3-Fc	721
FasL	Clone  ID 4849770,   Invitrogen	SEQ ID   NO：181	SEQ ID   NO：182	EcoRV,   EcoRI	pIRESbleo3   (Cat.No. 6989-1, BD   Biosciences)	1130

(c) expression OX40-Fc DNA construct is prepared

The DNA sequence dna of coding OX40 extracellular domains (ECD) is connected to the upstream of IgGI Fc sequences with two step cloning process.The first step is included with forward primer (SED ID NO：123) with reverse primer (SEQ ID NO：124) and EST cDNA libraries (5180287, Invitrogen) be used as template, expand OX40ECD first 292bp.Primer mixes restriction enzyme site EcoRV and BamHI respectively.The amplified production of purifying is cloned into the corresponding restriction enzyme site of pIRESbleo3-Fc expression vectors, the upstream of human IgG I Fc sequences.Second step includes, with forward primer (SEQID NO：125) with reverse primer (SEQ ID NO：126) the OX40 sequences and previously expanded are used as template, the remaining 355bp of amplification OX40ECD sequences.Primer all mixes restriction enzyme site BamHI at the two ends of amplified production.The amplified production of purifying is connected into BamHI sites, the downstream of first OX40 sequence and the upstream of Fc sequences.HpaII digestions prove the correct direction of second segment OX40 sequences.An artificial BamHI site is introduced in OX40 sequences does not cause the frameshit of amino acid sequence of influence translation albumen.

In addition, encoding the nucleotide sequence for the albumen for being cloned into carrier (such as pIRESbleo3 or pCEP4) can be expanded with primer, the restriction enzyme site mixed in primer allows the DNA sequence dna of coding Fc nucleotide sequence upstream proteins to be cloned into carrier-Fc (such as pIRESbleo3-Fc or pCEP4-Fc) upstream) in, therefore albumen and Fc nucleotide sequences are to be merged directly or by connexon in structure.

(d) Megaprep carriers-albumen or carrier-albumen-Fc are prepared

The Escherichia coli that the 750 μ l evening before yesterday were transfected into carrier-albumen or carrier-albumen-Fc are inoculated in the 750ml sterile LB mediums containing ampicillin (100 μ g/ml).37 DEG C of shakings are incubated 16 hours.Plasmid is prepared according to Qiagen Endofree Plasmid Mega Kit (Qiagen MegaPrep Kit#12381).

Embodiment 2

(a) preparation, separation and the purifying of TNF-α of the invention

(i) preparation of TNF-α of the invention

0th day, in 5 500cm²Tissue culture dishes (Corning) upper berth 3 × 10⁷The cell of the human embryonic kidney cell line of individual conversion, such as HEK 293, HEK293c18, HEK 293T, 293CEN4, HEK 239F, HEK 293E, HEK 293FT, AD-293 (Stratagene) or 293A (Invitrogen).Each culture dish, cell adds 90ml Dulbecco ' s ModifiedEagle ' s Medium/Ham nutrition compounds F12 (DMEM/F12) (JRH Biosciences), (v/v) heat-inactivated fetal bovine serum of addition 10% (FCS is needed in culture medium, JRH Biosciences), 4mM Glus (Amresco), 10mM HEPES (Sigma), with 1% (v/v) Pen .- Strep (benzyl penicillin 5000U/ml, streptomycin sulphate 5mg/ml) (JRHBiosciences).Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 1st day, transfected with calcium phosphate.Before transfection, the culture medium in each plate is substituted for the fresh DMEM/F12 of 120ml, wherein the Glu containing 10% (v/v) heat inactivation FCS or DCS, 4mM, 10mM HEPES and 1% (v/v) Pen .- Strep.Calcium phosphate/DNA is prepared to be precipitated as, 1200 μ g are added in sterilized water (BD Biosciences) has pIRESbleo3 (Invitrogen) DNAs of humanTNF-α's gene and 3720 μ l 2M calcium chloride solution (BD Biosciences), final volume is 30ml (solution A), or, in 1 × TE, equal number of DNA is added to 3000 μ l 2.5M CaCl₂In, final volume is 30ml (solution A).Solution A is added dropwise in 2 × HEPES buffer solutions of 30ml (HBS) (solution B) (BD Biosciences) with 10ml pipette.During dropwise addition, bubble slow transits through solution B.Mixture is incubated 20min at 25 DEG C and is vortexed.12ml mixture is added dropwise in each plate.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.Or, after 4 hours, remove the culture medium containing transfection composite, 100ml DMEM/F12 are added per plate, wherein containing 10% (v/v) DCS, 4mM Glus, the HCl of 1% (v/v) Pen .- Strep and final concentration of 3.5-4.0mM, the final pH of culture medium is 7.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 2nd day, cells and supernatant is removed.Each plate cleans material therein twice with 50ml DMEM/F12 culture mediums, and the fresh serum-free DMEM/F12 culture mediums of 100ml are added in each plate, N-ACETYL-D- MANNOSAMIDE (New ZealandPharmaceuticals) containing 40mM, 7 or 10mM Glu, 15mM HEPES, 0.5 or 4.1g/L mannose (Sigma) and 1% (v/v) Pen .- Strep and ITS solution (5mg/L bovine insulins, the selenium of the human transferrin of 5mg/L parts iron saturation and 5 μ g/ml) (Sigma) (or, without ITS solution).Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 3rd day; collect cells and supernatant; and the fresh serum-free DMEM/F12 culture mediums of 100ml are added in each plate; N-ACETYL-D- MANNOSAMIDE containing 40mM, 7 or 10mM Glu, 15mM HEPES (or; without HEPES); the Pen .- Strep of 0.5 or 4.1g/L mannose and 1% (v/v), and ITS solution (or, no ITS solution).Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)), 4 DEG C of preservations of mixture are added in the cells and supernatant of collection.

At the 4th day, cells and supernatant is collected.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the cells and supernatant of collection, and it is mixed with the sample that the 3rd day collects.Particulate is removed with one 0.45 micron of low protein binding filter (Durapore, Millipore).By adding 1/10th volume 200mM MES/50mM MgCl₂(pH6) sample of mixing, is adjusted into pH to 6.- 70 DEG C of mixture is preserved or used immediately.

(ii) separation and purifying of TNF-α

With tangential flow filtration (TFF) device (Pelicon XL, Ultracell, Millipore), the cells and supernatant that 950ml is filtered concentrates 20 times or so.Sample injects 150cm with 150ml/min speed²Regenerated cellulose film on, 5KDa molecular weight is blocked, until the volume of sample concentration to 30ml.The sample of concentration adds 70ml 50mM HEPES (pH is 8.5) diafiltration, and concentration volume is still 30ml.The diafiltration step is repeated twice, and final volume is 50ml.Then, the concentration filter sample is filtered with one 0.45 micron of low protein binding filter (Durapore, Millipore).The purifying of TNF-α is that it is located at one in advance with the ion exchange column (Bio-Rad Laboratories, MacroPrep HS) of 50mM HEPES (pH is 8.5) balances by TFF by the cells and supernatant of concentration.Then, with the TNF-α of 50mM HEPES (pH is 8.5) to 100%50mM HEPES (pH is 8.5) NaCl containing 1M linear gradient solution elution of bound.Obtained composition analyzes apparent molecular weight and purity by 1D SDS PAGE with 4-20% gradient Tris-Glycine glue (Invitrogen), and is quantified with anti-TNF-ELISA (R＆D systems).The solution containing TNF-α is mixed, below 1ml is concentrated to, molecular-exclusion chromatography is carried out using centrifugal filter device (Amicon Ultra, Millipore).

Molecular-exclusion chromatography is carried out on the anion exchange component of combination, 16/70 post (Pharmacia, Uppsala, Sweden) prepared using a Superdex 75.The flow velocity of 1% ammonium hydrogen carbonate level pad is 1ml/min.Total flowing time is 120min, and peak elution time is 20 between 100min.Elution composition is analyzed with the Tris- glycine glue (Invitrogen) and TNF-α ELISA of the 4-20% gradients of Silver stain.It was found that 50min or so peak elution contains TNF-α.The solution containing TNF-α is mixed, and below 2ml is concentrated to using centrifugal filter device (Amicon Ultra, Millipore).

It was found that its apparent MW of the TNF-α of purifying is 17kDa or so, the SDS PAGE analyses of Silver stain obtain its purity and are at least 95%.It is measured according to 280nm trap, molar extinction coefficient is 21555M^-1cm^-1, obtain the final concentration of 157 μ g/ml of TNF-α.

(b) LT- α of the invention preparation, separation and purifying

(i) LT- α of the invention preparation

0th day, in 5 500cm²Tissue culture dishes (Corning) upper berth 3 × 10⁷The cell of individual next inverting human embryonic kidney cell line, such as HEK 293, HEK293 c18, HEK 293T, 293 CEN4, HEK 239F, HEK 293E, HEK 293FT, AD-293 (Stratagene) or 293A (Invitrogen).Addition 10% (v/v) heat-inactivated fetal bovine serum (FCS, JRH Biosciences), 4mM Glus (Amresco), 10mM HEPES (Sigma) and 1% (v/v) Pen .- Strep (benzyl penicillin 5000U/ml, streptomycin sulphate 5mg/ml) (JRHBiosciences) are needed in each culture dish, cell addition 90ml Dulbecco ' s ModifiedEagle ' s Medium/Ham nutrition compounds F12 (DMEM/F12) (JRH Biosciences), culture medium.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 1st day, transfected with calcium phosphate.Before transfection, the culture medium in each plate is substituted for the fresh DMEM/F12 of 120ml, wherein containing 10% (v/v) heat inactivation FCS, 4mM Glu, 10mM HEPES and 1% (v/v) Pen .- Strep.Calcium phosphate/DNA is prepared to be precipitated as, 1200 μ g are added in sterilized water (BD Biosciences) has pIRESbleo3 (Invitrogen) DNAs of people's LT- α genes and 3720 μ 12M calcium solution (BD Biosciences), and final volume is 30ml (solution A).Solution A is added dropwise in 2 × HEPES buffer solutions of 30ml (HBS) (solution B) (BD Biosciences) with 10ml pipette.During dropwise addition, bubble slow transits through solution B.Mixture is incubated 20min at 25 DEG C and is vortexed.12ml mixture is added dropwise in each plate.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 2nd day, cells and supernatant is removed.Each plate cleans material therein twice with 50ml DMEM/F12 culture mediums; and the fresh serum-free DMEM/F12 culture mediums of 100ml are added in each plate; N-ACETYL-D- MANNOSAMIDE (New ZealandPharmaceuticals) containing 40mM; 7mM Glu (Ameresco), 0.5g/L mannose (Sigma) and 1% (v/v) Pen .- Strep.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 3rd day; collect cells and supernatant, and fresh addition 100ml serum-free DMEM/F12 culture mediums in each plate, the N-ACETYL-D- MANNOSAMIDE containing 40mM; 7mM Glu, 0.5g/L mannose and 1% (v/v) Pen .- Strep.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)), 4 DEG C of preservations of mixture are added in the cells and supernatant of collection.

At the 4th day, cells and supernatant is collected.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the cells and supernatant of collection, and it is mixed with the sample that the 3rd day collects.The collection sample of mixing, before particulate is removed with 0.45 micron low protein binding filter (Durapore, Millipore), adds 1/10th volume 200mMMES/50mM MgCl₂(pH6) its pH to 6 is adjusted.4 DEG C of mixture is preserved or used immediately.During long term storage, supernatant is in -70 DEG C of preservations.

(ii) LT- α of the invention separation and purifying

Dye-ligand chromatogram (DLC) process is used for the first step that LT- α are purified.The reactive dye of some fixations can be used for shielding LT- α and avoid effective combination, and be discharged in the form of mass purification microtitre.Then, the dye-protein conjugate that detection matches in the form of small-scale post.

During small scale purification, in the case where pH is 6 or 7.3, the dye-ligand post that the cells and supernatant sample that 5ml dissolves passes through 0.5ml.In this optimization step, the optimal dyestuff-cell factor of selection and pH combinations make rate of recovery highest, exaggerated scale is used for large volume DLC.

For vast scale DLC, the High (Zymatrix) of selection reactive dye numbering 18 has optimal combination and elution property to LT- α as reactive dye.The cells and supernatant of filtering is under gravity by 4.0ml or 8.0ml shaft (Alltech, Extract Clean Filtercolumns), respectively containing advance with 50mM MES/5mM MgCl₂Equilibrate to pH6 3ml or 6ml DLC resins.Pillar buffer A (20mM MES/5mM MgCl₂, pH6) it is rinsed until eluate, do not monitor albumen with colourmetric analysis of protein (Biorad analysis of protein).LT- α are eluted in the following order with three kinds of elution buffers：

Eluent 1：Buffer solution C (50mM Tris-Cl/10mM EDTA pH 8)

Eluent 2：ENl.0(50mM Tris-Cl/10mM EDTA/1.0M NaCl pH 8)

Eluent 3：EN2.0(50mM Tris-Cl/10mM EDTA/2.0M NaCl pH 8)

Analyzed by the 1D SDS PAGE of Silver stain with 4-20% gradient Tris-Glycine glue (Invitrogen) and anti-LT- α ELISA (R＆D systems) part of elution.It was found that LT- α are incorporated into reactive dye 18High, and eluted in buffer solution C and buffer solution EN1.0.The contaminating protein that SDS PAGE analyses obtain 70% is removed in first step purification step.DLC parts containing LT- α are mixed, and utilize a centrifugal filter device (Ami con

Ultra, Millipore) it is concentrated to 5ml or so.

Then by ten times of dilutions of sample of concentration, before it is by an anion-exchange column (Bio-RadLaboratories, Uno S1), pillar equilibrates to pH6.5 with 50mM MES (pH6.5) (Sigma) in advance.With reference to LT- α eluted with 50mM MES (pH6.5) to the NaCl containing 1M 50mM MES (pH6.5) gradient solution.Obtained composition analyzes apparent molecular weight and purity by the 1D SDS PAGE of Silver stain with 4-20% gradient Tris-Glycine glue (Invitrogen).The solution of the α containing LT- is mixed, below 1ml is concentrated to, molecular-exclusion chromatography is carried out using centrifugal filter device (Amicon Ultra, Millipore).

Molecular-exclusion chromatography is carried out on the sample of concentration, 16/70 grade of (Pharmacia, Uppsala, the Sweden) post prepared using a Superdex 75.The flow velocity of 1% ammonium hydrogen carbonate level pad is 1ml/min.Total flowing time is 120min, and peak elution time is 20 between 100min.The SDS PAGE for carrying out Silver stain by 4-20% Tris- glycine glue (Invitrogen) are analyzed elution composition.It was found that 45min or so peak elution contains LT- α.The solution of the α containing LT- is mixed, and below 2ml is concentrated to using centrifugal filter device (Amicon Ultra, Millipore).

It was found that its apparent MW of the LT- α of purifying is 20-38Da or so, the SDS PAGE of Silver stain are carried out by 4-20% Tris- glycine glue (Invitrogen), analysis obtains its purity and is at least 95%.It is measured according to 280nm trap, molar extinction coefficient is 21430M^-1cm^-1, obtain LT- α final concentration of 78 μ g/ml.

(c) TNFRI-Fc of the invention preparation, separation and purifying

(i) TNFRI-FC of the invention preparation

0th day, in 5 500cm²Tissue culture dishes (Corning) upper berth 3 × 10⁷The cell of the human embryonic kidney cell line of individual conversion, such as HEK 293, HEK293 c18, HEK 293T, 293CEN4, HEK 239F, HEK 293E, HEK 293FT, AD-293 (Stratagene) or 293A (Invitrogen).Each culture dish, cell add 90ml Dulbecco ' s ModifiedEagle ' s Medium/Ham nutrition compounds F12 (DMEM/F12) (JRH Biosciences), (v/v) heat-inactivated fetal bovine serum of addition 10% (FCS is needed in culture medium, JRH Biosciences), 4mM Glus (Amresco), 10mM HEPES (Sigma) and 1% (v/v) Pen .- Strep (benzyl penicillin 5000U/ml, streptomycin sulphate 5mg/ml) (JRHBiosciences).Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 1st day, transfected with calcium phosphate.Before transfection, the culture medium in each plate is substituted for the fresh DMEM/F12 of 120ml, wherein containing 10% (v/v) heat inactivation FCS or DCS, 4mM Glu, 10mM HEPES and 1% (v/v) Pen .- Strep.Calcium phosphate/DNA is prepared to be precipitated as, 1200 μ g are added in sterilized water has pIRESbleo3 (Invitrogen) DNAs of people's TNFRI-Fc genes and 3720 μ l 2M calcium chloride solution (BDBiosciences), final volume is 30ml (solution A), or, in 1 × TE, equal number of DNA is added to 3000 μ l 2.5M CaCl₂In, final volume is 30ml (solution A).Solution A is added dropwise in 2 × HEPES buffer solutions of 30ml (HBS) (solution B) (BD Biosciences) with 10ml pipette.During dropwise addition, bubble slow transits through solution B.Mixture is incubated 20mi n at 25 DEG C and is vortexed.12ml mixture is added dropwise in each plate.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.Or, after being incubated 4 hours, remove the culture medium containing transfection composite, 100mlDMEM/F12 is added per plate, wherein contain 10% (v/v) DCS, 4mM Glus, 1% (v/v) Pen .- Strep and final concentration of 3.5-4.0mM HCl, the final pH of culture medium is 7.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 2nd day, cells and supernatant is removed.Each plate cleans material therein twice with 50ml DMEM/F12 culture mediums, and the fresh serum-free DMEM/F12 culture mediums of 100ml are added in each plate, N-ACETYL-D- MANNOSAMIDE (New ZealandPharmaceuticals) containing 40mM, 7 or 10mM Glu, 15mM HEPES, 0.5 or 4.1g/L mannose (Sigma), 1% (v/v) Pen .- Strep and ITS solution (5mg/L bovine insulins, the selenium of the human transferrin of 5mg/L parts iron saturation and 5 μ g/ml) (Sigma) (or, without ITS solution).Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 3rd day; collect cells and supernatant; and the fresh serum-free DMEM/F12 culture mediums of 100ml are added in each plate; N-ACETYL-D- MANNOSAMIDE containing 40mM, 7 or 10mM Glu, 15mM HEPES (or, without HEPES), 0.5 or 4.1g/L mannose, 1% (v/v) Pen .- Strep ITS solution (or, without ITS solution).Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)), 4 DEG C of preservations of mixture are added in the cells and supernatant of collection.

At the 4th day, the DMEM/F12 culture mediums that serum is free of in culture dish are collected.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in culture medium is collected, and it is mixed with the serum-free samples that the 3rd day collects.The cell raise machine of mixing is collected sample and filtered with the low protein binding filters (Durapore, Millipore) of 0.45mm.Mixture freezes in -70 DEG C or used immediately.

(ii) TNFRI-FC of the invention separation and purifying

Culture medium is collected, 2M Tris-HCl (pH8) (Sigma) is added, makes its final concentration of 100mM, pH is adjusted to pH8, is filtered (Durapore, 0.45 μm, Millipore).The culture medium containing TNFRI-Fc after 1 liter of pH adjustment passes through the protein A Sepharose columns (Pharmacia) with 1ml bed volumes under gravity, and pillar equilibrates to pH8 with 100mM Tris-Cl (pH8) in advance.With post buffer solution (the 100mM Tris-Cl of 20 times of column volumes, pH8 after) rinsing, TNFRI-Fc is eluted with 0.1M citric acid (Sigma) (pH4.4), then eluted with 0.1M citric acid (Sigma) (pH2.2), and add 100 μ l and 400 μ l 2M Tris-HCl (pH9) (Sigma) immediately respectively and neutralized.By the SDS PAGE of Silver stain, analyzed with 4-20% gradient Tris-glycine glue (Invitrogen).The solution containing TNFRI-Fc of purifying is mixed, and is concentrated to below 1ml, molecular-exclusion chromatography is carried out using centrifugal filter device (AmiconUltra, Millipore).

Molecular-exclusion chromatography is carried out on the sample of concentration, 16/70 grade of (Pharmacia, Uppsala, the Sweden) post prepared using Superdex 200.The flow velocity of 1% ammonium hydrogen carbonate level pad is 1ml/min.Total flowing time is 120min, and peak elution time is 20 between 100min.The SDS PAGE for carrying out Silver stain by 4-20% Tris- glycine glue (Invitrogen) are analyzed elution composition.The solution containing TNFRI-Fc is mixed, and below 2ml is concentrated to using centrifugal filter device (Amicon Ultra, Millipore).

It was found that its apparent MW of the TNFRI-Fc of purifying is between 45-95Da, obtaining its purity by the SDSPAGE analyses of Silver stain is at least 99%.It is measured according to 280nm trap, molar extinction coefficient is 51725M^-1cm^-1, obtain TNFRI-Fc final concentration of 213.86 μ g/ml.

(d) TNFRII-Fc of the invention preparation, separation and purifying

(i) TNFRII-Fc of the invention preparation

0th day, in 5 500cm²Tissue culture dishes (Corning) upper berth 3 × 10⁷The cell of individual next inverting human embryonic kidney cell line, such as HEK 293, HEK 293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293E, HEK 293FT, AD-293 (Stratagene) or 293A (Invitrogen).Addition 10% (v/v) heat-inactivated fetal bovine serum (FCS, JRH Biosciences), 10mM HEPES (Sigma), 4mM Glus (Ameresco) and 1% (v/v) Pen .- Strep (JRH) are needed in each culture dish, cell addition 90ml Dulbecco ' s ModifiedEagle ' s Medium/Ham nutrition compounds F12 (DMEM/F12) (JRH Biosciences), culture medium.

At the 1st day, transfected with calcium phosphate.Before transfection, the culture medium in each plate is substituted for the fresh DMEM/F12 of 120ml (JRH Biosciences), wherein containing 10% hyclone (JRH).Prepare calcium phosphate/DNA to be precipitated as, 1200 μ g are added in sterilized water has pIRESbleo3 (Clonetech, the BD Biosciences) DNAs and 3720 μ l CaCl of people's TNFRII-Fc genes₂, final volume is 30ml (solution A).Solution A is added dropwise in 2 × HEPES buffer solutions of 30ml (HBS) (solution B) with 10ml pipette.During dropwise addition, bubble slow transits through solution B.Mixture is incubated 20min at 25 DEG C and is vortexed.With the mixture that 12ml is added dropwise in each plate of pipette.After 4 hours, remove the culture medium containing transfection composite, 100ml DMEM/F12 (pH7) are added per plate, wherein contain 10% (v/v) heat-inactivated fetal bovine serum (JRH Biosciences), 10mM HEPES, 4mM Glus, 1% (v/v) Pen .- Strep and 3.5 or 4.0mM HCl, are incubated overnight.

At the 2nd day; collect cells and supernatant; and the fresh serum-free DMEM/F12 culture mediums of 100ml, the Pen .- Strep of the Glu of N-ACETYL-D- MANNOSAMIDE, 7mM, 0.5g/L mannose and 1% (v/v) containing 40mM are added in each plate.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)), 4 DEG C of preservations of mixture are added in the cells and supernatant of collection.

At the 3rd day, the DMEM/F12 of serum-free, and the DMEM/F12 of fresh addition 100ml serum-free in each plate are collected.100mM PMSF, 1% (v/v) and 500mM EDTA, 1% (v/v), 4 DEG C of preservations of mixture are added in the cell culture medium of collection.

At the 4th day, the DMEM/F12 of serum-free in plate is collected.100mM PMSF, 1% (v/v) and 500mM EDTA are added in the cell culture medium of collection according to 1% (v/v), and mixed the mixture with the serum-free samples that first time collects, the cells and supernatant of mixing is filtered with 0.45 micron low protein binding filter (Durapore, Millipore).- 70 DEG C of mixture is preserved or used immediately.

(ii) TNFRII-Fc of the invention separation and purifying

Culture medium is collected, 2M Tris-HCl (pH8) (Sigma) is added, makes its final concentration of 100mM, pH is adjusted to pH8, is filtered (Durapore, 0.45 μm, Millipore).The culture medium containing TNFRII-Fc after 1 liter of pH adjustment passes through the protein A Sepharose columns (Pharmacia) with 1ml bed volumes under gravity, and pillar equilibrates to pH8 with 100mM Tris-Cl (Sigma) in advance.With post buffer solution (the 100mM Tris-Cl of 20 times of column volumes, pH8 after) rinsing, TNFRII-Fc is eluted with 0.1M citric acid (Sigma) (pH4.4), then eluted with 0.1M citric acid (Sigma) (pH2.2), and add 100 μ l and 400 μ l 2M Tris-HCl (pH9) (Sigma) immediately respectively and neutralized.By the SDS PAGE of Silver stain, analyzed with 4-20% gradient Tris-glycine glue (Invitrogen).The solution containing TNFRII-Fc of purifying is mixed, and is concentrated to below 1ml, molecular-exclusion chromatography is carried out using centrifugal filter device (Amicon Ultra, Millipore).

Molecular-exclusion chromatography is carried out on the sample of concentration, 16/70 grade of (Pharmacia, Uppsala, the Sweden) post prepared using Superdex 200.The flow velocity of 1% ammonium hydrogen carbonate level pad is 1ml/min.Total flowing time is 120min, and peak elution time is 20 between 100min.The SDS PAGE for carrying out Silver stain by 4-20% Tris- glycine glue (Invitrogen) are analyzed elution composition.The solution containing TNFRII-Fc is mixed, and below 2ml is concentrated to using centrifugal filter device (Amicon Ultra, Millipore).

It was found that its apparent MW of the TNFRII-Fc of purifying is between 45-100kDa, obtaining its purity by the SDS PAGE analyses of Silver stain is at least 99%.It is measured according to 280nm trap, molar extinction coefficient is 61110M^-1cm^-1, obtain TNFRII-Fc final concentration of 1321 μ g/ml.

(e) BAFF of the invention preparation, separation and purifying

(i) BAFF of the invention preparation

0th day, in 5 500cm²Tissue culture dishes (Corning) upper berth 3 × 10⁷The cell of individual next inverting human embryonic kidney cell line, such as HEK 293, HEK 293c 18, HEK 293T, 293 CEN4, HEK 239F, HEK 293E, HEK 293FT, AD-293 (Stratagene) or 293A (Invitrogen).Each culture dish, cell adds 90ml Dulbecco ' s ModifiedEagle ' s Medium/Ham nutrition compounds F12 (DMEM/F12) (JRH Biosciences), addition 10% (v/v) donor calf serum (DCS is needed in culture medium, JRH Biosciences), 4mM Glus (Amresco) and 1% (v/v) Pen .- Strep (benzyl penicillin 5000U/ml, streptomycin sulphate 5mg/ml) (JRH Biosciences).Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 1st day, transfected with calcium phosphate.Before transfection, the culture medium in each plate is substituted for the fresh DMEM/F12 of 120ml, wherein FCS or DCS, 4mM Glu containing 10% heat fire extinguishing, 10mM HEPES, and 1% (v/v) Pen .- Strep.Calcium phosphate/DNA is prepared to be precipitated as, 1200 μ g are added in sterilized water has pIRESbleo3 (Invitrogen) DNAs of people's BAFF genes and 3720 μ l 2M calcium chloride solution (BD Biosciences), final volume is 30ml (solution A), or, the DNA of identical quantity is added to 3000 μ l 2.5M CaCl in 1 sterile × TE₂In.Solution A is added dropwise in 2 × HEPES buffer solutions of 30ml (HBS) (solution B) (BD Biosciences) with 10ml pipette.During dropwise addition, bubble slow transits through solution B.Mixture is incubated 20min at 25 DEG C and is vortexed.It is added dropwise to by 12ml mixtures in each plate.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.Or, after being incubated 4 hours, remove the culture medium containing transfection composite, 100mlDMEM/F12 is added per plate, wherein contain 10% (v/v) DCS, 4mM Glus, 1% (v/v) Pen .- Strep and final concentration of 3.5-4.0mM HCl, the final pH of culture medium is 7.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 2nd day, cells and supernatant is removed.Each plate clean material therein twice with 50ml DMEM/F12 culture mediums, and serum-free DMEM/F12 culture mediums fresh addition 100ml in each plate, the N-ACETYL-D- MANNOSAMIDE (New ZealandPharmaceuticals) containing 40mM, 7 or 10mM Glu, 0.5 or 4.1g/L Mannose (Sigma) and 1% (v/v) Pen .- Strep.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 3rd day; collect cells and supernatant; and add the fresh serum-free DMEM/F12 culture mediums of 100ml in each plate, the N-ACETYL-D- MANNOSAMIDE containing 40mM, 7 or 10mM Glu, 0.5 or 4.1g/L Mannose and 1% (v/v) Pen .- Strep.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)), 4 DEG C of preservations of mixture are added in the cells and supernatant of collection.

At the 4th day, cells and supernatant is collected.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the cells and supernatant of collection, and mix it with the sample that the 3rd day collects, the collection sample of mixing, particulate is being removed with 0.45 micron low protein binding filter (Durapore, Millipore).- 70 DEG C of mixture is preserved or used immediately.

(ii) BAFF of the invention separation and purifying

With tangential flow filtration (TFF) device (Pelicon XL, Ultracell, Millipore), the cells and supernatant that 950ml is filtered concentrates 20 times or so.Sample injects 150cm with 150ml/min speed²Regenerated cellulose film on, 5KDa molecular weight is blocked, until the volume of sample concentration to 30ml.The sample of concentration adds 70ml 50mM HEPES (pH is 8) diafiltration, and concentration volume is still 30ml.The diafiltration step is repeated twice, and final volume is 50ml.Then, the concentration filter sample is filtered with one 0.45 micron of low protein binding filter (Durapore, Millipore).

BAFF purifying is that it is located at one in advance with the ion exchange column (Bio-Rad Laboratories, MacroPrep HS) of 50mM HEPES (pH is 8.5) balances by TFF by the cells and supernatant of concentration.Then, with the BAFF of 50mM HEPES (pH is 8) to 100%50mM HEPES (pH is 8) NaCl containing 1M linear gradient solution elution of bound.Obtained composition analyzes apparent molecular weight and purity by 1D SDS PAGE with 4-20% gradient Tris-Glycine glue (Invitrogen), and is quantified with anti-BAFF ELISA (R＆D systems).It was found that BAFF is eluted with two kinds of different ionic species from anion-exchange column.The solution containing BAFF is mixed, below 1ml is concentrated to, molecular-exclusion chromatography is carried out using centrifugal filter device (Amicon Ultra, Millipore).

Molecular-exclusion chromatography on the anion exchange component of combination on carry out, 16/70 grade of (Pharmacia, Uppsala, the Sweden) post prepared using Superdex75.The flow velocity of 1% ammonium hydrogen carbonate level pad is 1ml/min.Total flowing time is 120min, and peak elution time is 20 between 100min.The part of elution is analyzed with 4-20%Tris- glycine glue (Invitrogen) and BAFFELISA.The part containing BAFF is mixed, and below 2ml is concentrated to using centrifugal filter device (Amicon Ultra, Millipore).

It was found that its apparent MW of the BAFF of purifying is between 16-17kDa.It is measured according to 280nm trap, molar extinction coefficient is 14565M^-1cm^-1, obtain BAFF final concentration of 50 μ g/ml.

(f) NGFR-Fc of the invention preparation, separation and purifying

(i) NGFR-Fc of the invention preparation

0th day, in 5 500cm²Tissue culture dishes (Corning) upper berth 3 × 10⁷The cell of the human embryonic kidney cell line of individual conversion, such as HEK 293, HEK 293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293E, HEK 293FT, AD-293 (Stratagene) or 293A (Invitrogen).Each culture dish, cell adds 90ml Dulbecco ' s ModifiedEagle ' s Medium/Ham nutrition compounds F12 (DMEM/F12) (JRH Biosciences), (v/v) heat-inactivated fetal bovine serum of addition 10% (FCS is needed in culture medium, JRH Biosciences), 4mM Glus (Amresco) and 1% (v/v) Pen .- Strep (benzyl penicillin 5000U/ml, streptomycin sulphate 5mg/ml) (JRH Biosciences).Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 1st day, transfected with calcium phosphate.Before transfection, the culture medium in each plate is substituted for the fresh DMEM/F12 of 120ml, wherein containing 10% (v/v) heat inactivation FCS, 4mM Glus, and 1% (v/v) Pen .- Strep.Prepare calcium phosphate/DNA to be precipitated as, 1200 μ g are added in sterilized water has pIRESbleo3 (Invitrogen) DNAs of people's NGFR-Fc genes and 3720 μ l 2.5M CaCl₂Solution, final volume is 30ml (solution A).Solution A is added dropwise in 2 × HEPES buffer solutions of 30ml (HBS) (solution B) with 10ml pipette.During dropwise addition, bubble slow transits through solution B.Mixture is incubated 20min at 25 DEG C and is vortexed.12ml mixture is added dropwise in each plate.After 4 hours, the culture medium containing transfection composite is removed, 100mlDMEM/F12 is added per plate, wherein contain 10% (v/v) heat-inactivated FCS, 4mM Glus, 1% (v/v) Pen .- Strep and final concentration of 3.5mM HCl, the final pH of culture medium is 7.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 2nd day, cells and supernatant is removed.Each plate cleans material therein twice with 50ml DMEM/F12 culture mediums; and the fresh serum-free DMEM/F12 culture mediums of 100ml are added in each plate; N-ACETYL-D- MANNOSAMIDE (New ZealandPharmaceuticals) containing 40mM; 10mM Glu, 0.5g/L mannose (Sigma) and 1% (v/v) Pen .- Strep.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 3rd day; collect cells and supernatant, and fresh addition 100ml serum-free DMEM/F12 culture mediums in each plate, the N-ACETYL-D- MANNOSAMIDE containing 40mM; 10mM Glu, 0.5g/L mannose and 1% (v/v) Pen .- Strep.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)), 4 DEG C of preservations of mixture are added in the cells and supernatant of collection

At the 4th day, cells and supernatant is collected.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the cells and supernatant of collection, and it is mixed with the sample that the 3rd day collects.The collection sample of mixing, before particulate is removed with 0.45 micron low protein binding filter (Durapore, Millipore), adds 2M Tris-HCl (pH8) (Sigma) and adjusts its pH to 8, final concentration of 100mM.- 70 DEG C of mixture is preserved or used immediately.

(ii) NGFR-Fc of the invention separation and purifying

Culture medium is collected, 2M Tris-HCl (pH8) (Sigma) is added, makes its final concentration of 100mM, pH is adjusted to pH8, is filtered (Durapore, 0.45 μm, Millipore).The culture medium containing NGFR-Fc after 1 liter of pH adjustment passes through the protein A Sepharose columns (Pharmacia) with 1ml bed volumes under gravity, and pillar equilibrates to pH8 with 100mM Tris-Cl (Sigma) in advance.With post buffer solution (the 100mM Tris-Cl of 20 times of column volumes, pH8 after) rinsing, NGFR-Fc is eluted with 0.1M citric acid (Sigma) (pH4.4), then eluted with 0.1M citric acid (Sigma) (pH2.2), and add 100 μ l and 400 μ l 2M Tris-HCl (pH9) (Sigma) immediately respectively and neutralized.By the SDS PAGE of Silver stain, analyzed with 4-20% gradient Tris-glycine glue (Invitrogen).The solution containing NGFR-Fc of purifying is mixed, and is concentrated to below 1ml, molecular-exclusion chromatography is carried out using centrifugal filter device (AmiconUltra, Millipore).

Molecular-exclusion chromatography is carried out on the sample of concentration, 16/70 grade of (Pharmacia, Uppsala, the Sweden) post prepared using Superdex 200.The flow velocity of 1% ammonium hydrogen carbonate level pad is 1ml/min.Total flowing time is 120min, and peak elution time is 20 between 100min.The SDS PAGE for carrying out Silver stain by 4-20% Tris- glycine glue (Invitrogen) are analyzed elution composition.The solution containing NGFR-Fc is mixed, and below 2ml is concentrated to using centrifugal filter device (Amicon Ultra, Millipore).

It was found that its apparent MW of NGFR-Fc of purifying is in 50-110kDa, obtaining its purity by the SDSPAGE analyses of Silver stain is at least 95%.It is measured according to 280nm trap, molar extinction coefficient is 55735M^-1cm^-1, obtain NGFR-Fc final concentration of 1259 μ g/ml.

(g) preparation, separation and the purifying of FasL of the invention

(i) preparation of FasL of the invention

0th day, in 5 500cm²Tissue culture dishes (Corning) upper berth 3 × 10⁷The cell of the human embryonic kidney cell line of individual conversion, such as HEK 293, HEK293 c18, HEK 293T, 293CEN4, HEK 239F, HEK 293E, HEK 293FT, AD-293 (Stratagene) or 293A (Invitrogen).Each culture dish, cell adds 90ml Dulbecco ' s ModifiedEagle ' s Medium/Ham nutrition compounds F12 (DMEM/F12) (JRH Biosciences), addition 10% (v/v) donor calf serum (DCS is needed in culture medium, JRH Biosciences), 4mM Glus (Amresco) and 1% (V/V) Pen .- Strep (benzyl penicillin 5000U/ml, streptomycin sulphate 5mg/ml) (JRH Biosciences).Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 1st day, transfected with calcium phosphate.Before transfection, the culture medium in each plate is substituted for the fresh DMEM/F12 of 120ml, wherein containing 10% (v/v) DCS, 4mM Glus, and 1% (v/v) Pen .- Strep.Prepare calcium phosphate/DNA to be precipitated as, 1200 μ g are added in sterilized water (BD Biosciences) has pIRESbleo3 (Invitrogen) DNAs of people's Fa s ligand genes and 3720 μ l 2.5M CaCl₂(BD Biosciences), final volume is 30ml (solution A).Solution A is added dropwise in 2 × HEPES buffer solutions of 30ml (HBS) (solution B) (BD Biosciences) with 10ml pipette.During dropwise addition, bubble slow transits through solution B.Mixture is incubated 20min at 25 DEG C and is vortexed.12ml mixture is added dropwise in each plate.After 4 hours, remove the culture medium containing transfection composite, 100ml DMEM/F12 (pH7) are added per plate, wherein contain 10% (v/v) DCS, 4mM Glus, the HCl of 1% (v/v) Pen .- Strep and final concentration of 3.5mM, the final pH of culture medium is 7.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 2nd day, cells and supernatant is removed.Each plate cleans material therein twice with 50ml DMEM/F12 culture mediums; and the fresh serum-free DMEM/F12 culture mediums of 100ml are added in each plate; N-ACETYL-D- MANNOSAMIDE (New ZealandPharmaceuticals) containing 40mM; 10mM Glu; 0.5g/L mannose (Sigma), and 1% (v/v) Pen .- Strep.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.

At the 3rd day, cells and supernatant, and fresh addition 100ml serum-free DMEM/F12 culture mediums in each plate are collected; N-ACETYL-D- MANNOSAMIDE containing 40mM; 10mM Glu, 0.5g/L mannose, and 1% (v/v) Pen .- Strep.Plate is in 37 DEG C and 5%CO₂Under conditions of be incubated overnight.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)), 4 DEG C of preservations of mixture are added in the cells and supernatant of collection.

At the 4th day, cells and supernatant is collected.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the cells and supernatant of collection, and it is mixed with the sample that the 3rd day collects.The collection sample of mixing, before particulate is removed with 0.45 micron low protein binding filter (Durapore, Millipore), adds 200mM MES/50mM MgCl₂PH6 adjusts its pH to 6,.- 70 DEG C of mixture is preserved or used immediately.

(ii) separation and purifying of FasL of the invention

Dye-ligand chromatogram (DLC) process is used for the first step that FasL is purified.A series of reactive dye can be used for shielding FasL and avoid effective combination, and be discharged in the form of mass purification microtitre.Then, the dye-protein conjugate that detection matches in the form of small-scale post.

During small scale purification, in the case where pH is 6 or 7.3, the dye-ligand post that the cells and supernatant sample that 5ml dissolves passes through 0.5ml.In this optimization step, the optimal reactive dye-cell factor of selection and pH combinations make rate of recovery highest, exaggerated scale is used for large volume DLC.

For vast scale DLC, selection reactive dye numbering 18High (Zymatrix) has optimal combination and elution property to FasL as reactive dye.The cells and supernatant of filtering is under gravity by 4.0ml or 8.0ml shaft (Alltech, Extract CleanFilter columns), respectively containing advance with 50mM MES/5mM MgCl₂Equilibrate to pH6 3ml or 6ml DLC resins.Pillar buffer A (20mM MES/5mM MgCl₂, pH6) it is rinsed until eluate with colourmetric analysis of protein (Biorad analysis of protein) does not monitor albumen.FasL is eluted in the following order with three kinds of elution buffers：

Eluent 1：Buffer solution C (50mM Tris-Cl/10mM EDTA pH 8)

Eluent 2：EN1.0(50mM Tris-Cl/10mM EDTA/1.0M NaCl pH8)

Eluent 3：EN2.0(50mM Tris-Cl/10mM EDTA/2.0M NaCl pH8)

The SDS PAGE and the ELISA (R＆D systems) by anti-FasL of Silver stain are carried out by 4-20%Tris- glycine glue (Invitrogen), elution fraction is analyzed.It was found that FasL is incorporated into reactive dye 8High and eluted in ENl.0 buffer solutions.The albumen that SDS PAGE analytical proofs 90% pollute is removed in the purifying of this first step.The salinity of the DLC solution containing FasL is removed with PD10 posts (Amersham Biosciences), and is mixed for anion-exchange chromatography.

The solution of desalination is reached into purifying from PD10 posts by anion-exchange column (Bio-RadLaboratories, Uno S1), ion exchange column equilibrates to pH6.5 with 50mM MES (pH6.5) (Sigma) in advance.Eluted with reference to the FasL of conjunction with 50mM MES (pH6.5) to the NaCl containing 1M 50mM MES (pH6.5) linear gradient solution from pillar.Obtained composition analyzes apparent molecular weight and purity by ELISA and 1D SDS PAGE with 4-20% gradient Tris-Glycine glue (Invitrogen).Mix the solution of factor-containing.

Molecular-exclusion chromatography is carried out on the sample of concentration, 16/70 grade of (Pharmacia, Uppsala, the Sweden) post prepared using Superdex 75.The flow velocity of 1% ammonium hydrogen carbonate level pad is 1ml/min.Total flowing time is 120min, and peak elution time is between 20 and 100min.The SDS PAGE for carrying out Silver stain by 4-20%Tris- glycine glue (Invitrogen) are analyzed elution composition.

It was found that its apparent MW of the FasL of purifying is between 25-36kDa.It is measured according to 280nm trap, molar extinction coefficient is 27515M^-1cm^-1, obtain the final concentration of 94.8 μ g/ml of FasL.

(h) TNFRII-Fc (lot numbers of the further present invention：003) preparation, separation and purification embodiment

(i) TNFRII-Fc (lot numbers of the invention：003) preparation

Freestyle 293F cell suspending liquids are prepared, TCS is at least 5 × 10⁷Individual cell.The density and TCS of Freestyle 293F cells are determined with trypan blue exclusion.In 125ml conical flask, by 3 × 10⁷Individual cell is added in 28ml Freestyle ExpressionMedium (Invitrogen).

Then, cell is incubated in 37 DEG C of shakings while transfection composite is prepared.There is 30 μ g the DNA (pCEP-4-TNFRII-Fc) of TNFRII-Fc sequences to be added to 25 μ l volume in 975 μ l Opti-MEM (Invitrogen) (solution A).40 μ l 293fectin (Invitrogen) is added in 960 μ l Opti-MEM (solution B).

Solution A and solution B were in incubation at room temperature 5 minutes, and then by its careful mixing, room temperature is incubated 30 minutes again.

The transfection composite is added in the 28ml suspension of 293F cells.It is maintained to express culture medium until TNFRII-Fc expression stops by being inoculated with.

The extensive expression of albumen is carried out in shaking flask or MantaRay blake bottles (Fisher Scientific).The Freestyle 293F cells that 500ml or 1000ml has transfected pCEP-4-TNFRII-Fc carriers are prepared with Freestyle Expression Medium, cell density is 4 × 10⁵Cell/ml.Then the cell of transfection is centrifuged into 10min in 1000rpm, the sterile PBS preheated with 5ml is washed, 1000rpm centrifugation 10min, and be resuspended with Feestyle ExpressionMedium fresh 10ml.Cell is with 4.0 × 10⁵Cell/ml density is added in MantaRay blake bottles or shaking flask, and cell is resuspended in the Freestyle ExpressionMedium of 500ml or 1000ml preheatings.In the humidified incubator with stirring, in 37 DEG C, 5%CO₂Under the conditions of incubated cell.

Every 24 hours, the survival rate of cell is assessed with trypan blue exclusion.Once cell density reaches 1.5 × 10⁶Cell/ml (in generally after inoculation 5 days), collects supernatant.

(ii) TNFRII-Fc (lot numbers of the invention：003) separation and purifying

(a) TNFRII-Fc (lot numbers of the invention：003) purifying is aseptically carried out, and is realized with two kinds of chromatographies.Centrifugation makes expression culture supernatant (lot number：003) become clarification, and be added to 5ml/min flow velocity on protein A Sepharose columns (RN040633, Repligen).Then it is rinsed with 10 times of column volume (200ml) 0.1M Tris-Cl (pH8.0).With reference to TNFRII-Fc (lot numbers：003) eluted with cold 0.1M citric acids (pH4.0), 20ml solution is collected into the 50ml Falcon pipes of 8 marks.Elution samples 4 DEG C be incubated make within 1 hour it is virally inactivated, then with 2M Tris-Cl (pH8.5) neutralize eluent.

The TNFRII-Fc eluted from albumin A post is further purified in Q Sepharose Column anion-exchange columns, and pillar is balanced with 80mM citric acid 400mM Tris-Cl (pH9.0) in advance.The flow velocity of Protein A eluant is 5ml/min, collects peak value sample, 4 DEG C of preservations.With reference to albumen eluted with the level pad of the NaCl containing 1M.

Concentrated by the solution of peak value with 4 Centriprep YM-10 centrifugal filters devices (Millipore) according to the specification of manufacturer.After 3 times of concentration, the buffer solution of solution is become into 1 × DPBS pH7.0 (2.7mM KCl, 1.5mM KH₂PO₄, 137mM NaCl and 8mMNa₂HPO₄, pH7.0)

UV₂₈₀TNFRII-Fc (lot numbers are measured with Fc ELISA：003) ultimate yield is the common 60ml of 1.45mg/ml (i.e. 87mg total proteins).Silver staining coloring agent shows its purity more than 95%.

Biological standardization structure shows TNFRII-Fc (lot numbers：003) cytotoxicity of TNF-α can effectively, be suppressed in the proliferation test of WEHI164 cells.

(b) or, TNFRII-Fc is purified with 2ml IPA-400HC rProtein A posts (RepligenRN040633).Expression culture supernatant containing TNFRII-Fc is added in post, room temperature continues 4 hours, and at 4 DEG C overnight.Pillar is rinsed with buffer solution (12.5ml 1M NaCl, 0.1M Tris, pH8.0), with reference to TNFRII-Fc eluted with 14ml 0.1M citric acid (pH4.0), and with 6ml pH for 9.0 Tris neutralize.Eluent is concentrated with Centriprep YM-10 centrifugal filters devices according to the specification of manufacturer.According to method detection TNFRII-Fc (3mg/ml or so) described above.

Embodiment 3

(a) sign of TNF-α of the invention

(i) two-dimensional polyacrylamide electrophoresis

The sample that example 2 (a) is collected changes its buffer solution into by dialysis or desalting column (the FastDesalting Column of Pharmacia HR 10/10) in repurity (18MOhm) water, and is dried with SpeedVac concentrating instruments.Or, the sample of collection carries out TCA work acetone precipitations with known method.Sample is in secondary MSD buffer solutions (the 5M urea for being dissolved in 240 μ l, 2M thiocarbamides, 65mM DTT, 2% (w/v) CHAPS, 2% (w/v) sulfobetaines 3-10,0.2% (v/v) carrier ampholyte, 40mM Tris, 0.002% (w/v) bromophenol blue, water) in, and centrifuged 8 minutes in 15000g.

Isoelectric focusing (IEF) is carried out with prefabricated 11cm or prefabricated 17cm pH of latex gel 3-10 retentive force I EF adhesive tape (BioRad).Rehydration at least 6 hours at room temperature in sample of the IEF adhesive tape in sealed tube.IEF adhesive tape is placed in focus cell and covered with liquid paraffin.IEF operation：100V 1 hour when there is 11cm bands, 200V 1 hour, 600V 2 hours, 1000V2 hours, 2000V 2 hours, 3500V 12 hours and 100V were up to 12 hours, or (identical voltage raises program) 85kV hours when there is 17cm bands

Ensuing isoelectric focusing, adhesive tape is reduced and is partially alkylated or alkylated, and is being applied to second to before gel.Adhesive tape is cultivated at least 20 minutes in 1 × Tris/HCl pH 8.8,6M urea, 2% (w/v) SDS, 2% (v/v) glycerine, 5mM tributylphosphine oxides (TBP), 2.5% (v/v) acrylamide.

11cm adhesive tape, to separation, passes through Criterion pre-filled (11 × 8cmx1mm is thick) 10-20% Trisglycine gradient gels (BioRad) second.17cm adhesive tape is separated into the Tris glycine gradient gels for the 10-20% that 17 × 17cm, 1.5mm are thick, irrigate certainly.Precision or Kaleidoscope molecular weight markers (BioRad) are also used for gel.Adhesive tape is put into groove, uses 0.5% agarose containing the bromophenol blue as trace dyestuff.

SDS-PAGE is carried out (for 11cm glue with Criterion or Protean II electrophoresis systems (BioRad), 200V, 1 hour (until buffer solution forward position will run out of buffings end), and for 17cm glue, every piece of glue 15mA constant current, 21 hours).Buffer solution used is 192mM glycine, and 0.1% (w/v) SDS, 24.8mM Tris alkali is under pH 8.3.

The two-dimentional glue of completion fixes 30 minutes with 10% methanol (MeOH) and 7% acetic acid (Hac)-overnight.Then, glue is dyed at least 3 hours with Sypro Ruby glue dyes (BioRad), and is decolourized at least 30 minutes with 10%MeOH and 7%HAc.Or, glue uses Deep Purple fluorescent dyeings after fixation.Then, by glue in 300mM Na₂CO₃, 35mM NaHCO ₃2 × 30min of middle immersion, then, lucifuge are immersed in the Deep Purple dyestuffs of 1: 20O dilution at least 1 hour.Then with 2 × 15min of 10%MeOH and 7%HAc decolourings.In the case of two kinds, glue is all imaged with FX laser photometers (BioRad) and suitable optical filter.

With image analysis software analysis of two-dimensional electrophoresis protein graphical spectrum

Software I mageJ (http://rsb.info.nih.gov/ij/) be used to analyze the relative intensities of protein spotses on each gel.Spectrodensitometry is carried out to the spot of every piece of glue selected areas, and background is deducted with the suitable glue region without protein spotses.Volume integral is carried out to destination protein spot and thus calculates spot quality center.The relative intensity percentage of each albumen is calculated, the additive value of all spot densities is standardized as 100%, so as to determine density of each protein spotses relative to other spots in every piece of glue.

The relative distance of from every piece matrix line of spot is determined, and Precision the or Kaleidoscope molecular weight mark that it is utilized with every piece of glue marks indicated distance to be compared, so that it is determined that the molecular weight of each spot.One four multivariate exponential function is respectively suitable for the protein spotses position of accurate scale designation and incorporation.

The electric charge (pKa value) of isoform is determined with ImageJ by measuring the distance of the difference on the left of spot and each gel.Because the pI values of band and the physical distance of every piece of glue have linear relationship, therefore, it is possible to determine the pI values corresponding to the isoform spot of different pKa values easily.

The unique isoform of each protein spotses correspondence TNF-α.The isoform of major protein spots correspondence TNF-α in every piece of glue.Low-density spot is probably TNF-α or low-level pollutant, but because density is low, PMF not can prove that.Observation to glue shows at least 10 to 30 isoforms of TNF-α of the present invention.Table 9 and 10 gives the critical nature of these isoforms：PI values (± 1.0), apparent molecular weight (± 20%), and relative intensity (± the 20% of actual number, or total ± 2%, any one is larger).Listed numerical value corresponds to, the Density Weighted center in the glue region containing spot of selection, therefore, and it only reacts the albumen pI and molecular weight of certain reading in the selected areas of every piece of glue.In view of the intrinsic variability and protein positions of 2D glue sizes, based on the numerical value listed by table 9 and 10, it is 4-8.5 or so to determine the scope of molecule pI values；Based on the numerical value listed by table 9 and 10, the apparent molecular weight scope for determining molecule is 10-30kDa.

Table 9

The molecular weight and pI values of TNF-α isoform

Spot is numbered	Isoelectric point   (pI)	Molecular weight   (kDa)	Relative intensity (%) (standardized value)
				2	5.80	15.71	3.25
3	6.11	15.69	3.61
				4	6.49	15.57	6.42
5	6.64	15.57	1.15
				6	6.94	15.33	4.27
7	5.82	14.75	2.23
				8	6.13	14.84	3.51
9	6.48	14.80	8.32
				10	6.95	14.47	26.96
11	7.17	14.53	2.68
				12	7.53	14.09	22.39
13	7.72	14.14	1.55
				14	5.32	12.99	0.65
15	5.39	13.00	0.94
				16	5.44	13.00	0.92
17	5.49	12.99	0.88
				18	5.70	13.01	5.27
19	6.13	13.26	1.97
				20	6.43	13.23	0.53
21	6.59	13.32	0.66
				22	6.63	13.31	0.80
23	6.67	13.34	0.64
				24	6.72	13.32	0.40

Table 10：The molecular weight and pI values of TNF-α

Spot is numbered	Isoelectric point   (pI)	Molecular weight   (kDa)	Relative intensity (%) (standardized value)
				2	5.31	17.31	0.61
3	5.63	17.41	1.79
				4	5.77	17.31	1.77
5	5.92	17.33	5.34
				6	6.21	17.22	8.10
7	6.41	17.30	2.30
				8	6.51	17.20	1.34
9	6.62	17.16	5.95
				10	5.31	15.93	0.64
11	5.63	15.99	0.78
				12	5.76	15.94	1.03
13	5.92	15.85	2.59
				14	6.21	15.67	5.00
15	6.39	15.94	1.45
				16	6.45	15.88	1.74
17	6.50	15.82	1.41
				18	6.55	15.77	1.83
19	6.59	15.73	1.7l
				20	6.65	15.81	3.19
21	6.71	15.86	1.09
				22	6.76	16.03	1.13
23	6.81	15.90	1.40
				24	6.90	15.83	5.22
25	7.02	16.00	5.08
				26	7.11	16.04	4.11
27	7.17	16.05	1.54
				28	7.23	16.01	1.56
29	7.29	15.87	1.83
				30	7.36	15.93	2.19
31	7.44	15.85	2.37
				32	7.54	15.72	2.93
33	7.66	15.65	7.62
				34	7.81	15.74	2.69
35	7.92	15.68	0.62
				36	5.31	12.99	0.76
37	5.56	13.01	2.15
				38	5.83	12.87	3.69
39	6.21	12.70	1.60
				40	6.73	13.63	0.62
41	6.73	11.97	1.25

(ii) one-dimensional polyacrylamide gel electrophoresis

The sample collected by embodiment 2 (a) is dried, (10% glycerine, 0.1%SDS, 10mM DTT, 63mM tris-HCl) is then redissolved in 60 μ l 1D sample buffers and is heated 5 minutes at 100 DEG C.For PNGaseF processing, 30 μ L portions of sample are taken, and add NP40 to final concentration 0.5%.5 μ L PNGaseF is added, then sample is cultivated 3 hours at 37 DEG C.For the glycosidase cocktail factures of sample, take a part and then add NP40 to final concentration 0.5%.Add the μ L and sialidase A (neuramidase) of PNGase F 1, O- dextranases, β (1-4)-galactosidase and each 1 μ L of β-N-acetylglucosaminidase.Processing and untreated sample is cultivated 3 hours at 37 DEG C.Processing and untreated sample electrophoresis in prefabricated Tris gels, such as Tris 4-20% gradient gels (BioRad) or Tris HCl gradient gels (Invitrogen).Accurate molecular weight mark (BioRad catalogues encode 161-0363) is also used for gel.Criterion 4-20% or 18% gel are used for 1D SDS-PAGE, and (BioRad catalogues are encoded：345-0033 or 345-0024).SDS-PAGE is run with Mini Protean II or Criterion electrophoresis systems (BioRad), 200V is run 1 hour or so, or until buffer solution forward position will run out of buffings end.Buffer solution used is 192mM glycine, 0.1% (w/v) SDS, 24.8mM Tris alkali, pH 8.3.The glue of completion fixes at least 30 minutes with 10%MeOH and 7%HAc.Then, glue is dyed at least 3 hours with Sypro Ruby glue dyes (BioRad), and is decolourized at least 30 minutes with 10%MeOH and 7%HAc.Or, glue illustrates to be dyed with Deep Purple (Amersham) according to manufacturer after fixation.Glue is imaged with FX laser photometers (BioRad) and suitable optical filter.The apparent molecular weight (by SDS-PAGE it was observed that) of N- connections oligosaccharides (PNGase processing) TNF-α afterwards is removed 8 to 30kDa.The apparent molecular weight (by SDS-PAGE it was observed that) of N- connections and O- connections oligosaccharides (glucosides ferment treatment) TNF-α afterwards is removed 10 to 20kDa.

(iii) N- terminal sequences are determined

Protein band is cut from glue (from two-dimentional glue or one-dimensional glue) made above, is placed in a 0.5ml pipe, and add 100ml Extraction buffers (100mM sodium acetates, 0.1%SDS, 50mMDTT pH 5.5).Glue is incubated 16 hours in 37 DEG C of shakings.Supernatant is used for ProSorb films (ABI) according to manufacturer's technical specification and is sequenced according to manufacturer's technical specification with 494 protein sequencers of automation (Applied Biosystems).The sequence of acquisition is used for the homogeneity for determining protein.

(iv) peptide mapping fingerprinting

Protein band cuts (from two-way gel or unidirectional gel) and with 25 μ l lavation buffer solutions (in 50mM NH from the gel of above-mentioned preparation₄HCO₃In have 50% acetonitrile) washing.Gel slice retains at least 1 hour and through traditional vacuum drying in 30 minutes at room temperature.Gel slice and 12 μ l insulin solutions (20 μ g insulin, 1200 μ l NH₄HCO₃) be placed in each sample groove and cultivated 1 hour at 4 DEG C.Remove remaining insulin solutions and add 20 μ l 50mM NH₄HCO₃.Mixture is in 37 DEG C of gently shaken overnight cultures.Concentrate peptide sample and the prefabricated micro-column desalination with C18 Zip-Tips (Millipore, Bedford, MA) or containing Poros R2 (Perseptive Biosystems, Framingham, MA) chromatographic resin.Binding peptide is directly eluted in purpose flat board in 0.8 μ l matrix solutions (alpha-cyano -4- hydroxycinnamic acids (Sigma), 8mg/ml in 70% acetonitrile/1% formic acid).The peptide mapping fingerprinting of trypsase determines (MALDI-TOF MS) by using Perseptive Biosystems Voyager DE-STR matrix-assisted laser desorption/desorption ionization flight time mass spectrum and produced.Spectrogram is obtained by using the reflective-mode of 20kV accelerating potentials.Mass calibration is carried out using insulin from dissolved peak, 2211.11Da and 842.51Da as internal standard.The data produced by peptide mapping fingerprinting (PMF) are used to determine protein identity.Search for (main Homo sapien (mankind) and mammal entry) in database progress, such as the SWISS-PROT and TrEMBL, by PeptIdent (www.expasy.ch/tools/peptident.html) program.Identification parameter includes the peptide quality of 0.1Da allowable errors, and each peptide escapes the maximum segment of trypsase cracking, and methionine sulfoxide and cysteine-acrylamide modification.The percent of total for the amino acid sequence that peptide quality numbering and these peptides based on matching are covered is recognized, is compared in the input of other databases.General, the peptide quality with least 30% total sequential covering is for determining that homogeneity is necessary, but very low and high-quality protein, and the protein fragments obtained by these albumen, it is impossible to always meet these standards, it is therefore desirable to further identification.

It is inc or obtained without that can be analyzed by MALDI-TOF PMF in place of protein identity, the peptide mixer of reservation or from replicating identical spot that gel cuts through Trypsin Induced and analyzed by electrospray ionization tandem MS (ESI-MS/MS).For ESI-MS/MS, peptide uses the acetonitriles of 1-2 μ l 70% on Poros R2 micro-columns, and 1% formic acid is directly eluted to borosilicate electron spray syringe needle (Micromass, Manchester, UK).Series connection MS is carried out with Q-Tof mixed types three-level/orthogonal acceleration TOF mass spectrographs (Micromass).Electron spray syringe needle containing sample be embedded in source and with capillary voltage 900-1200V acquisition stationary flow in.Precursor ion scans are carried out to detect the quality of peptide and charge ratios (m/z) in mixture.The m/z of each other precursor ion is picked out for being broken and being collided with the argon gas of 18-30eV collision energies.The fragment ion missing of the amino acid from precursor peptide (correspondence) is recorded and with Mass Lynx Version

3.4 (Micromass) processing.Amino acid sequence is inferred to by using the mass discrepancy of y- or b- ions " gradient " series of Mass Seq (Micromass) program and determined by manual translation.Then, peptide sequence is used to search for NCBI and TrEMBL databases, uses BLASTP programs " short nearly exact matches ".The minimum value of two pairing peptides is necessary for providing given firmly believing for homogeneity.

The homogeneity of gum spot point is proved to be TNF-α.

(b) LT- α of the invention identification

(i) two-dimensional polyacrylamide electrophoresis

Sample being handled according to described by example 3 above (a) (i) that is collected into from example 2 (b) and analyzed.Major protein spots correspondence LT- α isoform in glue.Low-density spot is probably LT- α or low-level pollutants, but because density is low, PMF not can prove that.Observation to glue shows that the LT- α of the present invention have 7 to 33 isoforms.Table 11 and 12 gives the critical nature of these isoforms：PI values (± 1.0), apparent molecular weight (± 20%), and relative intensity (± the 20% of actual number, or total ± 2%, any one is larger).Listed numerical value corresponds to, the Density Weighted center in the glue region containing spot of selection, therefore, and it only reacts the albumen pI and molecular weight of certain reading in the selected areas of every piece of glue.In view of the intrinsic variability and protein positions of 2D glue sizes, based on the numerical value listed by table 11 and 12, it is 5-11 or so to determine the scope of molecule p I values；Based on the numerical value listed by table 11 and 12, the apparent molecular weight scope for determining molecule is 15-32kDa.

Table 11

The molecular weight and pI values of LT- α isoforms

Spot is numbered	Isoelectric point   (pI)	Molecular weight   (kDa)	Relative intensity (%) (standardized value)
				2	6.39	25.97	0.26
3	6.65	25.63	1.38
				4	6.86	25.16	3.02
5	7.10	24.44	9.34
				6	7.36	23.71	13.38
7	7.72	23.32	14.69
				8	8.25	22.64	16.65
9	9.13	22.14	13.41
				10	8.96	19.08	3.64
11	9.43	21.36	0.32
				12	9.78	22.04	1.16
13	9.99	22.52	3.91
				14	9.98	20.78	1.09
15	7.17	18.35	0.43
				16	7.26	18.20	0.62
17	7.49	18.13	2.23
				18	7.61	18.08	2.79
19	7.65	16.40	1.01
				20	7.90	18.00	1.92
21	8.02	18.46	3.15
				22	8.15	17.79	1.75
23	8.36	16.39	0.71
				24	6.40	15.77	0.15
25	6.82	15.46	0.32
				26	7.17	15.26	0.73
27	7.61	14.02	0.19
				28	8.29	14.44	0.22
29	8.78	15.10	0.29
				30	8.95	15.98	0.11
31	8.33	12.14	0.08
				32	8.46	12.05	0.18
33	8.88	12.24	0.26
				34	7.53	10.36	0.63

Table 12

The molecular weight and pI values of LT- α isoforms

Spot is numbered	Isoelectric point   (pI)	Molecular weight   (kDa)	Relative intensity (%) (standardized value)
				2	6.34	23.35	1.81
3	6.52	23.06	4.13
				4	6.71	22.65	7.95
5	6.92	22.40	11.51
				6	7.21	22.98	8.84
7	7.23	20.84	9.05
				8	7.58	22.99	6.59
9	7.59	21.07	7.74
				10	7.61	19.53	3.90
11	8.12	22.61	6.73
				12	8.12	21.06	6.13
13	8.09	19.71	5.73
				14	8.97	21.95	4.57
15	8.95	20.08	6.61
				16	9.94	20.82	3.12
17	7.03	20.60	4.01
				18	7.229	19.56	1.58

(ii) one-dimensional polyacrylamide gel electrophoresis

Progress of the sample being collected into from example 2 (b) according to described by example 3 above (a) (ii) is handled.N- connections oligosaccharides (by PNGase processing) LT- α afterwards apparent molecular weight (by SDS-PAGE it was observed that) is removed between 12 and 25kDa.N- connections and O- connections oligosaccharides (by glucosides ferment treatment) LT- α afterwards apparent molecular weight (by SDS-PAGE it was observed that) are removed between 12 and 23kDa.

(iii) the N- terminal sequences of albumen are determined

The LT- α of present invention N- terminal sequences determine being accomplished according to described by embodiment 3 (a) (iii).

(iv) peptide quality fingerprinting

The LT- α of present invention peptide quality fingerprinting being accomplished according to described by embodiment 3 (a) (iv).

The homogeneity of gum spot point is proved to be LT- α.

(c) TNFRI-Fc of the invention identification

(i) two-dimensional polyacrylamide electrophoresis

Sample being handled according to described by example 3 above (a) (i) that is collected into from example 2 (c) and analyzed.Major protein spots correspondence TNFRI-Fc isoform in glue.Low-density spot is probably TNFRI-Fc or low-level pollutants, but because density is low, PMF not can prove that.Observation to glue shows that the TNFRI-Fc of the present invention has 8 to 16 isoforms.Table 13 and 14 gives the critical nature of these isoforms：PI values (± 1.0), apparent molecular weight (± 20%), and relative intensity (± the 20% of actual number, or total ± 2%, any one is larger).Listed numerical value corresponds to, the Density Weighted center in the glue region containing spot of selection, therefore, and it only reacts the albumen pI and molecular weight of certain reading in the selected areas of every piece of glue.In view of the intrinsic variability and protein positions of 2D glue sizes, the numerical value listed by table 13 and 14 is determined, it is 5.5-9.5 or so to measure the scope of molecule pI values；Based on the numerical value listed by table 13 and 14, the apparent molecular weight scope for determining molecule is 45-75kDa.

Table 13：

The molecular weight and pI values of TNFRI-Fc isoforms

Spot is numbered	Isoelectric point   (pI)	Molecular weight   (kDa)	Relative intensity (%) (standardized value)
				9	7.20	46.09	7.34
10	7.39	45.86	6.37
				11	7.57	45.67	9.59
12	7.78	45.21	9.31
				13	8.09	44.88	10.58
14	8.41	44.33	8.96
				15	8.89	43.98	13.90
16	9.19	44.53	4.44
				17	9.46	44.93	4.75
18	9.77	45.58	4.11

Table 14：

The molecular weight and pI values of TNFRI-Fc isoforms

Spot is numbered	Isoelectric point   (pI)	Molecular weight   (kDa)	Relative intensity (%) (standardized value)
				2	6.31	57.87	0.860
3	6.40	57.04	2.170
				4	6.52	55.85	6.105
5	6.63	55.63	8.895
				6	6.78	54.51	8.934
7	6.89	54.15	7.654
				8	7.01	53.74	9.788
9	7.13	53.87	5.845
				10	7.22	53.79	4.220
11	7.30	53.69	6.510
				12	7.42	53.81	4.953
13	7.55	53.24	2.485
				14	7.65	53.43	1.360
15	7.72	53.53	0.753
				16	8.04	52.94	1.934
17	8.45	52.59	1.095

(ii) one-dimensional polyacrylamide gel electrophoresis

Progress of the sample being collected into from embodiment 2 (c) according to described by example 3 above (a) (ii) is handled.N- connections oligosaccharides (by PNGase processing) TNFRI-Fc afterwards apparent molecular weight (by SDS-PAGE it was observed that) is removed between 36 and 60kDa.N- connections and O- connections oligosaccharides (by glucosides ferment treatment) TNFRI-Fc afterwards apparent molecular weight (by SDS-PAGE it was observed that) are removed between 36 and 60kDa.

(iii) the N- terminal sequences of albumen are determined

The TNFRI-Fc of present invention N- terminal sequences determine being accomplished according to described by embodiment 3 (a) (iii).

(iv) peptide quality fingerprinting

The TNFRI-Fc of present invention peptide quality fingerprinting being accomplished according to described by embodiment 3 (a) (iv).

The homogeneity of gum spot point is proved to be TNFRI-Fc.

In addition, it was observed that the molecular weight of tryptic peptide has 1Da movement, this shows that the asparagine residue (N) of 1NX (S/T/C) motif in people TNFRI-Fc amino acid sequences in theory is modified to asparatate (D), and this meets PNGase F induction N to D residues after related N- connection oligosaccharides is removed and modifies property.Therefore, the N- glycosylation sites that one of TNFRI-Fc of the invention is proven are N-299 (from the section start open numbering of signal peptide)

(d) TNFRII-Fc of the invention identification

(i) two-dimensional polyacrylamide electrophoresis

From embodiment 2 (d) or 2 (h) sample being handled according to described by example 3 above (a) (i) being collected into and analyzed.

Major protein spots correspondence TNFRII-Fc isoform in glue.Low-density spot is probably TNFRI I-Fc or low-level pollutants, but because density is low, PMF not can prove that.Observation to glue shows that the TNFRII-Fc of the present invention has 10 to 40 isoforms.Table 15 and 15 (a) gives the critical nature of these isoforms：PI values (± 1.0), apparent molecular weight (± 20%), and relative intensity (± the 20% of actual number, or total ± 2%, any one is larger).Listed numerical value corresponds to, the Density Weighted center in the glue region containing spot of selection, therefore, and it only reacts the albumen pI and molecular weight of certain reading in the selected areas of every piece of glue.In view of the intrinsic variability and protein positions of 2D glue sizes, based on the numerical value listed by table 15 and 15 (a), it is 4-10 or so to determine the scope of molecule pI values；The numerical value listed by table 15 and 15 (a) is determined, the apparent molecular weight scope for measuring molecule is 46-118kDa.

Table 15：The molecular weight and pI values of TNFRII-Fc isoforms

Spot is numbered	Isoelectric point   (pI)	Molecular weight   (kDa)	Relative intensity (%) (standardized value)
				2	5.52	64.99	1.08
3	5.60	64.96	1.41
				4	5.70	64.62	1.82
5	5.82	64.74	2.92
				6	5.97	64.48	6.27
7	6.20	63.99	3.83
				8	6.36	63.76	4.70
9	6.51	63.60	4.33
				10	6.64	63.15	7.21
11	6.78	63.13	7.63
				12	6.93	62.87	4.11
13	7.09	62.39	7.95
				14	7.27	62.51	11.71
15	7.43	62.12	13.99
				16	7.52	62.30	3.79
17	7.65	61.45	5.82
				18	7.87	60.04	7.79
19	8.07	57.88	3.63

Table 15 (a)：

TNFRII-Fc (lot numbers：003) molecular weight of isoform and pI values

Spot is numbered	Isoelectric point   (pI)	Molecular weight   (kDa)	Relative intensity (%) (standardized value)
				2	5.29	84.18	0.96
3	5.36	83.43	1.28
				4	5.44	82.60	1.47
5	5.51	82.04	2.32
				6	5.59	81.27	2.52
7	5.66	81.05	2.61
				8	5.73	80.59	2.55
9	5.80	80.08	3.09

Spot is numbered	Isoelectric point   (pI)	Molecular weight   (kDa)	Relative intensity (%) (standardized value)
				10	5.87	79.88	2.97
11	5.96	79.40	4.19
				12	6.05	79.16	4.44
13	6.15	78.67	4.25
				14	6.26	77.66	5.05
15	6.37	76.38	5.07
				16	6.49	74.99	4.84
17	6.63	73.16	5.05
				18	6.75	72.24	3.62
19	6.87	71.37	3.80
				20	7.04	69.67	2.92
21	7.15	67.94	2.42
				22	7.27	65.86	2.91
23	7.44	64.81	2.79
				24	7.59	64.07	3.05
25	7.72	62.79	2.36
				26	7.87	61.84	1.26
27	7.95	61.44	1.61
				28	8.03	60.92	1.41
29	8.13	60.26	1.11
				30	8.34	58.18	2.28
31	8.44	58.27	1.02
				32	8.51	57.25	1.19
33	8.59	56.74	0.90
				34	8.77	56.18	1.40
35	8.93	56.46	2.34
				36	9.11	56.49	1.52
37	9.33	56.31	0.99
				38	9.45	56.16	1.14

(ii) one-dimensional polyacrylamide gel electrophoresis

From embodiment 2 (h) (ii) (b) (lot number：003) progress of the sample being collected into according to described by example 3 above (a) (ii) is handled.N- connections oligosaccharides (by PNGase processing) TNFRII-Fc afterwards apparent molecular weight (by SDS-PAGE it was observed that) is removed between 46 and 87kDa.N- connections and O- connections oligosaccharides (by glucosides ferment treatment) TNFRII-Fc afterwards apparent molecular weight (by SDS-PAGE it was observed that) are removed between 42 and 80kDa

(iii) the N- terminal sequences of albumen are determined

The TNFRII-Fc of present invention N- terminal sequences determine being accomplished according to described by example 3 above (a) (iii).The sequence (PAQVAFTPYA) of generation is used for the TNFRII-Fc proved homogeneity.

(iv) peptide quality fingerprinting

The TNFRII-Fc of present invention peptide quality fingerprinting being accomplished according to described by example 3 above (a) (iv).

The homogeneity of gum spot point is proved to be TNFRII-Fc.

In addition, it was observed that the molecular weight of tryptic peptide has 1Da movement, this shows that the asparagine residue (N) of one or more NX (S/T/C) motifs in people TNFRII-Fc amino acid sequences in theory is modified to asparatate (D), so as to demonstrate one or more N- glycosylation sites in TNFRII-Fc of the invention.

(e) OX40-Fc of the invention identification

(i) two-dimensional polyacrylamide electrophoresis

Sample being handled according to described by example 3 above (a) (i) that is collected into from embodiment 2 (e) and analyzed.

Major protein spots correspondence OX40-Fc isoform in glue.Low-density spot is probably OX40-Fc or low-level pollutants, but because density is low, PMF not can prove that.Observation to glue shows that the OX40-Fc of the present invention has 8 to 16 isoforms.Table 16 gives the critical nature of these isoforms：PI values (± 1.0), apparent molecular weight (± 20%), and relative intensity (± the 20% of actual number, or total ± 2%, any one is larger).Listed numerical value corresponds to, the Density Weighted center in the glue region containing spot of selection, therefore, and it only reacts the albumen pI and molecular weight of certain reading in the selected areas of every piece of glue.In view of the intrinsic variability and protein positions of 2D glue sizes, based on the numerical value listed by table 16, it is 4-9 or so to determine the scope of molecule pI values；Based on the numerical value listed by table 16, the apparent molecular weight scope for determining molecule is 46-75kDa.

Table 16

The molecular weight and pI values of OX40-Fc isoforms

Spot is numbered	Isoelectric point   (pI)	Molecular weight   (kDa)	Relative intensity (%) (standardized value)
				2	5.53	59.01	0.43
3	5.64	58.56	0.80
				4	5.75	58.27	1.50
5	5.88	58.23	2.31
				6	6.00	58.07	2.92
7	6.14	57.78	4.26
				8	6.29	57.38	4.89
9	6.43	57.12	5.42
				10	6.59	57.09	3.42
11	6.75	57.06	7.71
				12	6.90	57.06	4.78
13	7.06	57.16	7.31
				14	7.23	57.08	0.63
15	7.43	57.08	6.19
				16	7.63	56.92	9.80
17	7.86	56.79	7.66

(ii) one-dimensional polyacrylamide gel electrophoresis

Progress of the sample being collected into from embodiment 2 (e) according to described by example 3 above (a) (ii) is handled.N- connections oligosaccharides (by PNGase processing) OX40-Fc afterwards apparent molecular weight (by SDS-PAGE it was observed that) is removed between 44 and 72kDa.N- connections oligosaccharides (by PNGase processing) and O- connections oligosaccharides (by cocktail glucosides ferment treatment) OX40-Fc afterwards apparent molecular weight (by SDS-PAGE it was observed that) are removed between 41 and 70kDa.

(iii) N- terminal sequences are determined

The OX40-Fc of present invention N- terminal sequences determine being accomplished according to described by embodiment 3 (a) (iii).

(iv) peptide quality fingerprinting

The OX40-Fc of present invention peptide quality fingerprinting being accomplished according to described by example 3 above (a) (iv).

The homogeneity of gum spot point is proved to be OX40-Fc.

It was furthermore observed that the molecular weight of tryptic peptide has 1Da movement, this shows that the asparagine residue (N) of 2NX (S/T/C) motif in people OX40-Fc amino acid sequences in theory is modified to asparatate (D).Therefore, the N- glycosylation sites that OX40-Fc of the invention is proven include N-160 and N-298 (from the section start open numbering of signal peptide).

(f) BAFF of the invention identification

(i) two-dimensional polyacrylamide electrophoresis

Sample being handled according to described by example 3 above (a) (i) that is collected into from embodiment 2 (f) and analyzed.

Major protein spots correspondence BAFF isoform in glue.Low-density spot is probably BAFF or low-level pollutants, but because density is low, PMF not can prove that.Observation to glue shows that the BAFF of the present invention has 5 to 10 isoforms.Table 17 gives the critical nature of these isoforms：PI values (± 1.0), apparent molecular weight (± 20%), and relative intensity (± the 20% of actual number, or total ± 2%, any one is larger).Listed numerical value corresponds to, the Density Weighted center in the glue region containing spot of selection, therefore, and it only reacts the albumen p I and molecular weight of certain reading in the selected areas of every piece of glue.In view of the intrinsic variability and protein positions of 2D glue sizes, based on the numerical value listed by table 17, it is 4-8 or so to determine the scope of molecule pI values；Based on the numerical value listed by table 17, the apparent molecular weight scope for determining molecule is 10-22kDa.

Table 17

The molecular weight and pI values of BAFF isoforms

Spot is numbered	Isoelectric point   (pI)	Molecular weight   (kDa)	Relative intensity (%) (standardized value)
				2	4.91	17.07	3.34
3	5.01	16.97	9.32
				4	5.14	16.78	18.93
5	5.31	16.83	33.69
				6	5.48	16.85	8.76
7	4.99	14.95	4.50
				8	5.20	14.81	5.48
9	5.33	14.76	12.79
				10	5.47	14.86	3.19

(ii) one-dimensional polyacrylamide gel electrophoresis

Progress of the sample being collected into from embodiment 2 (f) according to described by example 3 above (a) (ii) is handled.N- connections oligosaccharides (by PNGase processing) BAFF afterwards apparent molecular weight (by SDS-PAGE it was observed that) is removed between 8 and 22kDa.N- connections oligosaccharides (by PNGase processing) and O- connections oligosaccharides (by cocktail glucosides ferment treatment) BAFF afterwards apparent molecular weight (by SDS-PAGE it was observed that) are removed in 8-22kDa.

(iii) N- terminal sequences are determined

The BAFF of present invention N- terminal sequences determine being accomplished according to described by example 3 above (a) (iii).

(iv) peptide quality fingerprinting

The BAFF of present invention peptide quality fingerprinting being accomplished according to described by embodiment 3 (a) (iv).

The homogeneity of gum spot point is proved to be BAFF

(g) NGFR-Fc of the invention identification

(i) two-dimensional polyacrylamide electrophoresis

Sample being handled according to described by example 3 above (a) (i) that is collected into from embodiment 2 (f) and analyzed.

Major protein spots correspondence NGFR-Fc isoform in glue.Low-density spot is probably NGFR-Fc or low-level pollutants, but because density is low, PMF not can prove that.Observation to glue shows that the NGFR-Fc of the present invention has 8 to 16 isoforms.Table 18 gives the critical nature of these isoforms：PI values (± 1.0), apparent molecular weight (± 20%), and relative intensity (± the 20% of actual number, or total ± 2%, any one is larger).Listed numerical value corresponds to, the Density Weighted center in the glue region containing spot of selection, therefore, and it only reacts the albumen pI and molecular weight of certain reading in the selected areas of every piece of glue.In view of the intrinsic variability and protein positions of 2D glue sizes, based on the numerical value listed by table 18, it is 3-6 or so to determine the scope of molecule pI values；Based on the numerical value listed by table 18, the apparent molecular weight scope for determining molecule is 55-105kDa.

Table 18

NGFR-Fc molecular weight and pI values

Spot is numbered	Isoelectric point   (pI)	Molecular weight   (kDa)	Relative intensity (%) (standardized value)
				2	4.23	85.48	6.50
3	4.36	85.33	6.92
				4	4.44	84.08	10.05
5	4.53	83.07	16.45
				6	4.62	82.77	16.20
7	4.70	82.71	11.33
				8	4.78	82.84	7.71
9	4.84	82.97	4.68
				10	4.90	83.31	3.97
11	4.98	82.91	3.77
				12	5.06	81.42	4.90
13	5.19	80.43	2.34
				14	5.27	80.59	1.47
15	5.19	74.02	1.36
				16	5.26	74.18	1.12
17	5.31	73.81	1.24

(ii) one-dimensional polyacrylamide gel electrophoresis

Progress of the sample being collected into from embodiment 2 (g) according to described by example 3 above (a) (ii) is handled.

NGFR-Fc apparent molecular weight (by SDS-PAGE it was observed that) is between 55 and 105kDa.The apparent molecular weight (by SDS-PAGE it was observed that) for removing NGFR-Fc after N- connection oligosaccharides is handled by PNGase between 48 and 90kDa.N- connections oligosaccharides (by PNGase processing) and O- connections oligosaccharides (by cocktail glucosides ferment treatment) NGFR-Fc afterwards apparent molecular weight (by SDS-PAGE it was observed that) are removed between 48 and 85kDa.

(iii) N- terminal sequences are determined

The NGFR-Fc of present invention N- terminal sequences determine being accomplished according to described by embodiment 3 (a) (iii).

(iv) peptide quality fingerprinting

The NGFR-Fc of present invention peptide quality fingerprinting being accomplished according to described by embodiment 3 (a) (iv).

The homogeneity of gum spot point is proved to be NGFR-Fc.

(h) identification of FasL of the invention

(i) two-dimensional polyacrylamide electrophoresis

Sample being handled according to described by example 3 above (a) (i) that is collected into from embodiment 2 (h) and analyzed.

(ii) one-dimensional polyacrylamide gel electrophoresis

Progress of the sample being collected into from embodiment 2 (h) according to described by example 3 above (a) (ii) is handled.The apparent molecular weight (by SDS-PAGE it was observed that) of FasL is between 15-35Da.The apparent molecular weight (by SDS-PAGE it was observed that) of N- connections oligosaccharides (by PNGase processing) FasL afterwards is removed between 12 and 21kDa.

(iii) N- terminal sequences are determined

The N- terminal sequences of the FasL of the present invention determine being accomplished according to described by embodiment 3 (a) (iii).

(iv) peptide quality fingerprinting

Peptide quality fingerprinting being accomplished according to described by embodiment 3 (a) (iv) of the FasL of the present invention.

The homogeneity of gum spot point is proved to be FasL.

It was observed that the molecular weight of tryptic peptide has 1Da movement, this shows that the asparagine residue (N) of 1NX (S/T/C) motif in people's FasL amino acid sequence in theory is modified to asparatate (D).Therefore, the N- glycosylation sites that one of FasL of the invention is proven include N-184 (from the section start open numbering of signal peptide).

Embodiment 4

(a) amino acid, monose, oligosaccharides, phosphate, sulfate and the isoform composition of the TNF-α of the present invention are analyzed.

(i) preparing sample is used for amino acid, monose, oligosaccharides, phosphate, sulfate and isoform analysis

In order to identify the glycosylation and phosphate of monose and oligosaccharides and the posttranslational modification of sulfate, sugar is removed from polypeptide backbone by the method for hydrolysis or enzyme first.Sample buffer component is also removed and changes water into should to avoid occurring before analysis starts hydrolysis and enzyme hair.The PBS solution of purifying TNF-α is dialysed with 4 liters of deionized ultrafiltration water (18MOhm), is continued 4 days, is changed daily 2 times, and the regenerated cellulose dialysis membrane (Spectrapore) that molecular weight (NMWC) is 5KDa is blocked with specified.After dialysis, solution is dried with Savant Speed Vac (New York, the U.S.).Dried sample is resuspended with 2ml deionization ultrafiltration water (18MOhm), is dispensed for different analyses.

(ii) analyzed by the amino acid composition of vapor phase hydrolysis method

Spread out with 6- aminoquinolines base-n-hydroxysuccinimide aminocarbamic acid ester (AQC) pre-column of physics and chemistry of amino acid in sample is analyzed.Separated with reverse (C18) HPLC and quantify stable Fluorescent amino acid derivative.Step used is to be based on Waters AccQTag amino acid analysis methods.

Take 3 part of 100 μ l TNF-α separation sample and dried in Speed Vac.Then, dry sample is hydrolyzed 24 hours at 110 DEG C.After hydrolysis, sample is dried again before the following physics and chemistry that spreads out is carried out.Dry sample is re-dissolved in 10 μ L vivo acid standard liquid (butyrine, AABA), is added 35 μ L borate buffer solutions, is added 15 μ L AQC derivative reagents.Reactant mixture is heated 12 minutes in a heat block at 50 DEG C.Derivative Freamine Ⅲ is transferred in the automatic sampler of HPLC system, HPLC system is entered long trap detector and sequentially constituted by the splitters of Wa ter sAlliance 2695, the fluorescence detectors of Waters 474 and the double waves of Waters 2487.Control and analysis software are Waters Empower ProModule (Waters Corporation, Milford.MA, USA).Sample is used chromatographic parameter known in the art (such as suitable eluent and gradient current) by WatersAccQTag posts (15cm × 3.9mm ID).

(iii) neutral and amino monosaccharide composition analysis

Take 2 part of 100 μ l TNF-α to prepare sample, handled to discharge monose with 2 kinds of methods.Every kind of processing, it is as follows, in triplicate.

1. 100 DEG C are heated to 4M trifluroacetic acid (TFA) to hydrolyze 4 hours to discharge neutral sugar (galactolipin, glucose, trehalose and mannose).

2. 100 DEG C are heated to 2M HCl to hydrolyze 4 hours to discharge alkaline sugared (N- acetyl-amine-galactose, N- acetyl-Glucosamine galactolipin).

All hydrolysates are lyophilized with Speed Vac system low-voltages, are re-dissolved in containing in the μ l water of target in 0.8nmol 200.For neutral and alkaline monose, internal standard is 1,5-anhydroglucitol.Then sample centrifuges 30 minutes to remove keratin debris in 10,000g.Supernatant is transferred in a new pipe, and analyzed with the high pH anion-exchange chromatographies of Dionex LC50 systems, it is made up of a GP50 pump and an ED50 pulse currents detector (Dionex Ltd).It is neutral sugared with alkalescence with the analysis of DionexCarboPac PA-20 posts.Eluted 20 minutes with 10mM hydroxyl concentration.This is completed with Dionex EG50 eluents generation system.

(iv) acid monosaccharide composition analysis

Take 100 μ l TNF-α to prepare sample, and handle to discharge sialic acid monosaccharide using the following method.In triplicate.

Sample hydrolyzes 40 minutes to discharge N- acetyl group and N- glycolyl neuraminic acids with 0.1M TFA at 80 DEG C.Hydrolysate Speed Vac lyophilizeds, are re-dissolved in containing in the μ l water of target in 0.8nmol 200.For the analysis of sialic acid, internal standard is lactobionic acid.Then sample centrifuges 30 minutes to remove keratin debris in 10,000g.Supernatant is transferred in a new pipe, and analyzed with the high pH anion-exchange chromatographies of Dionex LC50 systems, it is made up of a GP50 pump and an ED50 pulse current detector.Saliva acid analysis is carried out with DionexCarboPac PA1, is used chromatographic parameter known in the art (such as suitable eluent and gradient current).

(v) analysis of oligosaccharides composition

For analysis oligosaccharides composition, take 2 part of 300 μ l TNF-α to prepare sample, handled by a kind of following method, in triplicate：

1. the release of N- connection oligosaccharides is completed with peptide-N4- (N- acetyl group-β-D-Glucose amido) asparagine deamidase (PNGase).First, the denaturing soln (2%SDS (Sigma)/1M beta -mercaptoethanols (Sigma)) of 1/5th volumes is added in sample.Sample is heated to 100 DEG C 5 minutes.1/10^thThe 15%Triton-X100 (Sigma) of volume is added in sample.Sample is gently mixed and allows to cool to room temperature.Add the PNGase (Sigma) of 25 units and in 37 DEG C of cultures.

2. the release of O- connection oligosaccharides is completed by the method for β-elimination.4M Sodium Borohydrides (brand-new) (Sigma) solution of 1/2 volume is added in sample.The 0.4M NaOH (BDH, HPLC grade) of 1/2 volume are added in sample.Sample is cultivated 16 hours at 50 DEG C.Sample cools down in ice and the 0.4M acetic acid (Sigma) of 1/2 volume is added in sample.

N- connections and O- connections sample are all further handled to remove buffer components with Carbo Pac graphitised carbon SPE posts.Column equilibration and elution requirement are as follows：

First, pillar is balanced with the water of 80% acetonitrile (Sigma) followed by 2 times column volumes of 1 times of column volume.Loading under gravity, pillar is rinsed with the water of 2 times of volumes.2ml 50% acetonitrile is added in post to elute neutral oligosaccharides.The 2ml formic acid of 50% acetonitrile/0.1% is added in post to elute acidic oligosaccharide.The oligosaccharides of any residual is all eluted after the 2ml formic acid of 80% acetonitrile/0.1% is added.

Dried by the fraction containing neutral or amino N-connection oligosaccharides and neutral or amino O- connection oligosaccharides of SPE post separations with Speed Vac.Sample is re-dissolved in 200 μ l water, and by high pH anion exchange chromatographies, using the systems of Dionex LC 20 and GP50 pumps and ED50 pulse current detectors together.The analysis of neutral and amino-oligosacchride is carried out with CarboPac PA100 posts and chromatographic parameter known in the art (such as suitable eluent and gradient current).

(vi) analysis of sulfate and phosphate composition

Sulfate/Phosphate analysis is basically by by Harrison and Packer (Harrison and Packer Methods Mol Biol 125：The method of 211-216,2000) description is carried out.

Taking the sample of 100 μ l TNF-α products is used for sulfate/Phosphate analysis, and is hydrolyzed 4 hours at 100 DEG C in 4M HCl.Pass through the drying sample removing HCl in Speed Vac.Then, sample is re-dissolved in 200 μ l H₂O.In the systems of Dionex LC 50 of the 24 μ l samples injection with GP50 pumps and ED50 pulse current detectors.Separated, used chromatographic parameter known in the art (such as suitable eluent and gradient current) by Dionex IonPacIS11 anion-exchange columns.

(vii) the further separation of TNF-α isoform

The further separation of TNF-α isoform is carried out with pellicular anion exchange column.Suitable sample volume, for example, 24 μ l, are separated by ProPac SAX-10 posts (Dionex Ltd), use the Dionex SUMMIT systems with UV-Vis detectors (Dionex Ltd).Separation is carried out with suitable eluent and gradient known in the art.TNF-α isoform finds to be eluted in the peak of different shaped.

(b) the LT- α of present invention amino acid, monose, oligosaccharides, phosphate, sulfate and isoform composition is analyzed

(i) preparing sample is used for amino acid, monose, oligosaccharides, phosphate, sulfate and isoform analysis

Progress of the purifying LT- α PBS solution as described by example 4 above (a) (i) is handled.

(ii) constituted with vapor phase hydrolysis method (Gas Phase Hydrolysis Method) analysis of amino acid

Progress of the LT- α samples of preparation as described by example 4 above (a) (ii) is handled.

(iii) composition of neutral and alkaline monose is analyzed

Progress of the LT- α samples of preparation as described by example 4 above (a) (iii) is handled.

(iv) composition of acid monose is analyzed

Progress of the LT- α samples of preparation as described by example 4 above (a) (iv) is handled.

(v) composition of oligosaccharides is analyzed

Progress of the LT- α samples of preparation as described by example 4 above (a) (v) is handled.

(vi) analysis sulfate and phosphatic composition

Progress of the LT- α samples of preparation as described by example 4 above (a) (vi) is handled.

(vii) further protein isolate isoform

Progress of the LT- α samples of preparation as described by example 4 above (a) (vii) is handled.It was found that LT- α isoforms are eluted in the form of a kind of particular peaks.

(c) TNFRI-Fc of present invention amino acid, monose, oligosaccharides, phosphate, sulfate and isoform composition is analyzed

(i) preparing sample is used for amino acid, monose, oligosaccharides, phosphate, sulfate and isoform analysis

Progress of the TNFRI-Fc PBS solution as described by example 4 above (a) (i) is purified to handle.

(ii) constituted with vapor phase hydrolysis method (Gas Phase Hydrolysis Method) analysis of amino acid

Progress of the TNFRI-Fc samples of preparation as described by example 4 above (a) (ii) is handled.

(iii) composition of neutral and alkaline monose is analyzed

Progress of the TNFRI-Fc samples of preparation as described by example 4 above (a) (iii) is handled.

(iv) composition of acid monose is analyzed

Progress of the TNFRI-Fc samples of preparation as described by example 4 above (a) (iv) is handled.

(v) composition of oligosaccharides is analyzed

Progress of the TNFRI-Fc samples of preparation as described by example 4 above (a) (v) is handled.

(vi) analysis sulfate and phosphatic composition

Progress of the TNFRI-Fc samples of preparation as described by example 4 above (a) (vi) is handled.

(vii) further protein isolate isoform

Progress of the TNFRI-Fc samples of preparation as described by example 4 above (a) (vii) is handled.It was found that TNFRI-Fc isoforms are eluted in the form of a kind of particular peaks.

(viii) result

Amino acid is constituted

TNFRI-Fc is hydrolyzed, derives and is analyzed with described RPLC, following amino acid composition (table 19) is obtained.As a result it is expressed as the percentage of amino acid occurrence number in given sequence

Table 19

Monose and sulfate

Single monose and sulfate are hydrolyzed from TNFRI-Fc amino acid backbone and obtained, and are analyzed with described high pH anion-exchange chromatographies (HP AEC), obtain following components analysis.Standard (table 20-22) is used as from sample from obtained result with GalNAc and 3 times of mannose respectively.Table 23 is the summary to the result from 3 samples.

Table 20

Table 21

Table 22

Table 23

In view of in chromatographic process described above in variability, during with GalNAc as standard, it is determined that the trehalose that the TNFRI-Fc of the present invention monose, sialic acid and sulphates content are about 1: 1-4.5,1: 10-18 GlcNAc, 1: 3-9 galactolipin, 1: 4-11 mannose, 1: 0.1-2 NeuNAc and 1: 1.5-14 sulfate；During with 3 times of mannoses as standard, it is defined as 3: 0.1-1.5 trehalose, 3: 0.1-1 Gal NAc, 3: 3-11 GlcNAc, 3: 1-2.5 galactolipin, 3: 0-2 NeuNAc and 3: 0.5-4 sulfate.

Amino acid composition data combination monose and sulfate data, give different types of content (table 24).In view of in chromatographic process described above in variability, it is determined that the TNFRI-Fc of the present invention acid monose percentage ranges are about 0-10%, sulfate represents the TNFRI-Fc of present invention monose percentage, its scope about 10-16% is determined, it is determined that the acid percentage ranges of the TNFRI-Fc of present invention N- connection oligosaccharides are about the acid percentage ranges about 43-66% of 3-6% and the TNFRI-Fc of present invention O- connection oligosaccharides.

Table 24

(d) TNFRII-Fc of present invention amino acid, monose, oligosaccharides, phosphate, sulfate and isoform composition is analyzed

(i) preparing sample is used for amino acid, monose, oligosaccharides, phosphate, sulfate and isoform analysis

Progress of the TNFRII-Fc PBS solution as described by example 4 above (a) (i) is purified to handle.

(ii) constituted with vapor phase hydrolysis method (Gas Phase Hydrolysis Method) analysis of amino acid

Progress of the TNFRII-Fc samples of preparation as described by example 4 above (a) (ii) is handled.

(iii) composition of neutral and alkaline monose is analyzed

Progress of the TNFRII-Fc samples of preparation as described by example 4 above (a) (iii) is handled.

(iv) composition of acid monose is analyzed

Progress of the TNFRII-Fc samples of preparation as described by example 4 above (a) (iv) is handled.

(v) composition of oligosaccharides is analyzed

Progress of the TNFRII-Fc samples of preparation as described by example 4 above (a) (v) is handled.

(vi) analysis sulfate and phosphatic composition

Progress of the TNFRII-Fc samples of preparation as described by example 4 above (a) (vi) is handled.

(vii) further protein isolate isoform

Progress of the TNFRII-Fc samples of preparation as described by example 4 above (a) (vii) is handled.It was found that TNFRII-Fc isoforms are eluted in the form of a kind of particular peaks.

(viii) result

Amino acid is constituted

The TNFRII-Fc of preparation is hydrolyzed, derives and is analyzed with described RPLC, following amino acid composition (table 25) is obtained.As a result it is expressed as the percentage that weight amount and each amino acid occur in sequence (including SD).

Table 25

Monose and sulfate

Single monose and sulfate are hydrolyzed from TNFRII-Fc amino acid backbone and obtained, and are analyzed with described high pH anion-exchange chromatographies (HP AEC), obtain following components analysis.As a result it is used as standard (table 26-28) with GalNAc and 3 times of mannose respectively.Table 29 is the summary to the result from 3 samples.

Table 26

Table 27

Table 28

Table 29

In view of in chromatographic process described above in variability, during with Gal NAc as standard, it is determined that the trehalose that the TNFRII-Fc of the present invention monose, sialic acid and sulphates content are about 1: 0.01-2,1: 0.1-3 GlcNAc, 1: 0.1-2 galactolipin, 1: 0.1-2 mannose, 1: 0.01-2 NeuNAc and 1: 1-4 sulfate；During with 3 times of mannoses as standard, it is defined as 3: 0.1-2 trehalose, 3: 3-11 GalNAc, 3: 5-21 GlcNAc, 3: 3-6 galactolipin, 3: 0.1-2 NeuNAc and 3: 9-19 sulfate.

Amino acid composition data combination monose and phosphate and sulfate data, give different types of content (table 30), are expressed as percetage by weight.In view of in chromatographic process described above in variability, it is determined that the TNFRII-Fc of the present invention acid monose percentage ranges are about 1-10%, sulfate is expressed as the TNFRII-Fc of present invention monose percentage, its scope about 27-41% is determined, it is determined that the acid percentage ranges of the TNFRII-Fc of present invention N- connection oligosaccharides are about the acid percentage ranges about 51-78% of 16-26% and the TNFRII-Fc of present invention O- connection oligosaccharides.

Table 30

(e) OX40-Fc of present invention amino acid, monose, oligosaccharides, phosphate, sulfate and isoform composition is analyzed

(i) preparing sample is used for amino acid, monose, oligosaccharides, phosphate, sulfate and isoform analysis

Progress of the OX40-Fc PBS solution as described by example 4 above (a) (i) is purified to handle.

(ii) constituted with vapor phase hydrolysis method (Gas Phase Hydrolysis Method) analysis of amino acid

Progress of the OX40-Fc samples of preparation as described by example 4 above (a) (ii) is handled.

(iii) composition of neutral and alkaline monose is analyzed

Progress of the OX40-Fc samples of preparation as described by example 4 above (a) (iii) is handled.

(iv) composition of acid monose is analyzed

Progress of the OX40-Fc samples of preparation as described by example 4 above (a) (iv) is handled.

(v) composition of oligosaccharides is analyzed

Progress of the OX40-Fc samples of preparation as described by example 4 above (a) (v) is handled.

(vi) analysis sulfate and phosphatic composition

Progress of the OX40-Fc samples of preparation as described by example 4 above (a) (vi) is handled.

(vii) further protein isolate isoform

Progress of the OX40-Fc samples of preparation as described by example 4 above (a) (vii) is handled.It was found that OX40-Fc isoforms are eluted in the form of a kind of particular peaks.

(viii) result

Amino acid is constituted

The OX40-Fc of preparation is hydrolyzed, derives and is analyzed with described RPLC, following amino acid composition (table 31) is obtained.As a result be expressed as amino acid to sequence in the number that occurs, represented with percentage.Known glycine is a common contaminant in amino acid analysis, thus it is possible to vary amino acid is constituted.After this point, as a result there is comparativity with theoretical value.

Table 31

Monose and sulfate

Single monose, phosphate and sulfate are hydrolyzed from OX40-Fc amino acid backbone and obtained, and are analyzed with described high pH anion-exchange chromatographies (HP AEC), obtain following components analysis.Standard (table 32-34) is used as from sample from obtained result with GalNAc and 3 times of mannose respectively.Table 35 is the summary to the result from 3 samples.Glucose is the composition of a common contaminant and not usually N- connections or O- connection oligosaccharides.

Table 32

Table 33

Table 34

Table 35

In view of with chromatographic process described above in variability, during with GalNAc as standard, it is determined that the trehalose that the OX40-Fc of the present invention monose, sialic acid and sulphates content are about 1: 0.1-1,1: 2-3 GlcNAc, 1: 0.5-2 galactolipin, 1: 0.5-1 mannose, 1: 0-2 NeuNAc and 1: 0.30-2 sulfate；During with 3 times of mannoses as standard, it is defined as 3: 0.5-2 trehalose, 3: 3-5 GalNAc, 3: 6-10 GlcNAc, 3: 4-5 galactolipin, 3: 0-2 NeuNAc and 3: 1-5 sulfate.

For each OX40-Fc, amino acid composition data combination monose and sulfate data give different types of content (table 36).In view of in chromatographic process described above in variability, sialic acid is expressed as the OX40-Fc of present invention monose percentage, determine its scope about 0-10%, sulfate represents the OX40-Fc of present invention monose percentage, its scope about 9-15% is determined, it is determined that the acid percentage ranges of the OX40-Fc of present invention N- connection oligosaccharides are about the acid percentage ranges about 20-55% of 5-21% and the OX40-Fc of present invention O- connection oligosaccharides.

Table 36

(f) BAFF of present invention amino acid, monose, oligosaccharides, phosphate, sulfate and isoform composition is analyzed

(i) preparing sample is used for amino acid, monose, oligosaccharides, phosphate, sulfate and isoform analysis

Progress of the BAFF PBS solution as described by example 4 above (a) (i) is purified to handle.

(ii) constituted with vapor phase hydrolysis method (Gas Phase Hydrolysis Method) analysis of amino acid

Progress of the BAFF samples of preparation as described by example 4 above (a) (ii) is handled.

(iii) composition of neutral and alkaline monose is analyzed

Progress of the BAFF samples of preparation as described by example 4 above (a) (iii) is handled.

(iv) composition of acid monose is analyzed

Progress of the BAFF samples of preparation as described by example 4 above (a) (iv) is handled.

(v) composition of oligosaccharides is analyzed

Progress of the BAFF samples of preparation as described by example 4 above (a) (v) is handled.

(vi) analysis sulfate and phosphatic composition

Progress of the BAFF samples of preparation as described by example 4 above (a) (vi) is handled.

(vii) further protein isolate isoform

Progress of the BAFF samples of preparation as described by example 4 above (a) (vii) is handled.It was found that BAFF isoforms are eluted in the form of a kind of particular peaks.

(g) NGFR-Fc of present invention amino acid, monose, oligosaccharides, phosphate, sulfate and isoform composition is analyzed

(i) preparing sample is used for amino acid, monose, oligosaccharides, phosphate, sulfate and isoform analysis

Progress of the NGFR-Fc PBS solution as described by example 4 above (a) (i) is purified to handle.

(ii) constituted with vapor phase hydrolysis method (Gas Phase Hydrolysis Method) analysis of amino acid

Progress of the NGFR-Fc samples of preparation as described by example 4 above (a) (ii) is handled.

(iii) composition of neutral and alkaline monose is analyzed

Progress of the NGFR-Fc samples of preparation as described by example 4 above (a) (iii) is handled.

(iv) composition of acid monose is analyzed

Progress of the NGFR-Fc samples of preparation as described by example 4 above (a) (iv) is handled.

(v) composition of oligosaccharides is analyzed

Progress of the NGFR-Fc samples of preparation as described by example 4 above (a) (v) is handled.

(vi) analysis sulfate and phosphatic composition

Progress of the NGFR-Fc samples of preparation as described by example 4 above (a) (vi) is handled.

(vii) further protein isolate isoform

Progress of the NGFR-Fc samples of preparation as described by example 4 above (a) (vii) is handled.It was found that NGFR-Fc isoforms are eluted in the form of a kind of particular peaks.

(f) BAFF of present invention amino acid, monose, oligosaccharides, phosphate, sulfate and isoform composition is analyzed

(i) preparing sample is used for amino acid, monose, oligosaccharides, phosphate, sulfate and isoform analysis

Progress of the BAFF PBS solution as described by example 4 above (a) (i) is purified to handle.

(ii) constituted with vapor phase hydrolysis method (Gas Phase Hydrolysis Method) analysis of amino acid

Progress of the BAFF samples of preparation as described by example 4 above (a) (ii) is handled.

(iii) composition of neutral and alkaline monose is analyzed

Progress of the BAFF samples of preparation as described by example 4 above (a) (iii) is handled.

(iv) composition of acid monose is analyzed

Progress of the BAFF samples of preparation as described by example 4 above (a) (iv) is handled.

(v) composition of oligosaccharides is analyzed

Progress of the BAFF samples of preparation as described by example 4 above (a) (v) is handled.

(vi) analysis sulfate and phosphatic composition

Progress of the BAFF samples of preparation as described by example 4 above (a) (vi) is handled.

(vii) further protein isolate isoform

Progress of the BAFF samples of preparation as described by example 4 above (a) (vii) is handled.It was found that BAFF isoforms are eluted in the form of a kind of particular peaks.

(h) amino acid, monose, oligosaccharides, phosphate, sulfate and the isoform composition of the Fas aglucons of the present invention are analyzed

(i) preparing sample is used for amino acid, monose, oligosaccharides, phosphate, sulfate and isoform analysis

Progress of the PBS solution of FasL as described by example 4 above (a) (i) is purified to handle.

(ii) constituted with vapor phase hydrolysis method (Gas Phase Hydrolysis Method) analysis of amino acid

The Fas of preparation is handled with progress of the based specimen as described by example 4 above (a) (ii).

(iii) composition of neutral and alkaline monose is analyzed

The Fas of preparation is handled with progress of the based specimen as described by example 4 above (a) (iii).

(iv) composition of acid monose is analyzed

The Fas of preparation is handled with progress of the based specimen as described by example 4 above (a) (iv).

(v) composition of oligosaccharides is analyzed

The Fas of preparation is handled with progress of the based specimen as described by example 4 above (a) (v).

(vi) analysis sulfate and phosphatic composition

The Fas of preparation is handled with progress of the based specimen as described by example 4 above (a) (vi).

(vii) further protein isolate isoform

The Fas of preparation is handled with progress of the based specimen as described by example 4 above (a) (vii).It was found that Fas aglucons isoform is eluted in the form of a kind of particular peaks.

Embodiment 5

Saccharic amount dactylogram

(a) the sugared quality fingerprinting between albumen relatively more of the present invention and the corresponding human albumen of non-human cell's expression

2D gel electrophoresis technologies described by the albumen use-case 3 of the present invention are separated, and are blotted on polyethylene Difluoroethane (PVDF) film.Spot is dyed with the protein dye (Colloidal Coomassie Blue, Sypro Ruby or Deep Purple) of a standard volume, and the relative quantity optical density standard measure of isoform.Cut off each spot and handled with a series of deglycosylating enzymes and/or chemical method, depended on the circumstances, to remove the oligosaccharides of presence, the method described according to this specification.Once oligosaccharides is removed, it is separated and analyzed in the chromatography-electrospray-ionization/mass spectrometry system (LC-MS) using graphitized carbon post and organic solution (MeCN) gradient elution system.The peak shape of generation generates " dactylogram " of oligosaccharides present in isoform.Further, mass spectrometer system produces signal to various sugared quality present in sample, and it is by using the pattern match of GlycoSuite databases to recognize their structure.In addition, each mass peak can many times of fragmentations to provide MSn spectrum.These fragments can obtain the prediction of structure, using methods known in the art, for example, passing through the use of GlycosidIQ program bags.

Separation, deglycosylation and analytical procedure above is repeated with non-human system such as E.coli, yeast or expressing cho cell corresponding albumen, and discovery has significant difference.In structure level, such a result shows, there are various forms of oligosaccharide structures between the corresponding albumen of albumen of the present invention and non-human cell's expression.

(b) the sugared quality fingerprinting between comparison TNFRII-Fc of the invention and the TNFRII-Fc of expressing cho cell

The 2D gel electrophoresis technologies separation that the TNFRII-Fc use-cases 3 of the present invention are described, and blotted on polyethylene Difluoroethane (PVDF) film.Spot is dyed with the protein dyestuff (Colloidal Coomassie Blue, Sypro Ruby or Deep Purple) of a standard series, and the relative amount optical density calculating method of isoform is quantified.Single spot is taken out, is handled with a series of deglycosylating enzymes and/or chemical method, under the suitable conditions, the method according to described by example 4 above (a) (v) removes the oligosaccharide existed.

Once removing oligosaccharide, they are separated from each other, and are separated with liquid chromatogram-electrospray mass spectrometry system (LC-MS), utilize a graphitised carbon post and organic solvent (MeCN) gradient elution system.N- connections and " fingerprint " (being respectively Fig. 2 (a) and 2 (e)) of O- connection oligosaccharides that the TNFRII-Fc that the peak feature of generation represents the present invention is present.In addition, being divided into multiple fragments to obtain MSn collection of illustrative plates (Fig. 2 (b) and 2 (f)) single mass peak.Fragment is analyzed with GlycosidIQ softwares, can pre- geodesic structure (table 37 (a) and 37 (b)).

Table 37 (a)

The structure of N- connection oligosaccharides present in the TNFRII-Fc of the invention speculated with GlycosidIQ

Table 37 (b)

The structure of O- connection oligosaccharides present in the TNFRII-Fc of the invention speculated with GlycosidIQ

Molecular weight	Structure
		674	Gal b1-3GalNAc   +NeuAc(a2-)

The TNFRII-Fc that separation, deglycosylation and analytical procedure above is expressed with Chinese hamster ovary (or CHO) cell carries out repeating (Fig. 2 (c), 2 (d), 2 (g) and 2 (h) table 38 (a) and 38 (b)), then it and TNFRII-Fc of the invention described above accordingly result are compared.It was found that there is significant difference in respective sugared quality fingerprinting.In structure level, such a result shows, there are various forms of oligosaccharide structures between TNFRII-Fc of the invention and the people TNFRII-Fc of expressing cho cell.

Table 38 (a)

The structure of N- connection oligosaccharides present in the TNFRII-Fc of Chinese hamster ovary cell (Enbrel) expression speculated with GlycosidIQ

Table 38 (b)

The structure of O- connection oligosaccharides present in the TNFRII-Fc of Chinese hamster ovary cell (Enbrel) expression speculated with GlycosidIQ

Embodiment 6

Fluorescence assisted carbohydrate electrophoresis

The oligosaccharides profile that (FACE protocols) obtains target molecule is tested with fluorescence assisted carbohydrate electrophoresis.Oligosaccharides ammonium hydroxide from the aim cell factor is hydrolyzed from amino acid backbone, then with fluorescence 8- amino naphthyls -1,3,6- trisulfonic acids (ANTS) mark.Polyacrylamide gel electrophoresis is used to separate species and the standard for the distinctive oligosaccharides profile of identifying purpose molecule.Further, oligosaccharides is recognized with the substance assistant laser desorpted and ionisation-time of the flight mass spectrum (MALDI-TOF) dependent on fluorescence and to the special matrix of various sugared ionizations.Determine various sugared quality and with GlycoSuite database identification of protein structures.Potential sugar architectural feature is further described by tandem mass spectrum technology, and it make it that obtain oligosaccharides describes in the presence of part the or whole feature with their relative quantity.Further, reuse and recognize the method for isoform to produce the profile of oligosaccharides present in each isoform of separation by 2D gel electrophoresises.

Embodiment 7

QCM and SPR

With the binding characteristic and activity of quartz crystal microbalance (QCM) or surface plasma resonance (SPR) testing goal molecule.In two methods, the chemical action that suitable molecular receptor is described with manufacturer is attached on chip.Molecules of interest is dissolved in suitable biological buffer and is passed to buffer solution and the acceptor interaction on chip.By changing frequency of oscillation (in QCM methods) or measuring the change of the gross protein quality in wafer surface by changing the light scattering characteristic (in SPR methods) of chip.Then, only with biological buffer process chip to observe that molecules of interest is released back into solution.Then, the speed that acceptor reaches saturation and is kept completely separate is used for the binding curve for calculating molecules of interest.

Embodiment 8

The generation of genetically modified host cell system

(a) the genetically modified host cell system of α -2,6- sialyltransferases is contained

The cDNA of coding for alpha -2,6- sialyltransferase (α 2,6ST) is expanded by PCR by poly (A)-primed cDNA.PCR primer is connected in suitable carrier, such as pIRESpuro4 or pCEP4, to produce α 2,6ST plasmids.The cDNA of clone is sequenced and its homogeneity is confirmed by comparing with disclosed α -2,6ST cDNA sequences.DNA sequencing is carried out with known method.

Mammalian host cell, includes cell strain system (cell line-molecules of interest) α 2 of the identical pedigree of the high-level molecules of interest of expression, 6ST plasmid transfections, and it also carries antibiotic-resistance marker.By the selection that the transfectional cell that cell is stablized is cultivated in the presence of antibiotic；Collect the cell strain system that antibiotic-resistant is shown after transfection and be used for intracellular α 2, the inspection of 6ST activity.In order to separate expression 2,6ST individual cells strain, cell gleanings pass through such as Kronman (Gene 121：295-304,1992) description limitation dilution method clone.Single cell strain system is selected at random, and cell expands and detects the α 2 of strain, 6ST activity.

Cell precipitation is washed, is resuspended in dissolving buffer solution, and it is preposition in ice in ultrasonic degradation.Cellular lysate centrifuges and the supernatant of clarification is used for into protein concentration (via known method) and Sialyltransferase Activitychange In The Rat Mammary analysis.Sialyltransferase Activitychange In The Rat Mammary passes through known method, such as Datta et al. (J Biol Chem 270：1497-1500,1995) described by method analysis.

The molecules of interest of expression is purified from high expression 2,6ST cell lines-molecules of interest cell and external and/or Half-life in vivo biologicall test is carried out (see embodiment 10).From high express alpha 2,6ST cells molecules of interest compared with derived from no molecules of interest of identical parental cell line by any transgeneic procedure or the molecules of interest derived from other cell lines, show increased external and/or Half-life in vivo.

(b) the genetically modified host cell system containing fucosyltransferase

The cDNA of encoding trehalose based transferase (FT), such as FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, FUT10, FUT11, are expanded by PCR by poly (A)-primed cDNA.PCR primer is connected in suitable carrier, such as pIRESpuro4 or pCEP4, to produce α 2,6ST plasmids.The cDNA of clone is sequenced and confirms its homogeneity by the comparison with disclosed FT cDNA sequences.DNA sequencing is carried out with known method.

Human host cell, includes the cell strain system FT plasmid transfections of the identical pedigree of the high-level molecules of interest molecule (cell line-molecules of interest) of expression, it also carries antibiotic-resistance marker.Pass through the selection of the transfectional cell that the culture of cell is stablized in the presence of antibiotic；Collect the strain for the cell that antibiotic-resistant is shown after transfection and for the inspection of intracellular FT activity.In order to separate expression FT individual cells strain, cell gleanings are cloned by limiting dilution method, such as Kronman (Gene 121：295-304,1992) it is described；Individual cells strain is selected at random, and cell expands and detects the FT activity of strain.

Cell precipitation is washed, is resuspended in dissolving buffer solution, and it is preposition in ice in ultrasonic degradation.The supernatant that cellular lysate is centrifuged and clarified is used for protein concentration (through known method) and FT activity analysis.FT activity is analyzed by known method, such as Mas et al. (Glycobiology 8 (6)：605-13,1998) described by.

The molecules of interest of expression is by height expression FT cell lines-molecules of interest cell purification.Lewis x- specific antibodies, such as L5 and sialylated Lewis x- specific antibodies, such as KM93, HECA493,2H5 or CSLEX, presence for detecting Lewis x or sialylated Lewis x structures, according to methods known in the art, for example, such as Lucka et al. (Glycobiology 15 (1)：87,2005) it is described in detail.Or, the presence of Lewis x or sialylated Lewis x structures by using suitable glucosides ferment treatment sample detection and can detect influence of the glycosidase in parameter, such as with quality during MS or with retention time during HPLC.Saccharic amount dactylogram, as described in Example 5, can be used for predicting the presence of Lewis x or sialylated Lewis x structures.

Embodiment 9

Differential gene expression

The difference of gene expression can be analyzed with the aim cell system of molecules of interest.Aim cell grows into suitable density and molecules of interest or buffer control with a range of concentration handle multiple hours, for example, 72 hours.

In the different time, RNA harvests, purifying and reverse transcription are carried out, according to Affymetrix experimental programs.Then, prepare the cRNA (such as biotin labeling) of mark and hybridize with expression matrix, such as U133 GeneChips.It is washed out and signal amplifies, GeneChips GeneChip scanners (Affymetrix) is scanned, and intensity for hybridization and multiple change information GeneChip software (Affymetrix) acquisitions in different time points.

The unique gene expression of molecules of interest induction and cause with by separate sources, such as E.The different mRNA profiles of coli, the cell factor that yeast or Chinese hamster ovary celI are produced or receptor-inducible profile.

Embodiment 10

The measure of the half-life period of purpose of the present invention molecule

The half-life period of molecules of interest, system was measured in vitro.Composition comprising molecules of interest is mixed into human serum/blood plasma and certain temperature culture certain time (such as cultivating 4 hours, 12 hours 37 degree).The amount of the molecules of interest retained after this treatment is determined by ELISA method known in the art or dot blotting.The biological activity of the molecules of interest of reservation is determined by the progress of the suitable biological detection method selected by those skilled in the relevant art.Selected serum may come from a variety of human blood types (such as A, B, AB, O etc.).

The half-life period of molecules of interest is also measured in vivo system.Composition comprising molecules of interest by radioactive tracer mark (or other method) and through vein, it is subcutaneous, after eye socket, intramuscular or intraperitoneal injection to study in selected species, for example, mouse, rat, pig, primate or people.The time point blood sampling after injection and presence to molecules of interest is analyzed and (marked by ELISA method, dot blotting or by trichloroacetic acid (TCA)-precipitation, such as radiocounting).The comparison of composition containing the molecules of interest for being produced from separate sources, such as E.coli, yeast, or Chinese hamster ovary celI can be carried out as control.

Embodiment 11

(a) in vivo studies of purpose of the present invention molecule

The individual subjects of in vivo studies described herein are warm-blooded vertebrates, including people.

Clinical test is by strictly controlling to ensure that individual is not interposing in unnecessary risk and they are fully informed their effects under study for action.

It is preferred that, in order to compensate the psychological application for receiving treatment, treatment is carried out with double-blind fashion.Volunteer is randomly assigned into control or molecules of interest treatment group.Further, relevant clinician is also blind for the treatment system being administered to receptor, to prevent there is prejudice in being observed after their treatment.Use this random fashion, each volunteer has the identical chance for being given new treatment or control.

Volunteer receives molecules of interest or control agent for a period of time, simultaneously, starting (baseline before any treatment) with reference to the morbid state or the biological parameter of health status shown, terminating (after final treatment), and be measured in the interval of rule during studying.The measurement includes the level of molecules of interest in the body fluid compared with level before treatment, tissue or organ.Other measurements include, but not limited to morbid state or the index of treated health status, body weight, blood pressure, the serum titer of the pharmacology indicator of disease, such as special disease indicator or toxicity and ADME (absorb, distribution, metabolism and drain) measurement.

Record each patient information include the age (year), sex, the species of the family history (Yes/No) of height (cm), morbid state or disease, motivation grade (few/in/big) and numbering and the disease for showing or sick first therapy.

The volunteer for participating in the experiment is adult, the age at 18 to 65 years old and add experiment masculinity and femininity number approximate equality.Volunteer with some features is evenly distributed for control agent and molecules of interest treatment.General, the volunteer treated with control agent has seldom or not reacted to treatment, but in off-test, the volunteer treated with molecules of interest shows that their morbid state or the index of disease are intended to the positive.

(b) TNFRI-Fc preparation for treating people's Psoriatic skins are used with local

The subject of in vivo studies described here refers to temperature vertebrate, including the mankind.

Strict control clinical test, it is ensured that subject is without bearing unnecessary risk, and subject is informed their effects under study for action completely.In order to illustrate the psychological application for receiving treatment, volunteer is randomly divided into local placebo or local T NFRI-Fc treatment groups.In addition, in order to avoid prejudice of the doctor to treatment, not informing that the treatment that they give volunteer is part TNFRI-Fc or part placebo.Using the random device, the chance of the new treatment of each volunteer's acquisition or placebo is identical.

Volunteer receives local with TNFRI-Fc (no Distaval, formulation is shown in the description of embodiment 19 (c)) or one section of part placebo treatment suitable time, for example, in skin (total skin area 20cm of psoriatic lesion²) on single 0.8ml administrations, follow-up 7 days, or on identical target injured skin, multiple 0.8ml administrations gave 9 times (every other day) in 21 days.The biological parameter related to indicated morbid state or situation such as psoriasis, beginning (baseline determination before any treatment), end (after final treatment) in conceptual phase, and each same time interval are measured.This measure includes the TNF-α level in body fluid, tissue or organ, compared with the level before treatment.Other measure include, but not limited to the index body weight of treated morbid state or situation, blood pressure, the serum titration degree and ADME of disease or toxicity pharmacology indicator (absorb, distribution, metabolism and drain) measure.Especially, the local psoriasis volunteer patients with TNFRI-Fc for distributing in TNFRI-Fc treatment groups of the invention.

Record each patient information include the age (year), sex, the species of the family history (Yes/No) of height (cm), morbid state or disease, motivation grade (few/in/big) and numbering and the disease for showing or sick first therapy.

The volunteer for participating in this research is the adult of 18 to 65 years old, and the women and male of approximately the same number participate in this research.Local placebo is coequally assigned to the volunteer for determining feature and part TNFRI-Fc is treated.

Assessment to treatment is classified with 4 scopes, i.e. healing, significant effective, effective and invalid.The areas of inflammation that " healing " refers on spot thoroughly reduces, and pruritus disappears.The areas of inflammation that " significant effective " refers on spot reduces more than 60%, and pruritus weakens and relaxed.The areas of inflammation that " effective " refers on spot reduces 20 to 60%, and pruritus weakens and relaxed.The areas of inflammation that engineering noise refers on spot reduces less than 20%, or pruritus aggravation.

Or, treatment is assessed and is classified according to local speckle severity index (LPSI), each target spot point assess and erthema, scleroma and dessquamation has been scored, and is scored by clinic supervision doctor in specific clinical indagation every time with five graduation.Clinical indagation timetable the such as the 0th suitable for " multiple dosing " therapeutic scheme, 11 and 21 days.Five graduation are defined as：0=is asymptomatic；1=is slight；2=severes；3=is notable；4=highly significants.Erthema, scleroma and desquamation fraction are added.LPSI scope is 0 to 12, and fraction highest represents the morbid state of most serious.

In general, the volunteer for giving part placebo treatment is seldom to therapeutic response or reactionless, and the part for giving the present invention uses the volunteer of TNFRI-Fc cream for treating at the end of research, and positive trend is presented in their disease condition or state indices.Especially, the Most patients significant effective of topical preparation of the invention to TNFRI-Fc treatment groups.Obvious side effect is not observed.

(c) human rheumatoid arthritis is treated with TNFRI-Fc

The subject of in vivo studies described here refers to temperature vertebrate, including the mankind.

Strict control clinical test, it is ensured that subject is without bearing unnecessary risk, and subject is informed their effects under study for action completely.During clinical indagation, investigator will extract multiple blood samples；Extensive physical examination is carried out, including assesses the joint of swelling, tenderness and pain.In order to illustrate the psychological application for receiving treatment, volunteer is randomly divided into placebo or TNFRI-Fc treatment groups.In addition, in order to avoid prejudice of the doctor to treatment, not informing that the treatment that they give volunteer is TNFRI-Fc or placebo.Using the random device, the chance of the new treatment of each volunteer's acquisition or placebo is identical.

Volunteer receives TNFRI-Fc or one section of placebo treatment suitable time, the biological parameter related to the degree of indicated morbid state or situation such as arthroncus, beginning (baseline determination before any treatment), end (after final treatment) in conceptual phase, and each same time interval are measured.Measurement result includes the TNF-α in inflammatory parameters level such as body fluid, tissue or organ, compared with the level before treatment.Other measurement results include, but not limited to the index body weight of treated morbid state or situation, blood pressure, the serum titration degree and ADME of disease or toxicity pharmacology indicator (absorb, distribution, metabolism and drain) measure.Especially, the TNFRI-Fc of the present invention is used to distribute the rheumatoid arthritis volunteer patients in TNFRI-Fc treatment groups, TNFRI-Fc is subcutaneously injected twice weekly, continues eight weeks.

The information of every patient record include the age (year), sex, height (cm), the family history (with/without) of morbid state or situation, inducement grade (part/appropriateness/main) and indicated disease or situation go over the number and type of therapeutic scheme.

The volunteer for participating in this research is the adult of 18 to 65 years old, and equal number of women and male participate in this research.Placebo and TNFRI-Fc treatments are coequally assigned to the volunteer for determining feature.

Assessment to treatment is classified with 4 scopes, i.e. healing, significant effective, effective and invalid.Pain/tenderness the symptom of " healing " joint or joint without swelling or correlation." significant effective " refers to joint or the swelling in joint largely reduces (more than 60%) and with arthralgia and significantly reducing for touching a tender spot." effective " refers to joint or the swelling in joint reduces 20-60% and with related arthralgia and the slight reduction touched a tender spot.Engineering noise refers to joint or the swelling in joint reduces less than 20% and do not feel that related arthralgia makes moderate progress.

In general, the volunteer for giving placebo treatment is seldom to therapeutic response or reactionless, and the volunteers of the TNFRI-Fc treatments of the present invention is given at the end of research, positive trend is presented in their disease condition or state indices.Especially, the Most patients significant effective or effective of preparation of the invention to TNFRI-Fc treatment groups.Obvious side effect is not observed.

(d) TNFRII-Fc preparation for treating people's Psoriatic skins are used with local

The subject of in vivo studies described here refers to temperature vertebrate, including the mankind.

Progress of the clinical test as described by example 11 above (b), except non-placebo treatment group gives the TNFRII-Fc topical preparations composition without Distaval, the formulation is shown in the description of embodiment 19 (b).

In general, the volunteer for giving placebo treatment is seldom to therapeutic response or reactionless, and the volunteer of the TNFRII-Fc cream for treating of the present invention is given at the end of research, positive trend is presented in their disease condition or state indices (see description of embodiment 11 (b)).Especially, the Most patients significant effective of topical preparation of the invention to TNFRII-Fc treatment groups.Obvious side effect is not observed.

(e) human rheumatoid arthritis is treated with TNFRII-Fc

The subject of in vivo studies described here refers to temperature vertebrate, including the mankind.

Progress of the clinical test as described by example 1 above 1 (c), except non-placebo treatment group gives the TNFRII-Fc of the present invention.

In general, the volunteer for giving placebo treatment is seldom to therapeutic response or reactionless, and the volunteer of the TNFRII-Fc cream for treating of the present invention is given at the end of research, positive trend is presented in their rheumatoid arthritis (see description of embodiment 11 (c)).Especially, the Most patients significant effective or effective of preparation of the invention to TNFRII-Fc treatment groups.Obvious side effect is not observed.

(f) TNFRII-Fc preparation for treating people's pityriasis rubra pilaris is used with local

The TNFRII-Fc topical preparations of the present invention include TNFRII-Fc (250 μ g/ml) and Distaval (20mg/ml), for pityriasis rubra pilaris volunteer.Areas of inflammation is administered once for every two days, continues 2 weeks, each 2ml topical preparation.The hand of volunteer is shown in Fig. 3 (a) before treating first, and same value hand is shown in Fig. 3 (b) after the therapeutic scheme of 2 weeks terminates.Topical preparation substantially reduces crackle.Obvious side effect is not observed.

Embodiment 12

(a) bioactivity of TNF-α of the invention

TNF-α causes cytotoxicity and cell death in mouse fibrosarcoma cells system WEHI 164.The cells of WEHI 164 are pre-processed with the actinomycin D (2 μ g/ml) for suppressing transcription.Sensitiveness of this increase WEHI 164 to TNF-α.In 96 orifice plates, 0-10ng/ml TNF-α per 50,000, the hole cells of WEHI 164 at 37 DEG C with being incubated 18 hours.

Then the cytotoxicity of TNF-α is determined with the Aqueous One Solution Cell ProlifertionAssay (Promega) of CellTiter 96.In the analysis method, one tetrazolium salt composite MTS (3- (4,5- dimethylthiazole -2- bases) -5- (3- carboxymethoxyls phenyl) -2- (4- sulfophenyls) -2H- tetrazolium saltses) is reduced into first when there is electronics coupled agent (phenazine methosulfate) by cell biological

Product.The trap of solution is determined in 490nm with spectrophotometer (BioRad microplate readers), so as to determine first

Concentration.

A quadruplex parameters formula is obtained from the curve matching of trap and concentration, the ED50 of the present invention is calculated, is as a result 0.012-0.018ng/ml (Fig. 4).

(b) LT- α of the invention bioactivity

LT- α cause cytotoxicity and cell death in mouse fibrosarcoma cells system WEHI 164.The cells of WEHI 164 are pre-processed with transcription inhibitor actinomycin D (2 μ g/ml), are then inoculated in 96 orifice plates, and 0-400ng/ml LT- α are incubated 18 hours with being incubated altogether per 50,000, the hole cells of WEHI 164 at 37 DEG C.

Then cytotoxicity is determined with the Aqueous One Solution Cell ProliferationAssay (Promega) of CellTiter 96.In the analysis method, one tetrazolium salt composite MTS (3- (4,5- dimethylthiazole -2- bases) -5- (3- carboxymethoxyls phenyl) -2- (4- sulfophenyls) -2H- tetrazolium saltses) is reduced into first when there is electronics coupled agent (phenazine methosulfate) by cell biological

Product.The trap of solution is determined in 490nm with spectrophotometer (the maximum accurate microplate readers of E, Molecular Devices), so as to determine firstConcentration.

(c) TNFRI-Fc of the invention bioactivity

Using the ability of the cytotoxicity mediated in TNFRI-Fc with TNF-α in the cell lines of WEHI 164, its activity is determined.Serial dilution TNFRI-Fc, from 0.006 to 100ng/ml, is incubated 1 hour at 37 DEG C with 5ng/ml TNF-α, TNFRI-Fc is combined with TNF-α.Then 5 × 10 are added per hole⁴Individual WEHI-164 cells, cell is pre-processed with 2 μ g/ml actinomycin D, by suppressing sensitiveness of the transcription increase cell to TNF-α.Flat board is incubated 20 hours (37 DEG C, 5%CO₂), be subsequently added into 10% cellAQueous One solution reagents (Promega), wherein contain tetrazolium salt composite MTS [3- (4,5- dimethylthiazole -2- bases) -5- (3- carboxymethoxyls phenyl) -2- (4- sulfophenyls) -2H- tetrazolium saltses inner salt] and electronics coupled agent (phenazine ethaosulfate；PES).Cell number in 490nm determines trap, reacting hole.A quadruplex parameters formula is obtained from the curve matching of trap and concentration, ED50 is calculated, is as a result 14-20ng/ml (Fig. 6).

(d) TNFRII-Fc of the invention bioactivity

Using the ability of the cytotoxicity mediated in TNFRII-Fc with TNF-α in WEHI-164 cell lines, its activity is determined.Serial dilution TNFRII-Fc, from 0.006 to 100ng/ml, is incubated 1 hour at 37 DEG C with 5ng/ml TNF-α, TNFRII-Fc is combined with TNF-α.Then 5 × 10 are added per hole⁴Individual WEHI-164 cells, cell is pre-processed with 2 μ g/ml actinomycin D, by suppressing sensitiveness of the transcription increase cell to TNF-α.Flat board is incubated 20 hours (37 DEG C, 5%CO₂), be subsequently added into 10% cell

AQueous One solution reagents (Promega), wherein contain tetrazolium salt composite MTS [3- (4,5- dimethylthiazole -2- bases) -5- (3- carboxymethoxyls phenyl) -2- (4- sulfophenyls) -2H- tetrazolium saltses inner salt] and electronics coupled agent (phenazine ethaosulfate；PES).Cell number in 490nm determines trap, reacting hole.A quadruplex parameters formula is obtained from the curve matching of trap and concentration, the ED50 of the present invention is calculated, is as a result 14-20ng/ml (Fig. 7).

(e) comparison TNFRII-Fc of the invention and the TNFRII-Fc of non-human system's expression bioactivity

The TNFRII-Fc (Pepro Tech, Cat#310-12) of E.coli expression and the TNFRII-Fc (1500 μ g/ml) of the present invention bioactivity are measured according to the depression effect of its cytotoxicity mediated to TNF-α in mouse L-929 cells.Respective result is compared.

The TNFRII-Fc (Pepro Tech) of lyophilized is dissolved again and is made into original concentration, -80 DEG C of preservations.L-929 cells are resuspended in the medium.Re-suspension liquid is transferred in a block analysis flat board (8,000 cells/wells；Pass on #7), 100ml is added per hole.Flat board is in 37 DEG C of overnight incubations.

In independent one block of plate, by TNFRII-Fc (Pepro Tech) serial dilutions in analysis medium.Every kind of dilution adds analysis flat board in pairs, and 40 μ l every kind of dilution is added per hole.The analysis medium of TNF-α containing 4ng/ml (Pepro Tech) adds 60 μ l per hole.Cumulative volume per hole is 100 μ l.

TNF-α-TNFRII-Fc compounds are transferred to the analysis flat board kind of the cell containing L-929.100 μ l compounds are added per hole.Therefore, the final volume per hole is 200 μ l, the TNF-α containing 1ng/ml.

Flat board will be analyzed and material therein is incubated 17 hours.The Promega that 20 μ l are added per hole titrates the aqueous solution of 96 adherent cells.Mixture determines trap after 6 hours in 37 DEG C of incubations in 490nm.

Above-mentioned experiment is repeated with the TNFRII-Fc of the present invention.

Another control (not providing) is that analysis is repeated in the case of TNF-α is non-existent with the TNFRII-Fc of the present invention.L-929 cells are resuspended in the medium.Re-suspension liquid is transferred in a block analysis flat board (8,000 cells/wells；Pass on #7), 100 μ l are added per hole.Flat board is in 37 DEG C of overnight incubations.In independent one block of plate, by the TNFRII-Fc serial dilutions of the present invention in analysis medium, 100 μ l every kind of dilution is added per hole.Dilution is added in the L-929 cells of original position.Therefore, the final volume per hole is 200 μ l.Flat board is incubated 17 hours.The Promega that 20 μ l are added per hole titrates the aqueous solution of 96 adherent cells.Mixture determines trap after 6 hours in 37 DEG C of incubations in 490nm.

TNFRII (Pepro Tech) and the TNFRII-Fc of the present invention 50% (Conc.ED₅₀) effective dose concentration measure：Their A (490nm) is mapped to respective log concentration, and with GnuPlot by numerical fitting into the function of following form, GnuPlot is a kind of graphic package, by pattern fits into mathematical function and data (http://www.gnuplot.info)：

F (x)=cauchy (x)

And cauchy (x)=a0+a1 (a tan ((x-a2)/a3)/π

π=3.1416

ED₅₀Corresponding to the saddle point of cauchy functions, the corresponding y values (A (490nm)) of intermediate value between fitting parameter " a2 " and asymptotic minimum values and cauchy function maximas are consistent (Fig. 8).Obtain TNFRII-Fc ED₅₀For 3.2-4.8ng/ml.Obtain TNFRII (PeproTech) ED₅₀For 39-59ng/ml.

It was found that TNFRII-Fc activity, ED higher 8-18 times than TNFRII (Pepro Tech)₅₀It is relatively low, therefore bioactivity is higher.

(d) OX40-Fc of OX-40-Fc and non-human system expression relatively more of the present invention activity

It is reported that OX40 can suppress the IL-2 of OX40L inductions secretion in mouse T cell system CTLL2.IL-2 is a propagation and survival factors for CTLL2 cells.Therefore, OX40L multiplication effect can be suppressed by adding OX40.

In 96 orifice plates, the OX40-Fc of the 2ng/ml OX40L and 0-100ng/ml present invention is incubated 1 hour at 37 DEG C, combination is brought it about.Add 10000 CTLL2 cells/wells, 37 DEG C, 72 hours.Then cell number is determined with the Aqueous One Solution CellProliferation Assay (Promega) of CellTiter 96.In the analysis method, one tetrazolium salt composite MTS (3- (4,5- dimethylthiazole -2- bases) -5- (3- carboxymethoxyls phenyl) -2- (4- sulfophenyls) -2H- tetrazolium saltses) is reduced into first when there is electronics coupled agent (phenazine methosulfate) by cell biologicalProduct.The trap of solution is determined in 490nm with spectrophotometer (the maximum accurate microplate readers of E, Molecular Devices), so as to determine first

Concentration.

Above-mentioned analysis is repeated with the OX40-Fc of non-human system such as E.coli, yeast or expressing cho cell.Respective ED is calculated after curve matching trap₅₀, and obtain with a quadruplex parameters OX40-Fc concentration.It was found that ED₅₀In the presence of significant difference.

(e) comparison BAFF of the invention and the BAFF of non-human system's expression activity

It is reported that BAFF can induce the propagation of the cells of RPMI 2886.In 96 orifice plates, 10000 cells/wells of RPMI 2886 give 0-250ng/ml BAFF of the invention, 37 DEG C, 114 hours.

Then cell number is determined with the Aqueous One Solution Cell ProliferationAssay (Promega) of CellTiter 96.In the analysis method, one tetrazolium salt composite MTS (3- (4,5- dimethylthiazole -2- bases) -5- (3- carboxymethoxyls phenyl) -2- (4- sulfophenyls) -2H- tetrazolium saltses) is reduced into first when there is electronics coupled agent (phenazine methosulfate) by cell biological

Product.The trap of solution is determined in 490nm with spectrophotometer (the maximum accurate microplate readers of E, Molecular Devices), so as to determine first

Concentration.

The recombinant human B AFF molecules (Peprotech) expressed with E.coli repeat above-mentioned analysis.

Obtain the BAFF of present invention ED₅₀For 50-75ng/ml, and Peprotech (being expressed by E.coli) BAFF ED₅₀For 80-120ng/ml (Fig. 9).Therefore, the BAFF molecules of the breeding ratio E.coli expression of the BAFF of the invention induction cells of RPMI 8226 are high 1.1-2.4 times.

(e) NGFR-Fc of the invention bioactivity

It is reported that NFG- β can induce the propagation of TF-1 cells.NGFR-Fc combination NGF- β and Reverse transcriptase these molecules are incorporated into the NGF- beta receptors site of theirs in the cell, NGF- β is not showed biological activity, so as to inhibit NGF- β activity.Therefore, NGF- β and NGFR-Fc is incubated altogether will suppress the beta induced TF-1 cells propagation of NGF-.

In 96 orifice plates, 1ng/ml NGF- β and 0-100ng/ml NGFR-Fc of the invention are incubated 2 hours at 37 DEG C, combination is brought it about.18,000 TF-1 cells/well is added, 37 DEG C, is incubated 65 hours.Then cell number is determined with CellTiter 96Aqueous One SolutionCell Proliferation Assay (Promega).In the analysis method, one tetrazolium salt composite MTS (3- (4,5- dimethylthiazole -2- bases) -5- (3- carboxymethoxyls phenyl) -2- (4- sulfophenyls) -2H- tetrazolium saltses) is reduced into first when there is electronics coupled agent (phenazine methosulfate) by cell biological

Product.The trap of solution is determined in 490nm with spectrophotometer (the maximum accurate microplate readers of E, Molecular Devices), so as to determine first

Concentration.ED is calculated after curve matching trap₅₀, and obtain with a quadruplex parameters NGFR-Fc concentration.A quadruplex parameters formula is obtained from the curve matching of trap and concentration, the ED50 of the present invention is calculated, is as a result 670-1000ng/ml (Figure 10).

(f) bioactivity of comparison Fas aglucons of the invention and the Fas aglucons of non-human system's expression

It is reported that in the presence of 10 μ g/ml certain cross-linking antibody when, Fas aglucons can be apoptosis-induced in human T cell leukemia Jurkat cell system.In 96 orifice plates, 0-1 μ g/ml Fas aglucons of the invention are given in 10000 Jurkat cell/holes, 37 DEG C, are incubated 65 hours.

Then cell number is determined with the Aqueous One Solution Cell ProliferationAssay (Promega) of CellTiter 96.In the analysis method, one tetrazolium salt composite MTS (3- (4,5- dimethylthiazole -2- bases) -5- (3- carboxymethoxyls phenyl) -2- (4- sulfophenyls) -2H- tetrazolium saltses) is reduced into first when there is electronics coupled agent (phenazine methosulfate) by cell biological

Product.The trap of solution is determined in 490nm with spectrophotometer (the maximum accurate microplate readers of E, Molecular Devices), so as to determine firstConcentration.

ED50 is calculated after curve matching trap, and obtains with a quadruplex parameters concentration of Fas aglucons.

Above-mentioned analysis is repeated with non-human system such as E.coli, yeast or expressing cho cell Fas aglucons, ED is found₅₀In the presence of significant difference.

Embodiment 13

(a) the ion vitro immunization response feature of the humanTNF-α of TNF-α of the invention and non-human system's expression compares

The TNF-α albumen of the present invention is with R＆D system humanTNF-αs

ELISA kit (Cat#DY210) is measured according to manufacturer's explanation.

The TNF-α of the present invention is standardized with ELISA measurement results, and R＆D system humanTNF-αs are used according to manufacturer's explanation

ELISA kit is diluted and determined.Above mentioned ELISA kit is with the humanTNF-α expressed in E.coli as standard.The humanTNF-α (Cat#210-TA) of R＆D systems E.coli expression and the humanTNF-α (Cat#87/650) of WHO E.coli expression are also measured.

From R＆D systems

TNF-α ELISA kit result obtains the concentration curve of the humanTNF-α of the TNF-α of the present invention at OD450nm, the humanTNF-α of R＆D systems E.coli expression and WHOE.coli expression, while also obtaining the standard curve (Figure 11) of the internal DuoSet TNF-αs of E.coli expression.

These results show by R＆D system humanTNF-αsThe concentration for the TNF-α of the invention that ELISA kit is determined is relatively low, the commercial reagents box is using the E.coli humanTNF-α's standards expressed and humanTNF-α's antibody of E.coli expression, and this is the TNF-α for assessing human body expression natural in laboratory sample and people patient sample.

These results show that the TNF-α of the present invention and humanTNF-α's molecule of non-human cell's expression have different immune response features.

(a) LT- α of the invention and the people LT- α of non-human system's expression ion vitro immunization response feature LT- α albumen more of the invention are measured using suitable absorptivity (ε) and based on the molecular weight that SDS-PAGE analyses are measured according to A280 trap methods.

The LT- α of the present invention are standardized with protein estimation result described above, are said according to manufacturer

Bright use R＆D systems people's TNF-β

ELISA kit (Cat#DY211) be diluted and

Determine.The albumen that the people LT- α (TNF-β) that above mentioned ELISA kit is expressed with E.coli are calibrated is as standard.

From R＆D systems

TNF-β ELISA kit result obtains the LT- α of present invention concentration for 360pg/ml or so, and this is that the people restructuring LT- α expressed using 0.22 OD450nm (Figure 12) according to E.coli standard curve is assessed and obtained.And the LT- α of the present invention are 1000pg/ml or so (Figure 12) in the actual concentrations of identical OD450nm values.

These results show by R＆D system people's TNF-βs

Relatively low more than 2 times of the concentration for the LT- α of the invention that ELISA kit is determined, the commercial reagents box is using the E.coli people's LT- α standards expressed and people's LT- Alpha antibodies of E.coli expression, and this is the LT- α for assessing human body expression natural in laboratory sample and people patient sample.

These results show that the LT- α of the present invention and people's LT- alpha molecules of non-human cell's expression have different immune response features.

(c) the ion vitro immunization response feature of TNFRI-Fc of the invention and the soluble human TNFRI molecules of non-human system's expression compares

The TNFRI-Fc albumen of the present invention is measured with a kind of suitable protein concentration appraisal procedure, for example, assess albumen as standard Lowry methods with human IgG.

The TNFRI-Fc of the present invention is standardized with above mentioned protein estimation result, according to manufacturer explanation R＆D system soluble human TNF RI

ELISA kit (Cat#DY225) is diluted and determined.The albumen that the soluble human TNFRI that above mentioned ELISA kit is expressed with E.coli cells is calibrated is as standard.

The TNFRI-Fc (as monomer) of the invention determined according to commercial ELISA Kit protein concentration will differ from the concentration determined according to standard protein analysis method, capture used in commercial ELISA Kit and/or immunologic detection method and/or detection antibody, are the soluble human TNFRI albumen expressed for non-human cell.It should be noted that the TNFRI-Fc of the present invention is expressed as homodimer.

The result shows that the TNFRI-Fc of the present invention and the soluble human TNFRI molecules of non-human cell's expression have different immune response features.

(d) the ion vitro immunization response feature of TNFRII-Fc of the invention and the soluble human TNFRII molecules of non-human system's expression compares

The TNFRII-Fc albumen of the present invention is measured with a kind of suitable protein concentration appraisal procedure, for example, assess albumen as standard Lowry methods with human IgG.

The TNFRII-Fc of the present invention is standardized with above mentioned protein estimation result, according to manufacturer explanation R＆D system soluble human TNF RII

ELISA kit (Cat#DY726) is diluted and determined.The albumen that the soluble human TNF RII that above mentioned ELISA kit is expressed with E.coli cells are calibrated is as standard.

The TNFRII-Fc (as monomer) of the invention determined according to commercial ELISA Kit protein concentration will differ from the concentration determined according to standard protein analysis method, capture used in commercial ELISA Kit and/or immunologic detection method and/or detection antibody, are the soluble human TNFRII albumen expressed for non-human cell.It should be noted that the TNFRII-Fc of the present invention is expressed as homodimer.

The result shows that the TNFRII-Fc of the present invention and the soluble human TNFRII molecules of non-human cell's expression have different immune response features.

(e) the ion vitro immunization response feature of OX40-Fc of the invention and the soluble human OX40 molecules of non-human system's expression compares

OX40-Fc albumen of the present invention is measured with a kind of suitable protein concentration appraisal procedure, for example, assessing albumen as standard Lowry methods with human IgG.

OX40-Fc of the present invention is diluted and determined with IBL-Hamburg soluble human OX40ELISA kits (Cat#BE59401) according to manufacturer's explanation with above mentioned protein estimation methodological standardization.The albumen that the soluble human OX40 that above mentioned ELISA kit is expressed with non-human cell's cell is calibrated is as standard.

The OX40-Fc of the present invention (as monomer) determined according to commercial ELISA Kit protein concentration will differ from the concentration determined according to standard protein analysis method, and capture used in commercial ELISA Kit and/or immunologic detection method and/or detection antibody, it is the soluble human OX40 albumen expressed for non-human cell.It should be noted that OX40-Fc of the present invention is expressed as homodimer.

The result shows that OX40-Fc of the present invention and the soluble human OX40 molecules of non-human cell's expression have different immune response features.

(f) BAFF of the invention and the people BAFF of non-human system's expression ion vitro immunization response feature compare

The BAFF albumen of the present invention is with a kind of standard protein analytical technology for example, Bradford analysis of protein (Bradford Anal Biochem 72：248-254,1976), or be measured using suitable absorptivity (ε) and based on the molecular weight that SDS-PAGE analyses are measured according to A280 trap methods.

The BAFF of the present invention is standardized with mark analysis of protein result, is diluted and is determined with commercial ELISA Kit according to manufacturer's explanation, for example R＆D systems people BAFF

ELISA kit (Cat#DBLYS0).The soluble human BAFF that above mentioned ELISA kit is expressed with E.coli cells carries out standard.

The BAFF of the invention determined according to commercial ELISA Kit protein concentration will differ from the concentration determined according to standard protein analysis method, capture used in commercial ELISA Kit and/or detection antibody, be for non-human cell express can people's BAFF albumen.

In a structure level, such a result will indicate that BAFF and people's BAFF molecules of non-human cell's expression have different immune response features.

(g) the ion vitro immunization response feature of NGFR-Fc of the invention and the NGFR-Fc molecules of non-human system's expression compares

The NGFR-Fc albumen of the present invention is measured with a kind of suitable protein analytical methods, for example, assess albumen as standard Lowry methods with human IgG.

The NGFR-Fc of the present invention, after being standardized with standard protein analysis result, quantitative immunological measure is carried out with reagent obtained by commercial source.For example, anti- NGFR-Fc-Fc ELISA is developed into by the use of people NGFR-Fc Mab (R＆D system Cat#MAB367) and is used as capture antibody, biotinylation people NGFR-Fc Pab (R＆D system Cat#BAF367) are used as standard protein as detection antibody, and the recombined human NGFR-Fc-Fc (R＆D system Cat#367-NR-050/CF) of Sf21 insect cell expressions.The NGFR-Fc of the present invention, is standardized with standard protein analysis result, is analyzed with above-mentioned reagent using known ELISA method.

It is the people's NGFR-Fc chimeric proteins expressed for non-human cell that the NGFR-Fc of the invention measured using the reagent for having source according to quantitative immunological determination method protein concentration, which will differ from capturing used in the concentration measured with standard protein analysis method, immunoassay and/detection antibody,.

In a structure level, such a result will indicate that the NGFR-Fc of the present invention and people's NGFR-Fc chimeric molecules of non-human cell's expression have different immune response features.

(h) the ion vitro immunization response feature of Fas aglucons of the invention and people's Fas aglucons of non-human system's expression compares

The present invention Fas ligand proteins with a kind of standard protein analytical technology for example, Bradford analysis of protein (supra of Bradford 1976), or be measured using suitable absorptivity (ε) and based on the molecular weight that SDS-PAGE analyses are measured according to A280 trap methods.

The Fas aglucons of the present invention are standardized with mark analysis of protein result, are diluted and are determined with commercial ELISA Kit according to manufacturer's explanation, for example R＆D systems people Fas aglucons

ELISA kit (Cat#DY126).People's Fas aglucons of above mentioned ELISA kit expressing cho cell are used as standard.

The protein concentration of the Fas aglucons of the invention determined according to commercial ELISA Kit will differ from the concentration determined according to standard protein analysis method, capture used in commercial ELISA Kit and/or detection antibody, are the people's Fas ligand proteins expressed for non-human cell.

In a structure level, such a result shows people's Fas ligand molecules of the Fas aglucons of the present invention and non-human cell's expression having different immune response features.

Embodiment 14

Purpose of the present invention molecule is further purified and with ESI-MS/MS peptide mapping fingerprinting

In addition to the purification process described in embodiment 2, the purifying of purpose of the present invention molecule is further carried out by RP-HPLC, uses commercially available post.Elution protein is monitored and is collected by 215 or 280nm absorbance, while in order to be corrected due to the delay that the conduit volume between flow cell and collecting terminal is brought.

The gel piece containing protein example from 1D or 2D gels digests in insulin solutions, as described in Example 3.Or, the solution insulin containing protein sample digests in ammonium bicarbonate buffers (10-25mM, pH 7.5-9).Solution incubated overnight at 37 DEG C.Then by add acetic acid until pH in the range of 4-5 with stopped reaction.Peptide sample is concentrated and with C18 Zip-Tips (Millipore, Bedford, MA) or as described in Example 3, the prefabricated microtrabeculae desalination containing Poros R2 chromatographic resins (Perspetive Biosystems, Framingham, MA).

Rinsed with 0.1% formic acid in protein sample (2-5 μ l) injection C18 pre-columns and under 30 μ l/min with concentration and desalination.After rinsing 3 minutes, pre-column is converted to the analytical column containing C18 RP silica (Atlantis, 75 μ m 100mm, Waters Corporation) in column.Peptide linear solvent gradient is eluted in post, step by step, from H₂O∶CH₃CN(95∶5；+ 0.1% formic acid) arrive H₂O∶CH₃CN (20: 80 ,+0.1% formic acid) is carried out more than 40 minutes with 200nl/min.LC is eluted in Micromass QTOF Ultima mass spectrographs (Micromass, Manchester, UK) to be analyzed through cation nanoflow electron sprays.

Series connection MS is carried out with Q-Tof mixing level Four/orthogonal acceleration TOF mass spectrographs (Micromass).QTOF is operated according to the data of drainage pattern (DDA).TOFMS detection scannings (m/z 400-2000,1.0s) are obtained, while three kinds of most multiple-charged ions (counting ＞ 15) are continuously analyzed in detection scanning through MS/MS.MS/MS spectrums collect 8s (m/z 50-2000).

LC/MS/MS data are searched for Mascot (Matrix Science, London, UK) and ProteinLynx Global Server (" PLGS ") (Micromass).Protein sample is contemplated to molecules of interest.

Embodiment 15

(a) immunogenicity in non-human animal

(i) with the animal immune of target protein

The group of non-human animal respectively, for example, the protein that mouse is expressed with the protein of the 1-100ug present invention and in non-human cell is through subcutaneously, intraperitoneal or intramuscular (IP) are immune.Animal received secondary immunity at immune latter month.Before immune, protein is emulsified in adjuvant, for example, complete Freud ' the s adjuvants for initial immunity and incomplete Freud ' the s adjuvants for secondary immunity.

(ii) to the identification of the antibody of target protein

In order to determine antibody response, the animal from each group takes blood from afterbody and collects serum.Protein specific antibody passes through of the invention protein determinations of the solid phase ELISA with 50ng/ holes.Different immune immunoglobulin isotypes by using mark show anti-igg 1, IgG2, IgG2b, IgG3, IgM, IgA, IgD detection TPPA.Or, Resistant reaction is measured by the protein of the invention being adsorbed onto on the film for dot blotting or western blot.The measure of the isotype of different immunoglobulins is according to detection described above.It is expected that the protein of the present invention causes the antibody response of the albumen different from being expressed in non-human cell.

(iii) T cell proliferation assay

By immune animal euthanasia and prepare splenocyte.The splenocyte of suitable quantity, for example, 5 × 10⁵Cell, come the animal of the Western Immuno for the present invention that uses by oneself, cultivated together a period of time with the protein of the invention of various concentrations, and identical quantity is cultivated come the splenocyte of animal of Western Immuno expressed in the non-human cell that uses by oneself and the albumen expressed in the non-human cell of various concentrations together.For T cell proliferation assay, spleen cell cultures 96 hours and last 16 hours with 1 μ Ci [³H] thymidine (6-7 μ Ci/umol) processing.Harvesting on filter and incorporation is determined with standard method [³H] thymidine.Compared with the protein expressed in non-human cell, it is contemplated that protein of the invention causes different breeder reactions.

(iv) IFN gamma are analyzed

In order to which IFN gamma are analyzed, and the protein of the present invention or spleen cell cultures supernatant cultivated together of protein of non-human cell's expression was harvested at 96 hours and IFN gamma products are detected by sandwich ELISA, for example, the anti-IFN gamma of R＆D systemsELISA kit (Cat#DIF50), according to manufacturer's technical specification.It is expected that it is different that the IFN gamma products for the cell that the IFN gamma products of the culture supernatant for the cell cultivated together come the albumen for the present invention that uses by oneself are cultivated together with the protein expressed in come the non-human cell that uses by oneself, which are compared,.

(b) external people's Analysis of Immunogenicity

(i) human T-cell's response analysis

Human dendritic cell and CD4⁺T cell is prepared from people's whole blood, such as Stickler et al.Toxicological Sciences 77：280-289, described in 2004.BMDC and CD4⁺T cell is placed in 96 orifice plates and co-cultured, and includes 2 × 10⁴BMDC and 2 × 10⁵ CD4⁺T cell.The protein and the protein expressed in non-human cell of the present invention is fragments of peptides through enzymic digestion, with the suitable enzyme identified by cleavage site forecasting software, for example, PeptideCutter (http://au.expasy.org/tools/peptidecutter).Obtained fragments of peptides is purified by suitable technology, for example, liquid chromatogram, and add in coculture to final concentration 5ug/ml.Culture culture 5 days, then by 0.5uCi³H thymidines are added in each culture.By cell harvesting into filter and by [³H] thymidine incorporation measure cell propagation.

It is expected that the protein peptide derived from the present invention can cause weaker breeder reaction, relative to the peptide for the protein expressed in non-human cell.

(ii) human antibody response analysis

Blood is taken to people's donor of the protein therapeutic through being expressed with non-human cell and blood plasma is prepared.Protein specific antibody is determined by the solid phase ELISA for the protein expressed in anti-50ng/well protein of the invention and non-human cell.Different Immunoglobulin Isotypes by using show anti-human igg 1, IgG2, IgG3, IgG4, IgM, IgA, IgD mark detection TPPA.

Or, by adsorbed when dot blotting or Western blot film on protein of the invention and the protein determination antibody response expressed in non-human cell.The measure of different Immunoglobulin Isotypes is according to detection described above.

It is expected that the immunoglobulin being present in the serum of the people for the protein for treatment expressed with non-human cell can combine the protein expressed in non-human cell, and combined weak with the protein of the present invention or do not combined.

Embodiment 16

The preparation of protein of the invention from recombination group construct

TNF-α in the PCR amplification codings present invention, (SEQID NOs are respectively for LT- α or FasL genome sequence：191,192,193), clone into suitable expression vector, such as pI RESbleo3, pCMV-SPORT6, pUMCV3, pORF, pORF9, pcDNA 3.1/GS, pCEP4, pIRESpuro3, pIRESpuro4, pcDNA3.1/Hygro (+), pcDNA3.1/Hygro (-), pEF6/V5-His.These recombination structures prepare the people's cell conversion for being used to be stated in embodiment 1 (c).Recombinant DNA structure GM-CSF, IL-3, IL-4 and IL-5 generation and purifying are carried out as described in Example 2 in the present invention.

Embodiment 17

Compare in vivo it is of the invention in OX40-Fc and expressing cho cell suppression of the OX40-Fc to colitis

(the Taylor Journalof Leukocyte Biology 72 in the Murine models that TNB (TNBS) induces colitis：522-525,2002) evaluate the present invention in OX40-Fc effectiveness and expressing cho cell OX40-Fc suppress vivo immunization reaction effect.TNBS containing 2mg in the final concentration of 75 μ l cumulative volumes of TNBS is diluted in 50% ethanol solution.Mouse induces colitis by slightly benumbs with the μ l TNBS solution of plastics catheter intrarectal injection 75.Control mice injects 50% ethanol water.At the 4-6 days, to TNBS induce colitis mice and Ethanol Treatment control all inject proper amount of, such as 100 μ g, or in the present invention OX40-Fc or expressing cho cell OX40-Fc.Put to death mouse within 7th day, be colored because CD4+T cells penetrate into lamina propria colon.The CD4+T cell quantities that the mouse of OX40-Fc processing in the present invention penetrates into lamina propria are significantly reduced.

Transcriptional levels of the TNF-α mRNA in above-mentioned mouse Colon tissue is determined with real time RT PCR (RT-PCR).Total serum IgE is illustrated to extract with RNeasy Mini Kit (Qiagen, Australia) according to manufacturer, RNA concentration metric measurements.After extraction, sample is stored in -80 DEG C until using.Real-time RT-PCR is carried out with TaqMan One-Step RT-PCR Master MixReagents Kit (PE Applied Biosystems).Adding 1 × Master Mix, 1 × Multi Scribe and Rnase Inhibitor Mix, primer before 300nM TNF-αs, 300nMTNF- α anti-primers, synthesized in 100nM TNF-α probes, 25 μ l of 1 × 18srRNA Primer and Probe Mix and 100ng total serum IgEs reaction system.RT-PCR reactions are carried out in the Sequence Detectors of ABI Prism 7700 (PE Applied Biosystems).Thermal cycle conditions include 48 DEG C of reverse transcriptions 30 minutes, and 95 DEG C are denatured 10 minutes, and 95 DEG C 40 circulate for 15 seconds and 60 DEG C for 1 minute.The data of this reaction are collected and analyzed with suitable computer software.For expressing cho cell OX40-Fc, OX40-Fc is expressed TNF-α mRNA and acted on by reduction to a greater extent in the present invention.

Embodiment 18

(a) DNA structure of the sialyltransferase of express alpha 2,6 is prepared

PCR expands the DNA sequence dna of the sialyltransferases (a2,6ST) of α 2,6 from EST cDNA libraries (clone 3090115, Invitrogen), with preceding primer (SEQ ID NO：194) with anti-primer (SEQ ID NO：195) Not 1 and BamH1 restriction site are separately constituted.After amplification, with Not 1 and BamH1 enzymic digestion sequences, clone into restriction site corresponding expression vector pIRESbleo3 to prepare carrier pIRESbleo3- α 2,6ST.With Not 1 and BamHl enzymic digestion pIRESbleo3- α 2, it is estimated 1315bp that 6ST, which obtains segment,.

Or, PCR amplification a2,6ST DNA sequence dna, with preceding primer (SEQ ID NO from EST cDNA libraries (clone 3090115, Invitrogen)：196) with anti-primer (SEQ ID NO：197) BamH1 and Not 1 restriction site are separately constituted.After amplification, with BamH1 and the enzymic digestion sequences of Not 1, clone into restriction site corresponding expression vector pIRESpuro 3 to prepare carrier pIRESpuro3-a2,6ST.With Not 1 and BamH1 enzymic digestions pIRESpuro3-a2,6

It is estimated 1310bp that ST, which obtains segment,.

(b) 2,6 sialyltransferase expression vectors are largely prepared

750 are inoculated with the sterile LB medium that 750ml contains ampicillin (120 μ g/ml)

PIRESbleo3-a2,6ST or the pIRESpuro3-a2 of μ l overnight incubations, 6ST.Culture is shaken at 37 DEG C 16 hours.Plasmid is prepared with Qiagen Endofree Plasmid Mega Kit (Qiagen catalog number (Cat.No.)s 12381).

(c) highly sialylated TNFRI-Fc generation and purifying

Contain a2 in 1: 3 ratio mixing, Plasmid pIRES bleo3-a2,6ST or the pIRESpuro3-a2 of 6ST genes, 6ST and the Plasmid pIRES bleo3-TNFRI-Fc containing TNFRI-Fc genes.By mixture transfectional cell, obtained supernatant purification process and example 2 (c) unanimously, except the resulting segment containing TNFRI-Fc need not be concentrated further.

Research finds that the sialylated TNFRI-Fc of the height of purifying approximate molecular weight scope is 45-85kDa, and argentation identification purity is at least 99%.

(d) highly sialylated TNFRI-Fc characteristics

Polyacrylamide dielectrophoresis is carried out to highly sialylated TNFRI-Fc according to embodiment 3 (c).Table 39 shows the apparent molecular weight of TNFRI-Fc isoforms, pI values and relative intensity.Data are listed according to the intensity weighted center in the selected gel area comprising point, so can most reflect the pI and molecular weight of protein.

Table 39

The molecular weight and DI values of highly sialylated TNFRI-Fc isoforms

Spot is numbered	Isoelectric point (pI)	Molecular weight (kDa)	Relative intensity (%) (standardized value)
				2	6.19	62.65	0.28
3	6.28	61.81	1.10
				4	6.37	61.20	1.71
5	6.46	59.96	3.32
				6	6.57	58.95	6.87
7	6.69	57.91	12.04
				8	6.81	57.46	10.08
9	6.94	56.68	10.27
				10	7.04	56.60	3.84
11	7.10	56.27	4.08
				12	7.18	55.75	6.59
13	7.30	55.52	5.64
				14	7.46	53.95	7.46
15	7.60	52.81	1.59
				16	7.68	55.42	1.30
17	7.77	52.39	0.83
				18	7.86	52.48	0.43
19	7.96	54.38	0.61

Embodiment 19

(a) the topical ceram formula containing protein in the present invention

By the method described in example 2, the segment of target protein of the present invention is collected with sleeve pipe in a syringe.Proper amount of protein solution is penetrated into Falcon pipes, low protein binding pipe is transferred into, is mixed with proper amount of topical ceram, such as Cetaphil moisturizings creme (Galderma), whole destination protein concentration is 10-1000 μ g/ml.Slowly stirring is distributed creme in falcon pipes.Divide and mixture is transferred to syringe with blending ingredients from Falcon pipes several times.Creme is transferred to 60ml syringes, and proper amount of creme is taken out from syringe to be used to analyze.Remaining uniform homogeneous blend is then transferred into syringe.In order to avoid creme is directly contacted with rubber end socket, certain space is left before creme is transferred to.

(b) the topical ceram formula of the TNFRII-Fc containing the present invention

By the method described in example 2 (d) or 2 (h), the TNFRII-Fc of present invention segment is collected with sleeve pipe in a 20ml syringe.14.0mL 0.22 μm of 1mg/mL protein solutions are penetrated into 50mLFalcon pipes；0.5mL is transferred into low protein binding pipe as analysis sample.43mL Cetaphil moisturizings cremes (Galderma) are transferred into 60mL syringes with sleeve pipe, whole TNFRII-Fc concentration is 250 μ g/ml.Creme is slowly stirred and is distributed into falcon pipes.Divide and mixture is transferred to syringe with blending ingredients from Falcon pipes several times.By 0.5mL, this mixture is transferred in 1mL syringes for analyzing (sample 1).10mL syringes are then transferred into per 8mL uniform homogeneous blends.In order to avoid creme is directly contacted with rubber end socket, certain space is left before creme is transferred to.The creme of half is transferred into 60mL syringes.1.0g Distavals are added in Falcon pipes, are mixed with remaining creme.Repeat creme being transferred to all components of the process of syringe up to being thoroughly mixed creme from pipe.By 0.5mL, this mixture is transferred in 1mL syringes for analyzing (sample 2).As described above, being then transferred into 10mL syringes per 8mL uniform homogeneous blends.

(c) TNFRI-Fc of the present invention topical ceram formula is contained

By the method described in example 2 (c), TNFRI-Fc of the present invention segment is collected, is filtered by the method illustrated in embodiment 19 (b) and mixing Cetaphil moisturizings creme to whole TNFRI-Fc concentration is 250 μ g/ml.By what is illustrated in embodiment 19 (b), the uniform homogeneous blend containing the μ g/ml TNFRI-Fc of this research 250 is separated, or is not added with Distaval or configuration 20mg/mL Distavals, 10mL syringes are transferred to per 8mL.

Embodiment 20

(a) local application contains the bio distribution of TNFRII-Fc after TNFRII-Fc pharmaceutical composition

TNFRII-Fc in the present invention is carried out with chloramine T method¹²⁵I- marks.In brief, 20 μ l 3.5mg/ml TNFRII-Fc solution is added in 20 μ l 0.5M pH7.4 phosphate buffer.Sequentially add 2 μ l Na¹²⁵- I (0.2mCi), the mixing of 10 μ l toluene-sodium-sulfonchloramides (10mg/ml).10 μ l sodium pyrosulfites (10mg/ml) terminating reactions are added after 30 seconds.Remove what is dissociated in reaction system with the Sephadex G10 column chromatographies for adding 0.1M pH7.4 phosphate buffers¹²⁵I.Wash out material and 4 DEG C are stored in before for biodistribution research.¹²⁵The TNFRII-Fc of I- marks is mixed with the dissolving of 0.2mg/ml concentration with 1: 10 ratio with following creme, entitled Alpha Keri moisturizing lotions (Mentholatum), DermaVeen moisturizing lotions (DermaTech laboratories), QV skin lotions (Lision Hong), Cetaphil moisturizing lotions (Galderma laboratories, L.P.).Topical remedy's composition is placed in the skin area of anesthesia Balb/C mouse 2 × 1cm shavings.Local prescription be placed on mouse after upper 180 minutes mouse sentence euthanasia, remove all organs and counted with X-ray counter X.Figure 13 shows that permeate through epidermis gives the local prescription of the present invention¹²⁵After the TNFRII-Fc of I- marks,¹²⁵Distributions of the TNFRII-Fc of I- marks in mouse, wherein A is in Alpha Keri moisturizing lotions (Mentholatum)¹²⁵The TNFRII-Fc of I- marks local prescription；B is in DermaVeen moisturizing lotions (DermaTech laboratories)¹²⁵The TNFRII-Fc of I- marks local prescription；C is in QV skin lotions¹²⁵The TNFRII-Fc of I- marks local prescription；D is in Cetaphil moisturizing lotions (Galderma laboratories, L.P.)¹²⁵The TNFRII-Fc of I- marks local prescription.

As seen in figure 13,¹²⁵I-TNFRII-Fc is quickly present in the stripped region of skin, muscle and skin.

It will be appreciated by those skilled in the art that invention described here can be modified to the difference being particularly described with those by change XOR.It the present invention should be understood to include all such change XOR modifications.The present invention also includes respectively or fully all steps that are being related in specification or pointing out, feature, and composition and compound and any step or feature described in two or more are optionally combined.

Bibliography

Ackland et al.Chromatogr 540：187-198,1991

Aloj et al.J Biol Chem 247：1146-1151,1971

Altschul et al.Nucl Acids Res 25：389,1997

Antibodies：A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY, (1988)

Aronsson et al.FEBS Lett 411：359-364,1997

Atherton and Shephard Synthetic Vaccines 9：Blackwell ScientificPublications

Ausubel et al.In：Current Protocols in Molecular Biology John Wiley& Sons Inc.1994-1998)

Baneyx Current Opinion in Biotechnology, 10：411-421,1999

Bernstein Methods Mol Biol 237：195-204,2004

Bird Science 242：423,1988

Blenis and Resh Curr Opin Cell Biol 5(6)：984-9,1993

Bonner and Laskey Eur J Biochem 46：83,1974

Bradford Anal Biochem 72：248-254,1976

Caprioli et al.Biochem Biophys Res Commun 146：291-299,1987

Carr et al.Anal Biochem 175：492-499,1988

Carr et al.Anal Chem 63：2802-2824,1991

Carr et al.J Biol Chem 264(35)：21286-21295,1989

Clackson et al.Nature  352：624-628,1991

Clarke Curr Opin Cell Biol 5：977 983,1993

Datta et al.J Biol Chem 270：1497-1500,1995

Edman Mol Biol Biochem Biophys 8：211-55,1970

Erickson et al.Science 249：527-533,1990

Evereklioglu Expert Opin Pharmacother 5(2)：317-28,2004

Farruggia et al.Int J Biol Macromol 20：43-51,1997

Figeys and Aeber sold, Electrophoresis 19：885-892,1998

Frank et al.Characterization of proteins, Humana Press, Clifton, NJ, 1988

Fritz et al.PNAS 95：12283-12288,1998

Fukuhara et al.J Biol Chem 260：10487-10494,1985

Gelb et al.Curr Opin Chem Biol 2(1)：40-8,1998

Gramer et al.Biotechnology 13(7)：692-8,1995

Guedez et al.Am J Pathol 162：1431-1439,2003

Harrison and Packer Methods Mol Biol 125：211-216,2000

Hearn et al.Methods in Enzymol 104：190-212,1984

Herscovics et al.FASEB J 7：540-550,1993

Herzberg et al.Infrared and Raman Spectra of Polyatomic Molecules, Van Nostrand Reinhold, New York, NY, 1945

Hodgson Bio/Technology 9：19-21,1991

Holzwarth et al.J Am Chem.Soc 178：350,1965

Honroe et al.Biochem J 258：99-204,1989

Huston et al.Proc Natl Acad Sci USA 85：5879,1988

J Biochem 336：647-658,1998

J Biochem 363：619-631,2002

James and Bottomley Arch Biochem Biophy 356：296-300,1998

Jones et al.Nature 321：522-525,1986

Kennet et al.Monoclonal Antibodies, Hybridomas：A New Dimensionin Biological Analyses, Plenum Press, New York, 1980

Kivirikko et al.FASEB Journal 3：1609-1617,1989

Kohler et al.Nature 256：495,1975

Kortt et al.Protein Engineering 10：423,1997

Krimm and Bandekar Adv Protein Chem 38：181-364,1986

Kronman Gene 121：295-304,1992

Kurochkin et al.J Mol Biol 248：414-430,1995

Kwon and Yu Biophim Biophys Acta 1335：265-272,1997

Larrick et al.Bio/Technology 7：934,1989

Larsen et al.J Biol Chem 265：7055-7061,1990

Li et al.Biochemistry 34：5762-5772,1995

Lipozecic et al.Acta Dermatovenerol Croat 12(1)：35-41,2004

Liu et al.J Immunol 158：604-613,1994

Lucka et al.Glycobiology 15(1)：87-100,2005

Mahe et al.Ann Dermatol Venereol 129(12)：1374-9,2002Marks et al.J Mol Biol 222：581-597,1991

Marmur and Doty J Mol Biol 5：109,1962

Martin et al.Nature Medicine 11(2)：228-232,2005

Mas et al.Glycobiology 8(6)：605-13,1998

McGettrick et al.Methods Mol Biol 244：151-72004

Mire-Sluis et al.J Immunol Methods 289(1-2)：1-16,2004

Moore J Biol Chem 278(27)：24243-24246,2003

Morrison et al.Proc Natl Acad Sci USA 81：6851-6855,1984

Nguyen et al.J Chromatogr A.705：21-45,1995

Nickoloff et al.J Clin Invest.113：1664-1675,2004

Packer et al.Glycoconj J 5(8)：737-47,1998

Phillies Anal Chem 62：1049A-1057A, 1990

Pikal et al.Pharm Res 8：427-436,1991

Presta, Curr Op Struct Biol 2：593-596,1992

Rando Biochim Biophys Acta 1300(1)：5-16,1996

Reichmann et al.Nature 332：323-329,1988

Sambrook et al.Molecular Cloning-A Laboratory Manual, Cold SpringHarbour, New York, USA, 1990

Schmid et al.Protein structure, a practical approach, CreightonEd., IRI Press, Oxford, England, 1989

Shepherd et al.Arterioscler Thromb Vasc Biol 24：898-904,2004

Stickler et al.Toxicological Sciences 77：280-289,2004

Taylor Journal of Leukocyte Biology 72：522-525,2002

Teo et al.Microbes Infect 4(11)：1193-202,2002

Triguero et al.J of Neurochemistry 54：1882-1888 1990

Ward et al.Nature 334：544,1989

Wells Methods Enzymol 202：2699-2705,1991

Wilkinson Annu Rev Nutr 15：161-89,1995

Winter and Harris TIPS 14：139,1993

Yoshioka et al.Pharm Res 10：103-108,1993

Sequence table

<110>Apollo Life Sciences Ltd.

PRIEST, John D (only to the U.S.)

WATTS, Alan D (only to the U.S.)

WHITTAKER, Jason S (only to the U.S.)

DOMAGALA, Teresa A (only to the U.S.)

PILKINGTON, Glenn R (only to the U.S.)

BOEHM, Ingrid (only to the U.S.)

LEE, Carol M Y (only to the U.S.)

LIM, Mei Ann (only to the U.S.)

THOMAS, Nikolien S (only to the U.S.)

<120>Molecule and its chimeric molecule

<130>12715050/EJH/HPM

<150>60/670,715

<151>2005-04-12

<150>60/647,758

<151>2005-01-28

<150>60/662,465

<151>2005-03-15

<150>60/653,284

<151>2005-02-14

<150>60/665,556

<151>2005-03-24

<150>60/648,158

<151>2005-01-28

<150>60/648,190

<151>2005-01-28

<150>60/676,046

<151>2005-04-29

<150>60/677,088

<151>2005-05-02

<150>2005906366

<151>2005-11-16

<150>2005906750

<151>2005-12-01

<160>199

<170>PatentIn version 3.1

<210>1

<211>714

<212>DNA

<213>People

<400>1

aaggtggaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc     60

ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac    120

accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa    180

gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca    240

aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg    300

caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca    360

gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac    420

accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc    480

aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac    540

aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag    600

ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat    660

gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaa          714

<210>2

<211>238

<212>PRT

<213>People

<400>2

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

1               5                   10                  15

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

            20                  25                  30

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

        35                  40                  45

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

    50                  55                  60

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

65                  70                  75                  80

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

                85                  90                  95

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

            100                 105                 110

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

        115                 120                 125

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

    130                 135                 140

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

145                 150                 155                 160

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

                165                 170                 175

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

            180                 185                 190

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

        195                 200                 205

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

    210                 215                 220

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

225                 230                 235

<210>3

<211>714

<212>DNA

<213>People

<400>3

aaggtggaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc     60

ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac    120

accctcatga tetcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa    180

gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca    240

aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg    300

caccaggact ggctgaatgg caaggagtac aagtgcaggg tctccaacaa agccctccca    360

gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac    420

accctgcccc catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc    480

aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac    540

aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag    600

ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat    660

gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaa          714

<210>4

<211>238

<212>PRT

<213>People

<400>4

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

1               5                   10                  15

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

            20                  25                  30

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

        35                  40                  45

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

    50                  55                  60

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

65                  70                  75                  80

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

                85                  90                  95

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

            100                 105                 110

Arg Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

        115                 120                 125

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

    130                 135                 140

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

145                 150                 155                 160

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

                165                 170                 175

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

            180                 185                 190

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

        195                 200                 205

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

    210                 215                 220

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

225                 230                 235

<210>5

<211>702

<212>DNA

<213>People

<400>5

aaggtggaca agacagttga gcgcaaatgt tgtgtcgagt gcccaccgtg cccagcacca     60

cctgtggcag gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc    120

tcccggaccc ctgaggtcac gtgcgtggtg gtggacgtga gccacgaaga ccccgaggtc    180

cagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccacgggag    240

gagcagttca acagcacgtt ccgtgtggtc agcgtcctca ccgttgtgca ccaggactgg    300

ctgaacggca aggagtacaa gtgcaaggtc tccaacaaag gcctcccagc ccccatcgag    360

aaaaccatct ccaaaaccaa agggcagccc cgagaaccac aggtgtacac cctgccccca    420

tcccgggagg agatgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctac    480

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc    540

acacctccca tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac    600

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac    660

aaccactaca cgcagaagag cctctccctg tctccgggta aa                       702

<210>6

<211>234

<212>PRT

<213>People

<400>6

Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro

1               5                   10                  15

Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro

            20                  25                  30

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

        35                  40                  45

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp

    50                  55                  60

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

65                  70                  75                  80

Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val

                85                  90                  95

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

            100                 105                 110

Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

        115                 120                 125

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

    130                 135                 140

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

145                 150                 155                 160

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

                165                 170                 175

Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe

            180                 185                 190

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

        195                 200                 205

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

    210                 215                 220

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

225                 230

<210>7

<211>765

<212>DNA

<213>People

<400>7

aaggtggaca agagagttga gctcaaaacc ccacttggtg acacacctcc cccatgccca     60

cggtgcccag agcccaaatc ttgtgacaca cctcccccgt gcccaaggtg cccagcacct    120

gaactcctgg gaggaccgtc agtcttcctc ttccccccaa aacccaagga tacccttatg    180

atttcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccacga agaccccgag    240

gtccagttca agtggtacgt ggacggcgtg gaggtgcata atgccaagac aaagctgcgg    300

gaggagcagt acaacagcac gttccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac    360

tggctgaacg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc    420

gagaaaacca tctccaaagc caaaggacag ccccgagaac cacaggtgta caccctgccc    480

ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc    540

taccccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaac    600

accacgcctc ccatgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg    660

gacaagagca ggtggcagca ggggaacatc ttctcatgct ccgtgatgca tgaggctctg    720

cacaaccgct acacgcagaa gagcctctcc ctgtctccgg gtaaa                    765

<210>8

<211>255

<212>PRT

<213>People

<400>8

Lys Val Asp Lys Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Pro

1               5                   10                  15

Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro

            20                  25                  30

Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

        35                  40                  45

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

    50                  55                  60

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

65                  70                  75                  80

Val Gln Phe Lys Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

                85                  90                  95

Thr Lys Leu Arg Glu Glu Gln Tyr Asn Ser Thr Phe Arg Val Val Ser

            100                 105                 110

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

        115                 120                 125

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

    130                 135                 140

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

145                 150                 155                 160

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

                165                 170                 175

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

            180                 185                 190

Gly Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser

        195                 200                 205

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

    210                 215                 220

Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu

225                 230                 235                 240

His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

                245                 250                 255

<210>9

<211>705

<212>DNA

<213>People

<400>9

aaggtggaca agagagttga gtccaaatat ggtcccccat  gcccatcatg cccagcacct    60

gagttcctgg ggggaccatc agtcttcctg ttccccccaa aacccaagga cactctcatg    120

atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccagga agaccccgag    180

gtccagttca actggtacgt ggatggcgtg gaggtgcata atgccaagac aaagccgcgg    240

gaggagcagt tcaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac    300

tggctgaacg gcaaggagta caagtgcaag gtctccaaca aaggcctccc gtcctccatc    360

gagaaaacca tctccaaagc caaagggcag ccccgagagc cacaggtgta caccctgccc    420

ccatcccagg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc    480

taccccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag    540

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcag gctaaccgtg    600

gacaagagca ggtggcagga ggggaatgtc ttctcatgct ccgtgatgca tgaggctctg    660

cacaaccact acacacagaa gagcctctcc ctgtctctgg gtaaa                    705

<210>10

<211>235

<212>PRT

<213>People

<400>10

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser

1               5                   10                  15

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

            20                  25                  30

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr PTo Glu Val Thr

        35                  40                  45

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

    50                  55                  60

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

65                  70                  75                  80

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

                85                  90                  95

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

            100                 105                 110

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

        115                 120                 125

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

    130                 135                 140

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

145                 150                 155                 160

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

                165                 170                 175

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

            180                 185                 190

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

        195                 200                 205

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

    210                 215                  220

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

225                 230                 235

<210>11

<211>819

<212>DNA

<213>People

<400>11

aagtccgtga catgccacgt gaagcactac acgaatccca gccaggatgt gactgtgccc     60

tgcccagttc cctcaactcc acctacccca tctccctcaa ctccacctac cccatctccc    120

tcatgctgcc acccccgact gtcactgcac cgaccggccc tcgaggacct gctcttaggt    180

tcagaagcga acctcacgtg cacactgacc ggcctgagag atgcctcagg tgtcaccttc    240

acctggacgc cctcaagtgg gaagagcgct gttcaaggac cacctgaccg tgacctctgt    300

ggctgctaca gcgtgtccag tgtcctgtcg ggctgtgccg agccatggaa ccatgggaag    360

accttcactt gcactgctgc ctaccccgag tccaagaccc cgctaaccgc caccctctca    420

aaatccggaa acacattccg gcccgaggtc cacctgctgc cgccgccgtc ggaggagctg    480

gccctgaacg agctggtgac gctgacgtgc ctggcacgtg gcttcagccc caaggatgtg    540

ctggttcgct ggctgcaggg gtcacaggag ctgccccgcg agaagtacct gacttgggca    600

tcccggcagg agcccagcca gggcaccacc accttcgctg tgaccagcat actgcgcgtg    660

gcagccgagg actggaagaa gggggacacc ttctcctgca tggtgggcca cgaggccctg    720

ccgctggcct tcacacagaa gaccatcgac cgcttggcgg gtaaacccac ccatgtcaat    780

gtgtctgttg tcatggcgga ggtggacggc acctgctac                           819

<210>12

<211>273

<212>PRT

<213>People

<400>12

Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp

1               5                   10                  15

Val Thr Val Pro Cys Pro Val Pro Ser Thr Pro Pro Thr Pro Ser Pro

            20                  25                  30

Ser Thr Pro Pro Thr Pro Ser Pro Ser Cys Cys His Pro Arg Leu Ser

        35                  40                  45

Leu His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser Glu Ala Asn

    50                  55                  60

Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly Val Thr Phe

65                  70                  75                  80

Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly Pro Pro Asp

                85                  90                  95

Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu Ser Gly Cys

            100                 105                 110

Ala Glu Pro Trp Asn His Gly Lys Thr Phe Thr Cys Thr Ala Ala Tyr

        115                 120                 125

Pro Glu Ser Lys Thr Pro Leu Thr Ala Thr Leu Ser Lys Ser Gly Asn

    130                 135                 140

Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser Glu Glu Leu

145                 150                 155                 160

Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg Gly Phe Ser

                165                 170                 175

Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln Glu Leu Pro

            180                 185                 190

Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro Ser Gln Gly

        195                 200                 205

Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala Ala Glu Asp

    210                 215                 220

Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly His Glu Ala Leu

225                 230                 235                 240

Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala Gly Lys Pro

                245                 250                 255

Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys

            260                 265                 270

Tyr

<210>13

<211>780

<212>DNA

<213>People

<400>13

aagtccgtga catgccacgt gaagcactac acgaatccca gccaggatgt gactgtgccc     60

tgcccagttc ccccacctcc cccatgctgc cacccccgac tgtcgctgca ccgaccggcc    120

ctcgaggacc tgctcttagg ttcagaagcg aacctcacgt gcacactgac cggcctgaga    180

gatgcctctg gtgccacctt cacctggacg ccctcaagtg ggaagagcgc tgttcaagga    240

ccacctgagc gtgacctctg tggctgctac agcgtgtcca gtgtcctgcc tggctgtgcc    300

cagccatgga accatgggga gaccttcacc tgcactgctg cccaccccga gttgaagacc    360

ccactaaccg ccaacatcac aaaatccgga aacacattcc ggcccgaggt ccacctgctg    420

ccgccgccgt cggaggagct ggccctgaac gagctggtga cgctgacgtg cctggcacgt    480

ggcttcagcc ccaaggatgt gctggttcgc tggctgcagg ggtcacagga gctgccccgc    540

gagaagtacc tgacttgggc atcccggcag gagcccagcc agggcaccac caccttcgct    600

gtgaccagca tactgcgcgt ggcagccgag gactggaaga agggggacac cttctcctgc    660

atggtgggcc acgaggccct gccgctggcc ttcacacaga agaccatcga ccgcttggcg    720

ggtaaaccca cccatgtcaa tgtgtctgtt gtcatggcgg aggtggacgg cacctgctac    780

<210>14

<211>260

<212>PRT

<213>People

<400>14

Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp

1               5                   10                  15

Val Thr Val Pro Cys Pro Val Pro Pro Pro Pro Pro Cys Cys His Pro

            20                  25                  30

Arg Leu Ser Leu His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser

        35                  40                  45

Glu Ala Asn Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly

    50                  55                  60

Ala Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly

65                  70                  75                  80

Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu

                85                  90                  95

Pro Gly Cys Ala Gln Pro Trp Asn His Gly Glu Thr Phe Thr Cys Thr

            100                 105                 110

Ala Ala His Pro Glu Leu Lys Thr Pro Leu Thr Ala Asn Ile Thr Lys

        115                 120                 125

Ser Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser

    130                 135                 140

Glu Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg

145                 150                 155                 160

Gly Phe Ser Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln

                165                 170                 175

Glu Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro

            180                 185                 190

Ser Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala

        195                 200                 205

Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val Gly His

    210                 215                 220

Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala

225                 230                 235                 240

Gly Lys Pro Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp

                245                 250                 255

Gly Thr Cys Tyr

            260

<210>15

<211>1545

<212>DNA

<213>People

<400>15

aagagtcgag tcaccatatc agtagacacg tccaagaagc agctctccct gaagttgagc    60

tctgtgaacg ccgcggacac ggctgtgtat tactgtgcga gagttattac tagggcgagt     120

cctggcacag acgggaggta cggtatggac gtctggggcc aagggaccac ggtcaccgtc     180

tcctcaggga gtgcatccgc cccaaccctt ttccccctcg tctcctgtga gaattccccg     240

tcggatacga gcagcgtggc cgttggctgc ctcgcacagg acttccttcc cgactccatc     300

actttctcct ggaaatacaa gaacaactct gacatcagca gcacccgggg cttcccatca     360

gtcctgagag ggggcaagta cgcagccacc tcacaggtgc tgctgccttc caaggacgtc     420

atgcagggca cagacgaaca cgtggtgtgc aaagtccagc accccaacgg caacaaagaa     480

aagaacgtgc ctcttccagt gattgccgag ctgcctccca aagtgagcgt cttcgtccca     540

ccccgcgacg gcttcttcgg caacccccgc aagtccaagc tcatctgcca ggccacgggt     600

ttcagtcccc ggcagattca ggtgtcctgg ctgcgcgagg ggaagcaggt ggggtctggc     660

gtcaccacgg accaggtgca ggctgaggcc aaagagtctg ggcccacgac ctacaaggtg     720

accagcacac tgaccatcaa agagagcgac tggctcagcc agagcatgtt cacctgccgc     780

gtggatcaca ggggcctgac cttccagcag aatgcgtcct ccatgtgtgt ccccgatcaa     840

gacacagcca tccgggtctt cgccatcccc ccatcctttg ccagcatctt cctcaccaag     900

tccaccaagt tgacctgcct ggtcacagac ctgaccacct atgacagcgt gaccatctcc     960

tggacccgcc agaatggcga agctgtgaaa acccacacca acatctccga gagccacccc    1020

aatgccactt tcagcgccgt gggtgaggcc agcatctgcg aggatgactg gaattccggg    1080

gagaggttca cgtgcaccgt gacccacaca gacctgccct cgccactgaa gcagaccatc    1140

tcccggccca agggggtggc cctgcacagg cccgatgtct acttgctgcc accagcccgg    1200

gagcagctga acctgcggga gtcggccacc atcacgtgcc tggtgacggg cttctctccc    1260

gcggacgtct tcgtgcagtg gatgcagagg gggcagccct tgtccccgga gaagtatgtg    1320

accagcgccc caatgcctga gccccaggcc ccaggccggt acttcgccca cagcatcctg    1380

accgtgtccg aagaggaatg gaacacgggg gagacctaca cctgcgtggt ggcccatgag    1440

gccctgccca acagggtcac cgagaggacc gtggacaagt ccaccggtaa acccaccctg    1500

tacaacgtgt ccctggtcat gtccgacaca gctggcacct gctac                    1545

<210>16

<211>515

<212>PRT

<213>People

<400>16

Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Lys Gln Leu Ser

1               5                   10                  15

Leu Lys Leu Ser Ser Val Asn Ala Ala Asp Thr Ala Val Tyr Tyr Cys

            20                  25                  30

Ala Arg Val Ile Thr Arg Ala Ser Pro Gly Thr Asp Gly Arg Tyr Gly

        35                  40                  45

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser

    50                  55                  60

Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser Cys Glu Asn Ser Pro

65                  70                  75                  80

Ser Asp Thr Ser Ser Val Ala Val Gly Cys Leu Ala Gln Asp Phe Leu

                85                  90                  95

Pro Asp Ser Ile Thr Phe Ser Trp Lys Tyr Lys Asn Asn Ser Asp Ile

            100                 105                 110

Ser Ser Thr Arg Gly Phe Pro Ser Val Leu Arg Gly Gly Lys Tyr Ala

        115                 120                 125

Ala Thr Ser Gln Val Leu Leu Pro Ser Lys Asp Val Met Gln Gly Thr

    130                 135                 140

Asp Glu His Val Val Cys Lys Val Gln His Pro Asn Gly Asn Lys Glu

145                 150                 155                 160

Lys Asn Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys Val Ser

                165                 170                 175

Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser

            180                 185                 190

Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln Ile Gln Val

        195                 200                 205

Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser Gly Val Thr Thr Asp

    210                 215                 220

Gln Val Gln Ala Glu Ala Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val

225                 230                 235                 240

Thr Ser Thr Leu Thr Ile Lys Glu Ser Asp Trp Leu Ser Gln Ser Met

                245                 250                 255

Phe Thr Cys Arg Val Asp His Arg Gly Leu Thr Phe Gln Gln Asn Ala

            260                 265                 270

Ser Ser Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg Val Phe Ala

        275                 280                 285

Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr Lys Leu

    290                 295                 300

Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr Ile Ser

305                 310                 315                 320

Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn Ile Ser

                325                 330                 335

Glu Ser His Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala Ser Ile

            340                 345                 350

Cys Glu Asp Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr Val Thr

        355                 360                 365

His Thr Asp Leu Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg Pro Lys

    370                 375                 380

Gly Val Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg

385                 390                 395                 400

Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu Val Thr

                405                 410                 415

Gly Phe Ser Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg Gly Gln

            420                 425                 430

Pro Leu Ser Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro

        435                 440                 445

Gln Ala Pro Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val Ser Glu

    450                 455                 460

Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val Val Ala His Glu

465                 470                 475                 480

Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly

                485                 490                 495

Lys Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr Ala Gly

            500                 505                 510

Thr Cys Tyr

        515

<210>17

<211>1026

<212>DNA

<213>People

<400>17

gtggcacaca ctccatcgtc cacagactgg gtcgacaaca aaaccttcag cgtctgctcc     60

agggacttca ccccgcccac cgtgaagatc ttacagtcgt cctgcgacgg cggcgggcac    120

ttccccccga ccatccagct cctgtgcctc gtctctgggt acaccccagg gactatcaac    180

atcacctggc tggaggacgg gcaggtcatg gacgtggact tgtccaccgc ctctaccacg    240

caggagggtg agctggcctc cacacaaagc gagctcaccc tcagccagaa gcactggctg    300

tcagaccgca cctacacctg ccaggtcacc tatcaaggtc acacctttga ggacagcacc    360

aagaagtgtg cagattccaa cccgagaggg gtgagcgcct acctaagccg gcccagcccg    420

ttcgacctgt tcatccgcaa gtcgcccacg atcacctgtc tggtggtgga cctggcaccc    480

agcaagggga ccgtgaacct gacctggtcc cgggccagtg ggaagcctgt gaaccactcc    540

accagaaagg aggagaagca gcgcaatggc acgttaaccg tcacgtccac cctgccggtg    600

ggcacccgag actggatcga gggggagacc taccagtgca gggtgaccca cccccacctg    660

cccagggccc tcatgcggtc cacgaccaag accagcggcc cgcgtgctgc cccggaagtc    720

tatgcgtttg cgacgccgga gtggccgggg agccgggaca agcgcaccct cgcctgcctg    780

atccagaact tcatgcctga ggacatctcg gtgcagtggc tgcacaacga ggtgcagctc    840

ccggacgccc ggcacagcac gacgcagccc cgcaagacca agggctccgg cttcttcgtc    900

ttcagccgcc tggaggtgac cagggccgaa tgggagcaga aagatgagtt  catctgccgt   960

gcagtccatg aggcagcgag cccctcacag accgtccagc gagcggtgtc tgtaaatccc   1020

ggtaaa                                                              1026

<210>18

<21l>342

<212>PRT

<213>People

<400>18

Val Ala His Thr Pro Ser Ser Thr Asp Trp Val Asp Asn Lys Thr Phe

1               5                   10                  15

Ser Val Cys Ser Arg Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln

            20                  25                  30

Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu

        35                  40                  45

Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu

    50                  55                  60

Glu Asp Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr

65                  70                  75                  80

Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln

                85                  90                  95

Lys His Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln

            100                 105                  110

Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn Pro

        115                 120                 125

Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe Asp Leu Phe

    130                 135                 140

Ile Arg Lys Ser Pro Thr Ile Thr Cys Leu Val Val Asp Leu Ala Pro

145                 150                 155                 160

Ser Lys Gly Thr Val Asn Leu Thr Trp Ser Arg Ala Ser Gly Lys Pro

                165                 170                 175

Val Asn His Ser Thr Arg Lys Glu Glu Lys Gln Arg Asn Gly Thr Leu

            180                 185                 190

Thr Val Thr Ser Thr Leu Pro Val Gly Thr Arg Asp Trp Ile Glu Gly

        195                 200                 205

Glu Thr Tyr Gln Cys Arg Val Thr His Pro His Leu Pro Arg Ala Leu

    210                 215                 220

Met Arg Ser Thr Thr Lys Thr Ser Gly Pro Arg Ala Ala Pro Glu Val

225                 230                 235                 240

Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr

                245                 250                 255

Leu Ala Cys Leu Ile Gln Asn Phe Met Pro Glu Asp Ile Ser Val Gln

            260                 265                 270

Trp Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg His Ser Thr Thr

        275                 280                 285

Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Ser Arg Leu

    290                 295                 300

Glu Val Thr Arg Ala Glu Trp Glu Gln Lys Asp Glu Phe Ile Cys Arg

305                 310                 315                 320

Ala Val His Glu Ala Ala Ser Pro Ser Gln Thr Val Gln Arg Ala Val

                325                 330                 335

Ser Val Asn Pro Gly Lys

            340

<210>19

<211>1044

<212>DNA

<213>People

<400>19

aaatgcgtgg tccagcacac cgccagcaag agtaagaagg agatcttccg ctggccagag     60

tctccaaagg cacaggcctc ctccgtgccc actgcacaac cccaagcaga gggcagcctc    120

gccaaggcaa ccacagcccc agccaccacc cgtaacacag gaagaggagg agaagagaag    180

aagaaggaga aggagaaaga ggaacaagaa gagagagaga caaagacacc agagtgtccg    240

agccacaccc agcctcttgg cgtctacctg ctaacccctg cagtgcagga cctgtggctc    300

cgggacaaag ccaccttcac ctgcttcgtg gtgggcagtg acctgaagga tgctcacctg    360

acctgggagg tggctgggaa ggtccccaca gggggcgtgg aggaagggct gctggagcgg    420

cacagcaacg gctcccagag ccagcacagc cgtctgaccc tgcccaggtc cttgtggaac    480

gcggggacct ccgtcacctg cacactgaac catcccagcc tcccacccca gaggttgatg    540

gcgctgagag aacccgctgc gcaggcaccc gtcaagcttt ctctgaacct gctggcctcg    600

tctgaccctc ccgaggcggc ctcgtggctc ctgtgtgagg tgtctggctt ctcgcccccc    660

aacatcctcc tgatgtggct ggaggaccag cgtgaggtga acacttctgg gtttgccccc    720

gcacgccccc ctccacagcc caggagcacc acgttctggg cctggagtgt gctgcgtgtc    780

ccagccccgc ccagccctca gccagccacc tacacgtgtg tggtcagcca cgaggactcc    840

cggactctgc tcaacgccag ccggagccta gaagtcagct acctggccat gacccccctg    900

atccctcaga gcaaggatga gaacagcgat gactacacga cctttgatga tgtgggcagc    960

ctgtggacca ccctgtccac gtttgtggcc ctcttcatcc tcaccctcct ctacagcggc   1020

attgtcactt tcatcaaggt gaag                                          1044

<210>20

<211>348

<212>PRT

<213>People

<400>20

Lys Cys Val Val Gln His Thr Ala Ser Lys Ser Lys Lys Glu Ile Phe

1               5                   10                  15

Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala

            20                  25                  30

Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala

        35                  40                  45

Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys

    50                  55                  60

Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

65                  70                  75                  80

Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala Val Gln

                85                  90                  95

Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly

            100                 105                 110

Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val

        115                 120                 125

Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly

    130                 135                 140

Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn

145                 150                 155                 160

Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro Pro

                165                 170                 175

Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro Val Lys

            180                 185                 190

Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala Ala Ser

        195                 200                 205

Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile Leu Leu

    210                 215                 220

Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro

225                 230                 235                 240

Ala Arg Pro Pro Pro Gln Pro Arg Ser Thr Thr Phe Trp Ala Trp Ser

                245                 250                 255

Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr

            260                 265                 270

Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg

        275                 280                285

Ser Leu Glu Val Ser Tyr Leu Ala Met Thr Pro Leu Ile Pro Gln Ser

    290                 295                 300

Lys Asp Glu Asn Ser Asp Asp Tyr Thr Thr Phe Asp Asp Val Gly Ser

305                 310                 315                 320

Leu Trp Thr Thr Leu Ser Thr Phe Val Ala Leu Phe Ile Leu Thr Leu

                325                 330                 335

Leu Tyr Ser Gly Ile Val Thr Phe Ile Lys Val Lys

            340                 345

<210>21

<211>35

<212>DNA

<213>Artificial sequence

<400>21

cccaggatcc ccaaggtgga caagaaagtt gagcc    35

<210>22

<211>30

<212>DNA

<213>Artificial sequence

<400>22

gggtacgtgc ccagcacact ggtgcgaccg          30

<210>23

<211>24

<212>DNA

<213>Artificial sequence

<400>23

aaaggatcca gcaacaccaa ggtg                    24

<210>24

<211>41

<212>DNA

<213>Artificial sequence

<400>24

aaattaattc cagcacactg gtcatttacc cggagacagg g 41

<210>25

<211>23

<212>DNA

<213>Artificial sequence

<400>25

ctgacaagat atcaggcagg ttc                     23

<210>26

<211>22

<212>DNA

<213>Artificial sequence

<400>26

tggtctccag aattccagat gt                      22

<210>27

<211>231

<212>DNA

<213>People

<400>27

atgagcactg aaagcatgat ccgggacgtg gagctggccg aggaggcgct ccecaagaag     60

acaggggggc cccagggctc caggcggtgc ttgttcctca gcctcttctc cttcctgatc    120

gtggcaggcg ccaccacgct cttctgcctg ctgcactttg gagtgatcgg cccccagagg    180

gaagagtccc ccagggacct ctctctaatc agccctctgg cccaggcagt c             231

<210>28

<211>77

<212>PRT

<213>People

<400>28

Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala

1               5                   10                  15

Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe

            20                  25                  30

Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe

        35                  40                  45

Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Ser Pro

    50                  55                  60

Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val

65                  70                  75

<210>29

<211>231

<212>DNA

<213>People

<400>29

atgagcactg aaagcatgat ccgggacgtg gagctggccg aggaggcgct ccccaagaag     60

acaggggggc cccagggctc caggcggtgc ttgttcctca gcctcttctc cttcctgatc    120

gtggcaggcg ccaccacgct cttctgcctg ctgcactttg gagtgatcgg cccccagagg    180

gaagagttcc ccagggacct ctctctaatc agccctctgg cccaggcagt c             231

<210>30

<211>77

<212>PRT

<213>People

<400>30

Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala

1               5                   10                  15

Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe

            20                  25                  30

Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe

        35                  40                  45

Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro

    50                  55                  60

Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val

65                  70                  75

<210>31

<211>468

<212>DNA

<213>People

<400>31

agatcatctt ctcgaacccc gagtgacaag cctgtagccc atgttgtagc aaaccctcaa     60

gctgaggggc agctccagtg gctgaaccgc cgggccaatg ccctcctggc caatggcgtg    120

gagctgagag ataaccagct ggtggtgcca tcagagggcc tgtacctcat ctactcccag    180

gtcctcttca agggccaagg ctgcccctcc acccatgtgc tcctcaccca caccatcagc    240

cgcatcgccg tctcctacca gaccaaggtc aacctcctct ctgccatcaa gagcccctgc    300

cagagggaga ccccagaggg ggctgaggcc aagccctggt atgagcccat ctatctggga    360

ggggtcttcc agctggagaa gggtgaccga ctcagcgctg agatcaatcg gcccgactat    420

ctcgactttg ccgagtctgg gcaggtctac tttgggatca ttgccctg                 468

<210>32

<211>156

<212>PRT

<213>People

<400>32

Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val

1               5                   10                  15

Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala

            20                  25                  30

Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val

        35                  40                  45

Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys

    50                  55                  60

Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser

65                  70                  75                  80

Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile

                85                  90                  95

Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro

            100                 105                 110

Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly

        115                 120                 125

Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala

    130                 135                 140

Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu

145                 150                 155

<210>33

<211>699

<212>PRT

<213>Artificial sequence

<400>33

Ala Thr Gly Ala Gly Cys Ala Cys Thr Gly Ala Ala Ala Gly Cys Ala

1               5                   10                  15

Thr Gly Ala Thr Cys Cys Gly Gly Gly Ala Cys Gly Thr Gly Gly Ala

            20                  25                  30

Gly Cys Thr Gly Gly Cys Cys Gly Ala Gly Gly Ala Gly Gly Cys Gly

        35                  40                  45

Cys Thr Cys Cys Cys Cys Ala Ala Gly Ala Ala Gly Ala Cys Ala Gly

    50                  55                  60

Gly Gly Gly Gly Gly Cys Cys Cys Cys Ala Gly Gly Gly Cys Thr Cys

65                  70                  75                  80

Cys Ala Gly Gly Cys Gly Gly Thr Gly Cys Thr Thr Gly Thr Thr Cys

                85                  90                  95

Cys Thr Cys Ala Gly Cys Cys Thr Cys Thr Thr Cys Thr Cys Cys Thr

            100                 105                 110

Thr Cys Cys Thr Gly Ala Thr Cys Gly Thr Gly Gly Cys Ala Gly Gly

        115                 120                 125

Cys Gly Cys Cys Ala Cys Cys Ala Cys Gly Cys Thr Cys Thr Thr Cys

    130                 135                 140

Thr Gly Cys Cys Thr Gly Cys Thr Gly Cys Ala Cys Thr Thr Thr Gly

145                 150                 155                 160

Gly Ala Gly Thr Gly Ala Thr Cys Gly Gly Cys Cys Cys Cys Cys Ala

                165                 170                 175

Gly Ala Gly Gly Gly Ala Ala Gly Ala Gly Thr Cys Cys Cys Cys Cys

            180                 185                 190

Ala Gly Gly Gly Ala Cys Cys Thr Cys Thr Cys Thr Cys Thr Ala Ala

        195                 200                 205

Thr Cys Ala Gly Cys Cys Cys Thr Cys Thr Gly Gly Cys Cys Cys Ala

    210                 215                 220

Gly Gly Cys Ala Gly Thr Cys Ala Gly Ala Thr Cys Ala Thr Cys Thr

225                 230                 235                 240

Thr Cys Thr Cys Gly Ala Ala Cys Cys Cys Cys Gly Ala Gly Thr Gly

                245                 250                 255

Ala Cys Ala Ala Gly Cys Cys Thr Gly Thr Ala Gly Cys Cys Cys Ala

            260                 265                 270

Thr Gly Thr Thr Gly Thr Ala Gly Cys Ala Ala Ala Cys Cys Cys Thr

        275                 280                 285

Cys Ala Ala Gly Cys Thr Gly Ala Gly Gly Gly Gly Cys Ala Gly Cys

    290                 295                 300

Thr Cys Cys Ala Gly Thr Gly Gly Cys Thr Gly Ala Ala Cys Cys Gly

305                 310                 315                 320

Cys Cys Gly Gly Gly Cys Cys Ala Ala Thr Gly Cys Cys Cys Thr Cys

                325                 330                 335

Cys Thr Gly Gly Cys Cys Ala Ala Thr Gly Gly Cys Gly Thr Gly Gly

            340                 345                 350

Ala Gly Cys Thr Gly Ala Gly Ala Gly Ala Thr Ala Ala Cys Cys Ala

        355                 360                 365

Gly Cys Thr Gly Gly Thr Gly Gly Thr Gly Cys Cys Ala Thr Cys Ala

    370                 375                 380

Gly Ala Gly Gly Gly Cys Cys Thr Gly Thr Ala Cys Cys Thr Cys Ala

385                 390                 395                 400

Thr Cys Thr Ala Cys Thr Cys Cys Cys Ala Gly Gly Thr Cys Cys Thr

                405                 410                 415

Cys Thr Thr Cys Ala Ala Gly Gly Gly Cys Cys Ala Ala Gly Gly Cys

            420                 425                 430

Thr Gly Cys Cys Cys Cys Thr Cys Cys Ala Cys Cys Cys Ala Thr Gly

        435                 440                 445

Thr Gly Cys Thr Cys Cys Thr Cys Ala Cys Cys Cys Ala Cys Ala Cys

    450                 455                 460

Cys Ala Thr Cys Ala Gly Cys Cys Gly Cys Ala Thr Cys Gly Cys Cys

465                 470                 475                 480

Gly Thr Cys Thr Cys Cys Thr Ala Cys Cys Ala Gly Ala Cys Cys Ala

                485                 490                 495

Ala Gly Gly Thr Cys Ala Ala Cys Cys Thr Cys Cys Thr Cys Thr Cys

            500                 505                 510

Thr Gly Cys Cys Ala Thr Cys Ala Ala Gly Ala Gly Cys Cys Cys Cys

        515                 520                 525

Thr Gly Cys Cys Ala Gly Ala Gly Gly Gly Ala Gly Ala Cys Cys Cys

    530                 535                 540

Cys Ala Gly Ala Gly Gly Gly Gly Gly Cys Thr Gly Ala Gly Gly Cys

545                 550                 555                 560

Cys Ala Ala Gly Cys Cys Cys Thr Gly Gly Thr Ala Thr Gly Ala Gly

                565                 570                 575

Cys Cys Cys Ala Thr Cys Thr Ala Thr Cys Thr Gly Gly Gly Ala Gly

            580                 585                 590

Gly Gly Gly Thr Cys Thr Thr Cys Cys Ala Gly Cys Thr Gly Gly Ala

        595                 600                 605

Gly Ala Ala Gly Gly Gly Thr Gly Ala Cys Cys Gly Ala Cys Thr Cys

    610                 615                 620

Ala Gly Cys Gly Cys Thr Gly Ala Gly Ala Thr Cys Ala Ala Thr Cys

625                 630                 635                 640

Gly Gly Cys Cys Cys Gly Ala Cys Thr Ala Thr Cys Thr Cys Gly Ala

                645                 650                 655

Cys Thr Thr Thr Gly Cys Cys Gly Ala Gly Thr Cys Thr Gly Gly Gly

            660                 665                 670

Cys Ala Gly Gly Thr Cys Thr Ala Cys Thr Thr Thr Gly Gly Gly Ala

        675                 680                 685

Thr Cys Ala Thr Thr Gly Cys Cys Cys Thr Gly

    690                 695

<210>34

<211>233

<212>PRT

<213>Artificial sequence

<400>34

Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala

1               5                   10                  15

Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe

            20                  25                  30

Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe

        35                  40                  45

Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Ser Pro

    50                  55                  60

Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser

65                  70                  75                  80

Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro

                85                  90                  95

Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu

            100                 105                 110

Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser

        115                 120                 125

Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly

    130                 135                 140

Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser ArgIle Ala

145                 150                 155                 160

Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro

                165                 170                 175

Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu

            180                 185                 190

Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu

        195                 200                 205

Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly

    210                 215                 220

Gln Val Tyr Phe Gly Ile Ile Ala Leu

225                 230

<210>35

<211>699

<212>DNA

<213>Artificial sequence

<400>35

atgagcactg aaagcatgat ccgggacgtg gagctggccg aggaggcgct ccccaagaag     60

acaggggggc cccagggctc caggcggtgc ttgttcctca gcctcttctc cttcctgatc    120

gtggcaggcg ccaccacgct cttctgcctg ctgcactttg gagtgatcgg cccccagagg    180

gaagagttcc ccagggacct ctctctaatc agccctctgg cccaggcagt cagatcatct    240

tctcgaaccc cgagtgacaa gcctgtagcc catgttgtag caaaccctca agctgagggg    300

cagctccagt ggctgaaccg ccgggccaat gccctcctgg ccaatggcgt ggagctgaga    360

gataaccagc tggtggtgcc atcagagggc ctgtacctca tctactccca ggtcctcttc    420

aagggccaag gctgcccctc cacccatgtg ctcctcaccc acaccatcag ccgcatcgcc    480

gtctcctacc agaccaaggt caacctcctc tctgccatca agagcccctg ccagagggag    540

accccagagg gggctgaggc caagccctgg tatgagccca tctatctggg aggggtcttc    600

cagctggaga agggtgaccg actcagcgct gagatcaatc ggcccgacta tctcgacttt    660

gccgagtctg ggcaggtcta ctttgggatc attgccctg                           699

<210>36

<211>233

<212>PRT

<213>Artificial sequence

<400>36

Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala

1               5                   10                  15

Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe

            20                  25                  30

Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe

        35                  40                  45

Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro

    50                  55                  60

Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser

65                  70                  75                  80

Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro

                85                  90                  95

Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu

            100                 105                 110

Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser

        115                 120                 125

Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly

    130                 135                 140

Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala

145                 150                 155                 160

Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro

                165                 170                 175

Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu

            180                 185                 190

Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu

        195                 200                 205

Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly

    210                 215                 220

Gln Val Tyr Phe Gly Ile Ile Ala Leu

225                 230

<210>37

<211>1428

<212>DNA

<213>Artificial sequence

<400>37

atgagcactg aaagcatgat ccgggacgtg gagctggccg aggaggcgct ccccaagaag     60

acaggggggc cccagggctc caggcggtgc ttgttcctca gcctcttctc cttcctgatc    120

gtggcaggcg ccaccacgct cttctgcctg ctgcactttg gagtgatcgg cccccagagg    180

gaagagtccc ccagggacct ctctctaatc agccctctgg cccaggcagt cagatcatct    240

tctcgaaccc cgagtgacaa gcctgtagcc catgttgtag caaaccctca agctgagggg    300

cagctccagt ggctgaaccg ccgggccaat gccctcctgg ccaatggcgt ggagctgaga    360

gataaccagc tggtggtgcc atcagagggc ctgtacctca tctactccca ggtcctcttc    420

aagggccaag gctgcccctc cacccatgtg ctcctcaccc acaccatcag ccgcatcgcc    480

gtctcctacc agaccaaggt caacctcctc tctgccatca agagcccctg ccagagggag    540

accccagagg gggctgaggc caagccctgg tatgagccca tctatctggg aggggtcttc    600

cagctggaga agggtgaccg actcagcgct gagatcaatc ggcccgacta tctcgacttt    660

gccgagtctg ggcaggtcta ctttgggatc attgccctgg gatccagcaa caccaaggtg    720

gacaagaaag ttgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca    780

cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc    840

atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct     900

gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg     960

cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag    1020

gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc    1080

atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg    1140

cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc    1200

ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac    1260

aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc    1320

gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct    1380

ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaa                 1428

<210>38

<211>476

<212>PRT

<213>Artificial sequence

<400>38

Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala

1               5                   10                  15

Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe

            20                  25                  30

Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe

        35                  40                  45

Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Ser Pro

    50                  55                  60

Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser

65                  70                  75                  80

Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro

                85                  90                  95

Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu

            100                 105                 110

Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser

        115                 120                 125

Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly

    130                 135                 140

Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala

145                 150                 155                 160

Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro

                165                 170                 175

Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu

            180                 185                 190

Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu

        195                 200                 205

Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly

    210                 215                  220

Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Ser Ser Asn Thr Lys Val

225                 230                 235                 240

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

                245                 250                 255

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pr0 Ser Val Phe Leu Phe

            260                 265                 270

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

        275                 280                 285

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

    290                 295                 300

Asn Trp Tyr Val Asp Gly Val Glu Val Hi s Asn Ala Lys Thr Lys Pro

305                 310                 315                 320

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

                325                 330                 335

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

            340                 345                 350

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

        355                 360                 365

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

    370                 375                 380

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

385                 390                 395                 400

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

                405                 410                 415

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

            420                 425                 430

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

        435                 440                 445

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

    450                 455                 460

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465                 470                 475

<210>39

<211>1428

<212>DNA

<213>Artificial sequence

<400>39

atgagcactg aaagcatgat ccgggacgtg gagctggccg aggaggcgct ccccaagaag     60

acaggggggc cccagggctc caggcggtgc ttgttcctca gcctcttctc cttcctgatc    120

gtggcaggcg ccaccacgct cttctgcctg ctgcactttg gagtgatcgg cccccagagg    180

gaagagttcc ccagggacct ctctctaatc agccctctgg cccaggcagt cagatcatct    240

tctcgaaccc cgagtgacaa gcctgtagcc catgttgtag caaaccctca agctgagggg    300

cagctccagt ggctgaaccg ccgggccaat gccctcctgg ccaatggcgt ggagctgaga    360

gataaccagc tggtggtgcc atcagagggc ctgtacctca tctactccca ggtcctcttc    420

aagggccaag gctgcccctc cacccatgtg ctcctcaccc acaccatcag ccgcatcgcc    480

gtctcctacc agaccaaggt caacctcctc tctgccatca agagcccctg ccagagggag    540

accccagagg gggctgaggc caagccctgg tatgagccca tctatctggg aggggtcttc    600

cagctggaga agggtgaccg actcagcgct gagatcaatc ggcccgacta tctcgacttt    660

gccgagtctg ggcaggtcta ctttgggatc attgccctgg gatccagcaa caccaaggtg    720

gacaagaaag ttgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca    780

cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc    840

atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct    900

gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg    960

cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag   1020

gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc   1080

atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg   1140

cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc   1200

ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac   1260

aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc   1320

gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct   1380

ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaa                1428

<210>40

<211>476

<212>PRT

<213>Artificial sequence

<400>40

Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala

1               5                   10                  15

Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe

            20                  25                  30

Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe

        35                  40                  45

Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro

    50                  55                  60

Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser

65                  70                  75                  80

Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro

                85                  90                  95

Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu

            100                 105                 110

Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser

        115                 120                 125

Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly

    130                 135                 140

Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala

145                 150                 155                 160

Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro

                165                 170                 175

Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu

            180                 185                 190

Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu

        195                 200                 205

Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly

    210                 215                 220

Gln Val Tyr Phe Gly Ile Ile Ala Leu Gly Ser Ser Asn Thr Lys Val

225                 230                 235                 240

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

                245                 250                 255

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

            260                 265                 270

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

        275                 280                 285

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

    290                 295                 300

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

305                 310                 315                 320

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

                325                 330                 335

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

            340                 345                 350

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

        355                 360                 365

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

    370                 375                 380

Asp Glu Leu Thr Lys Asn Gln ValSer Leu Thr Cys Leu Val Lys Gly

385                 390                 395                 400

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

                405                 410                 415

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

            420                 425                 430

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

        435                 440                 445

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

    450                 455                 460

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465                 470                 475

<210>41

<211>28

<212>DNA

<213>Artificial sequence

<400>41

gccgatatct tggttctccc catgacac    28

<210>42

<211>28

<212>DNA

<213>Artificial sequence

<400>42

tttctttctg gatccttcca agttctac                                       28

<210>43

<211>102

<212>DNA

<213>People

<400>43

atgacaccac ctgaacgtct cttcctccca agggtgtgtg gcaccaccct acacctcctc    60

cttctggggc tgctgctggt tctgctgcct ggggcccagg gg                      102

<210>44

<211>34

<212>PRT

<213>People

<400>44

Met Thr Pro Pro Glu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr

1               5                   10                  15

Leu His Leu Leu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala

            20                  25                  30

Gln Gly

<210>45

<211>513

<212>DNA

<213>People

<400>45

ctccctggtg ttggcctcac accttcagct gcccagactg cccgtcagca ccccaagatg     60

catcttgccc acagcaccct caaacctgct gctcacctca ttggagaccc cagcaagcag    120

aactcactgc tctggagagc aaacacggac cgtgccttcc tccaggatgg tttctccttg    180

agcaacaatt ctctcctggt ccccaccagt ggcatctact tcgtctactc ccaggtggtc    240

ttctctggga aagcctactc tcccaaggcc acctcctccc cactctacct ggcccatgag    300

gtccagctct tctcctccca gtaccccttc catgtgcctc tcctcagctc ccagaagatg    360

gtgtatccag ggctgcagga accctggctg cactcgatgt accacggggc tgcgttccag    420

ctcacccagg gagaccagct atccacccac acagatggca tcccccacct agtcctcagc    480

cctagtactg tcttctttgg agccttcgct ctg    513

<210>46

<211>171

<212>PRT

<213>People

<400>46

Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala Arg Gln

1               5                   10                  15

His Pro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala Ala His

            20                  25                  30

Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg Ala Asn

        35                  40                  45

Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn Asn Ser

    50                  55                  60

Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln Val Val

65                  70                  75                  80

Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro Leu Tyr

                85                  90                  95

Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe His Val

            100                 105                 110

Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln Glu Pro

        115                 120                 125

Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr Gln Gly

    130                 135                 140

Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val Leu Ser

145                 150                 155                 160

Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu

                165                 170

<210>47

<211>513

<212>DNA

<213>People

<400>47

ctccctggtg ttggcctcac accttcagct gcccagactg cccgtcagca ccccaagatg     60

catcttgccc acagcaacct caaacctgct gctcacctca ttggagaccc cagcaagcag    120

aactcactgc tctggagagc aaacacggac cgtgccttcc tccaggatgg tttctccttg    180

agcaacaatt ctctcctggt ccccaccagt ggcatctact tcgtctactc ccaggtggtc    240

ttctctggga aagcctactc tcccaaggcc acctcctccc cactctacct ggcccatgag    300

gtccagctct tctcctccca gtaccccttc catgtgcctc tcctcagctc ccagaagatg    360

gtgtatccag ggctgcagga accctggctg cactcgatgt accacggggc tgcgttccag    420

ctcacccagg gagaccagct atccacccac acagatggca tcccccacct agtcctcagc    480

cctagtactg tcttctttgg agccttcgct ctg                                 513

<210>48

<211>171

<212>PRT

<213>People

<400>48

Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala Arg Gln

1               5                   10                  15

His Pro Lys Met His Leu Ala His Ser Asn Leu Lys Pro Ala Ala His

            20                  25                  30

Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg Ala Asn

        35                  40                  45

Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn Asn Ser

    50                  55                  60

Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln Val Val

65                  70                  75                  80

Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro Leu Tyr

                85                  90                  95

Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe His Val

            100                 105                 110

Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln Glu Pro

        115                 120                 125

Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr Gln Gly

    130                 135                 140

Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val Leu Ser

145                 150                 155                 160

Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu

             165                 170

<210>49

<211>615

<212>PRT

<213>Artificial sequence

<400>49

Ala Thr Gly Ala Cys Ala Cys Cys Ala Cys Cys Thr Gly Ala Ala Cys

1               5                   10                  15

Gly Thr Cys Thr Cys Thr Thr Cys Cys Thr Cys Cys Cys Ala Ala Gly

            20                  25                  30

Gly Gly Thr Gly Thr Gly Thr Gly Gly Cys Ala Cys Cys Ala Cys Cys

        35                  40                  45

Cys Thr Ala Cys Ala Cys Cys Thr Cys Cys Thr Cys Cys Thr Thr Cys

    50                  55                  60

Thr Gly Gly Gly Gly Cys Thr Gly Cys Thr Gly Cys Thr Gly Gly Thr

65                  70                  75                  80

Thr Cys Thr Gly Cys Thr Gly Cys Cys Thr Gly Gly Gly Gly Cys Cys

                85                  90                  95

Cys Ala Gly Gly Gly Gly Cys Thr Cys Cys Cys Thr Gly Gly Thr Gly

            100                 105                 110

Thr Thr Gly Gly Cys Cys Thr Cys Ala Cys Ala Cys Cys Thr Thr Cys

        115                 120                 125

Ala Gly Cys Thr Gly Cys Cys Cys Ala Gly Ala Cys Thr Gly Cys Cys

    130                 135                 140

Cys Gly Thr Cys Ala Gly Cys Ala Cys Cys Cys Cys Ala Ala Gly Ala

145                 150                 155                 160

Thr Gly Cys Ala Thr Cys Thr Thr Gly Cys Cys Cys Ala Cys Ala Gly

                165                 170                 175

Cys Ala Cys Cys Cys Thr Cys Ala Ala Ala Cys Cys Thr Gly Cys Thr

            180                 185                 190

Gly Cys Thr Cys Ala Cys Cys Thr Cys Ala Thr Thr Gly Gly Ala Gly

        195                 200                 205

Ala Cys Cys Cys Cys Ala Gly Cys Ala Ala Gly Cys Ala Gly Ala Ala

    210                 215                 220

Cys Thr Cys Ala Cys Thr Gly Cys Thr Cys Thr Gly Gly Ala Gly Ala

225                 230                 235                 240

Gly Cys Ala Ala Ala Cys Ala Cys Gly Gly Ala Cys Cys Gly Thr Gly

                245                 250                 255

Cys Cys Thr Thr Cys Cys Thr Cys Cys Ala Gly Gly Ala Thr Gly Gly

            260                 265                 270

Thr Thr Thr Cys Thr Cys Cys Thr Thr Gly Ala Gly Cys Ala Ala Cys

        275                 280                 285

Ala Ala Thr Thr Cys Thr Cys Thr Cys Cys Thr Gly Gly Thr Cys Cys

    290                 295                 300

Cys Cys Ala Cys Cys Ala Gly Thr Gly Gly Cys Ala Thr Cys Thr Ala

305                 310                 315                 320

Cys Thr Thr Cys Gly Thr Cys Thr Ala Cys Thr Cys Cys Cys Ala Gly

                325                 330                 335

Gly Thr Gly Gly Thr Cys Thr Thr Cys Thr Cys Thr Gly Gly Gly Ala

            340                 345                 350

Ala Ala Gly Cys Cys Thr Ala Cys Thr Cys Thr Cys Cys Cys Ala Ala

        355                 360                 365

Gly Gly Cys Cys Ala Cys Cys Thr Cys Cys Thr Cys Cys Cys Cys Ala

    370                 375                 380

Cys Thr Cys Thr Ala Cys Cys Thr Gly Gly Cys Cys Cys Ala Thr Gly

385                 390                 395                 400

Ala Gly Gly Thr Cys Cys Ala Gly Cys Thr Cys Thr Thr Cys Thr Cys

                405                 410                 415

Cys Thr Cys Cys Cys Ala Gly Thr Ala Cys Cys Cys Cys Thr Thr Cys

            420                 425                 430

Cys Ala Thr Gly Thr Gly Cys Cys Thr Cys Thr Cys Cys Thr Cys Ala

        435                 440                 445

Gly Cys Thr Cys Cys Cys Ala Gly Ala Ala Gly Ala Thr Gly Gly Thr

    450                 455                 460

Gly Thr Ala Thr Cys Cys Ala Gly Gly Gly Cys Thr Gly Cys Ala Gly

465                 470                 475                 480

Gly Ala Ala Cys Cys Cys Thr Gly Gly Cys Thr Gly Cys Ala Cys Thr

                485                 490                 495

Cys Gly Ala Thr Gly Thr Ala Cys Cys Ala Cys Gly Gly Gly Gly Cys

            500                 505                 510

Thr Gly Cys Gly Thr Thr Cys Cys Ala Gly Cys Thr Cys Ala Cys Cys

        515                 520                 525

Cys Ala Gly Gly Gly Ala Gly Ala Cys Cys Ala Gly Cys Thr Ala Thr

    530                 535                 540

Cys Cys Ala Cys Cys Cys Ala Cys Ala Cys Ala Gly Ala Thr Gly Gly

545                 550                 555                 560

Cys Ala Thr Cys Cys Cys Cys Cys Ala Cys Cys Thr Ala Gly Thr Cys

                565                 570                 575

Cys Thr Cys Ala Gly Cys Cys Cys Thr Ala Gly Thr Ala Cys Thr Gly

            580                 585                 590

Thr Cys Thr Thr Cys Thr Thr Thr Gly Gly Ala Gly Cys Cys Thr Thr

        595                 600                 605

Cys Gly Cys Thr Cys Thr Gly

    610                 615

<210> 50

<211>205

<212>PRT

<213>Artificial sequence

<400>50

Met Thr Pro Pro Glu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr

1               5                   10                  15

Leu His Leu Leu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala

            20                  25                  30

Gln Gly Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala

        35                  40                  45

Arg Gln His Pro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala

    50                  55                  60

Ala His Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg

65                  70                  75                  80

Ala Asn Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn

                85                  90                  95

Asn Ser Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln

            100                 105                 110

Val Val Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro

        115                 120                 125

Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe

    130                 135                 140

His Val Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln

145                 150                 155                 160

Glu Pro Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr

                165                 170                 175

Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val

            180                 185                 190

Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu

        195                 200                 205

<210>51

<2ll>615

<212>DNA

<213>Artificial sequence

<400>5l

atgacaccac ctgaacgtct cttcctccca agggtgtgtg gcaccaccct acacctcctc     60

cttctggggc tgctgctggt tctgctgcct ggggcccagg ggctccctgg tgttggcctc    120

acaccttcag ctgcccagac tgcccgtcag caccccaaga tgcatcttgc ccacagcaac    180

ctcaaacctg ctgctcacct cattggagac cccagcaagc agaactcact gctctggaga    240

gcaaacacgg accgtgcctt cctccaggat ggtttctcct tgagcaacaa ttctctcctg    300

gtccccacca gtggcatcta cttcgtctac tcccaggtgg tcttctctgg gaaagcctac    360

tctcccaagg ccacctcctc cccactctac ctggcccatg aggtccagct cttctcctcc    420

cagtacccct tccatgtgcc tctcctcagc tcccagaaga tggtgtatcc agggctgcag    480

gaaccctggc tgcactcgat gtaccacggg gctgcgttcc agctcaccca gggagaccag    540

ctatccaccc acacagatgg catcccccac ctagtcctca gccctagtac tgtcttcttt    600

ggagccttcg ctctg                                            615

<210>52

<211>205

<212>PRT

<213>Artificial sequence

<400>52

Met Thr Pro Pro Glu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr

1               5                   10                  15

Leu His Leu Leu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala

            20                  25                  30

Gln Gly Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala

        35                  40                  45

Arg Gln His Pro Lys Met His Leu Ala His Ser Asn Leu Lys Pro Ala

    50                  55                  60

Ala His Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg

65                  70                  75                  80

Ala Asn Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn

                85                  90                  95

Asn Ser Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln

            100                 105                 110

Val Val Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro

        115                 120                 125

Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe

    130                 135                 140

His Val Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln

145                 150                 155                 160

Glu Pro Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr

                165                 170                 175

Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly lle Pro His Leu Val

            180                 185                 190

Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu

        195                 200                 205

<210>53

<211>1344

<212>DNA

<213>Artificial sequence

<400>53

atgacaccac ctgaacgtct cttcctccca agggtgtgtg gcaccaccct acacctcctc     60

cttctggggc tgctgctggt tctgctgcct ggggcccagg ggctccctgg tgttggcctc    120

acaccttcag ctgcccagac tgcccgtcag caccccaaga tgcatcttgc ccacagcacc    180

ctcaaacctg ctgctcacct cattggagac cccagcaagc agaactcact gctctggaga    240

gcaaacacgg accgtgcctt cctccaggat ggtttctcct tgagcaacaa ttctctcctg    300

gtccccacca gtggcatcta cttcgtctac tcccaggtgg tcttctctgg gaaagcctac    360

tctcccaagg ccacctcctc cccactctac ctggcccatg aggtccagct cttctcctcc    420

cagtacccct tccatgtgcc tctcctcagc tcccagaaga tggtgtatcc agggctgcag    480

gaaccctggc tgcactcgat gtaccacggg gctgcgttcc agctcaccca gggagaccag    540

ctatccaccc acacagatgg catcccccac ctagtcctca gccctagtac tgtcttcttt    600

ggagccttcg ctctgggatc cagcaacacc aaggtggaca agaaagttga gcccaaatct    660

tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca    720

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc    780

acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg    840

gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg   900

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac   960

aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc  1020

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc  1080

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg  1140

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac  1200

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag  1260

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag  1320

agcctctccc tgtctccggg taaa                                         1344

<210>54

<211>448

<212>PRT

<213>Artificial sequence

<400>54

Met Thr Pro Pto Glu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr

1               5                   10                  15

Leu His Leu Leu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala

            20                  25                  30

Gln Gly Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala

        35                  40                  45

Arg Gln His Pro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala

50                  55                  60

Ala His Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg

65                  70                  75                  80

Ala Asn Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn

                85                  90                  95

Asn Ser Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln

            100                 105                 110

Val Val Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro

        115                 120                 125

Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe

    130                 135                 140

His Val Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln

145                 150                 155                 160

Glu Pro Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr

                165                 170                 175

Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val

            180                 185                 190

Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu Gly Ser Ser

        195                 200                 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

    210                 215                 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225                 230                 235                 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

                245                 250                 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

            260                 265                 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

        275                 280                 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

    290                 295                 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305                 310                 315                 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

                325                 330                 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

           340                 345                 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

        355                 360                 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

    370                 375                 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385                 390                 395                 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

                405                 410                 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

            420                 425                 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

        435                 440                 445

<210>55

<211>1344

<212>DNA

<213>Artificial sequence

<400>55

atgacaccac ctgaacgtct cttcctccca agggtgtgtg gcaccaccct acacctcctc     60

cttctggggc tgctgctggt tctgctgcct ggggcccagg ggctccctgg tgttggcctc    120

acaccttcag ctgcccagac tgcccgtcag caccccaaga tgcatcttgc ccacagcaac   180

ctcaaacctg ctgctcacct cattggagac cccagcaagc agaactcact gctctggaga   240

gcaaacacgg accgtgcctt cctccaggat ggtttctcct tgagcaacaa ttctctcctg   300

gtccccacca gtggcatcta cttcgtctac tcccaggtgg tcttctctgg gaaagcctac   360

tctcccaagg ccacctcctc cccactctac ctggcccatg aggtccagct cttctcctcc   420

cagtacccct tccatgtgcc tctcctcagc tcccagaaga tggtgtatcc agggctgcag   480

gaaccctggc tgcactcgat gtaccacggg gctgcgttcc agctcaccca gggagaccag   540

ctatccaccc acacagatgg catcccccac ctagtcctca gccctagtac tgtcttcttt   600

ggagccttcg ctctgggatc cagcaacacc aaggtggaca agaaagttga gcccaaatct   660

tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca   720

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc   780

acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg   840

gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg   900

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac   960

aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc  1020

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc  1080

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg  1140

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac  1200

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag  1260

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag  1320

agcctctccc tgtctccggg taaa                                         1344

<210>56

<211>448

<212>PRT

<213>Artificial sequence

<400>56

Met Thr Pro Pro Glu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr

1               5                   10                  15

Leu His Leu Leu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala

            20                  25                  30

Gln Gly Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala

        35                  40                  45

Arg Gln His Pro Lys Met His Leu Ala His Ser Asn Leu Lys Pro Ala

    50                  55                  60

Ala His Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg

65                  70                  75                  80

Ala Asn Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn

                85                  90                  95

Asn Ser Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln

            100                 105                 110

Val Val Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro

        115                 120                 125

Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe

    130                 135                 140

His Val Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln

145                 150                 155                 160

Glu Pro Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr

                165                 170                 175

Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val

            180                 185                 190

Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu Gly Ser Ser

        195                 200                 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

    210                 215                 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225                 230                 235                 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

                245                 250                 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

            260                 265                 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

        275                 280                 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

    290                 295                 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305                 310                 315                 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

                325                 330                 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

            340                 345                 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

        355                 360                 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

    370                 375                 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385                 390                 395                 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

                405                 410                 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

            420                 425                 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

        435                 440                 445

<210>57

<211>28

<212>DNA

<213>Artificial sequence

<400>57

agtgatatcc catagctgtc tggcatgg                                    28

<210>58

<211>27

<212>DNA

<213>Artificial sequence

<400>58

agcactggga tccctgagtc ctcagtg                                     27

<210>59    

<211>57

<212>DNA

<213>People

<400>59

atgggcctct ccaccgtgcc tgacctgctg ctgccgctgg tgctcctgga gctgttg    57

<210>60

<211>19

<212>PRT

<213>People

<400>60

Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu

1               5                   10                  15

Glu Leu Leu

<210>61

<211>63

<212>DNA

<213>People

<400>61

atgggcctct ccaccgtgcc tgacctgctg ctgccgctgg tgctcctgga gctgttggtg    60

gga                                                                  63

<210>62

<211>21

<212>PRT

<213>People

<400>62

Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu

1               5                   10                  15

Glu Leu Leu Val Gly

            20

<210>63

<211>570

<212>DNA

<213>People

<400>63

gtgggaatat acccctcagg ggttattgga ctggtccctc acctagggga cagggagaag     60

agagatagtg tgtgtcccca aggaaaatat atccaccctc aaaataattc gatttgctgt    120

accaagtgcc acaaaggaac ctacttgtac aatgactgtc caggcccggg gcaggatacg    180

gactgcaggg agtgtgagag cggctccttc accgcttcag aaaaccacct cagacactgc    240

ctcagctgct ccaaatgccg aaaggaaatg ggtcaggtgg agatctcttc ttgcacagtg    300

gaccgggaca ccgtgtgtgg ctgcaggaag aaccagtacc ggcattattg gagtgaaaac    360

cttttccagt gcttcaattg cagcctctgc ctcaatggga ccgtgcacct ctcctgccag    420

gagaaacaga acaccgtgtg cacctgccat gcaggtttct ttctaagaga aaacgagtgt    480

gtctcctgta gtaactgtaa gaaaagcctg gagtgcacga agttgtgcct accccagatt    540

gagaatgtta agggcactga ggactcaggg                                     570

<210>64

<211>190

<212>PRT

<213>People

<400>64

Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro His Leu Gly

1               5                   10                  15

Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His

            20                  25                  30

Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr

        35                  40                  45

Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Glu

    50                  55                  60

Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys

65                  70                  75                  80

Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser

                85                  90                  95

Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln

            100                 105                 110

Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys Ser

        115                 120                 125

Leu Cys Leu Asn Gly Thr Val Hi s Leu Ser Cys Gln Glu Lys Gln Asn

    130                 135                 140

Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu Asn Glu Cys

145                 150                 155                 160

Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr Lys Leu Cys

                165                 170                 175

Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser Gly

            180                 185                 190

<210>65

<211>564

<212>DNA

<213>People

<400>65

atatacccct caggggttat tggactggtc cctcacctag gggacaggga gaagagagat     60

agtgtgtgtc cccaaggaaa atatatccac cctcaaaata attcgatttg ctgtaccaag    120

tgccacaaag gaacctactt gtacaatgac tgtccaggcc cggggcagga tacggactgc    180

agggagtgtg agagcggctc cttcaccgct tcagaaaacc acctcagaca ctgcctcagc    240

tgctccaaat gccgaaagga aatgggtcag gtggagatct cttcttgcac agtggaccgg    300

gacaccgtgt gtggctgcag gaagaaccag taccggcatt attggagtga aaaccttttc    360

cagtgcttca attgcagcct ctgcctcaat gggaccgtgc acctctcctg ccaggagaaa    420

cagaacaccg tgtgcacctg ccatgcaggt ttctttctaa gagaaaacga gtgtgtctcc    480

tgtagtaact gtaagaaaag cctggagtgc acgaagttgt gcctacccca gattgagaat    540

gttaagggca ctgaggactc aggg    564

<210>66

<211>188

<212>PRT

<213>People

<400>66

Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro His Leu Gly Asp Arg

1               5                   10                  15

Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His Pro Gln

            20                  25                  30

Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr

        35                  40                  45

Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu

    50                  55                  60

Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser

65                  70                  75                  80

Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys

                85                  90                  95

Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg

            100                 105                 110

His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys Ser Leu Cys

        115                 120                 125

Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu Lys Gln Asn Thr Val

    130                 135                 140

Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu Asn Glu Cys Val Ser

145                 150                 155                 160

Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu Pro

                165                 170                 175

Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser Gly

            180                 185

<210>67

<211>627

<212>DNA

<213>Artificial sequence

<400>67

atgggcctct ccaccgtgcc tgacctgctg ctgccgctgg tgctcctgga gctgttggtg     60

ggaatatacc cctcaggggt tattggactg gtccctcacc taggggacag ggagaagaga    120

gatagtgtgt gtccccaagg aaaatatatc caccctcaaa ataattcgat ttgctgtacc    180

aagtgccaca aaggaaccta cttgtacaat gactgtccag gcccggggca ggatacggac    240

tgcagggagt gtgagagcgg ctccttcacc gcttcagaaa accacctcag acactgcctc    300

agctgctcca aatgccgaaa ggaaatgggt caggtggaga tctcttcttg cacagtggac    360

cgggacaccg tgtgtggctg caggaagaac cagtaccggc attattggag tgaaaacctt    420

ttccagtgct tcaattgcag cctctgcctc aatgggaccg tgcacctctc ctgccaggag    480

aaacagaaca ccgtgtgcac ctgccatgca ggtttctttc taagagaaaa cgagtgtgtc    540

tcctgtagta actgtaagaa aagcctggag tgcacgaagt tgtgcctacc ccagattgag    600

aatgttaagg gcactgagga ctcaggg                                        627

<210>68

<211>209

<212>PRT

<213>Artificial sequence

<400>68

Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu

1               5                   10                  15

Glu Leu Leu Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro

            20                  25                  30

His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys

        35                  40                  45

Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys

    50                  55                  60

Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp

65                  70                  75                  80

Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu

                85                  90                  95

Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val

            100                 105                 110

Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg

        115                 120                 125

Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe

    130                 135                 140

Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu

145                 150                 155                 160

Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu

                165                 170                 175

Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr

            180                 185                 190

Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser

        195                 200                 205

Gly

<210>69

<211>1290

<212>DNA

<213>Artificial sequence

<400>69

gtgggaatat acccctcagg ggttattgga ctggtccctc acctagggga cagggagaag     60

agagatagtg tgtgtcccca aggaaaatat atccaccctc aaaataattc gatttgctgt    120

accaagtgcc acaaaggaac ctacttgtac aatgactgtc caggcccggg gcaggatacg    180

gactgcaggg agtgtgagag cggctccttc accgcttcag aaaaccacct cagacactgc    240

ctcagctgct ccaaatgccg aaaggaaatg ggtcaggtgg agatctcttc ttgcacagtg    300

gaccgggaca ccgtgtgtgg ctgcaggaag aaccagtacc ggcattattg gagtgaaaac    360

cttttccagt gcttcaattg cagcctctgc ctcaatggga ccgtgcacct ctcctgccag    420

gagaaacaga acaccgtgtg cacctgccat gcaggtttct ttctaagaga aaacgagtgt    480

gtctcctgta gtaactgtaa gaaaagcctg gagtgcacga agttgtgcct accccagatt    540

gagaatgtta agggcactga ggactcaggg atccccaagg tggacaagaa agttgagccc    600

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga    660

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct   720

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg   780

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac   840

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag   900

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc   960

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag  1020

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc  1080

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg  1140

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg  1200

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg  1260

cagaagagcc tctccctgtc tccgggtaaa                                   1290

<210>70

<211>430

<212>PRT

<213>Artificial sequence

<400>70

Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro His Leu Gly

1               5                   10                  15

Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His

            20                  25                  30

Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr

        35                  40                  45

Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Glu

    50                  55                  60

Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys

65                  70                  75                  80

Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser

                85                  90                  95

Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln

            100                 105                 110

Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys Ser

        115                 120                 125

Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu Lys Gln Asn

    130                 135                 140

Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu Asn Glu Cys

145                 150                 155                 160

Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr Lys Leu Cys

                165                 170                 175

Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser Gly Ile Pro

            180                 185                 190

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

        195                 200                 205

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

    210                 215                 220

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

225                 230                 235                 240

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

                245                 250                 255

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

            260                 265                 270

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

        275                 280                 285

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

    290                 295                 300

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

305                 310                 315                 320

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

                325                 330                 335

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

            340                 345                 350

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

        355                 360                 365

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

    370                 375                 380

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

385                 390                 395                 400

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

                405                 410                 415

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

            420                 425                 430

<210>71

<211>1284

<212>DNA

<213>Artificial sequence

<400>71

atatacccct caggggttat tggactggtc cctcacctag gggacaggga gaagagagat     60

agtgtgtgtc cccaaggaaa atatatccac cctcaaaata attcgatttg ctgtaccaag    120

tgccacaaag gaacctactt gtacaatgac tgtccaggcc cggggcagga tacggactgc   180

agggagtgtg agagcggctc cttcaccgct tcagaaaacc acctcagaca ctgcctcagc   240

tgctccaaat gccgaaagga aatgggtcag gtggagatct cttcttgcac agtggaccgg   300

gacaccgtgt gtggctgcag gaagaaccag taccggcatt attggagtga aaaccttttc   360

cagtgcttca attgcagcct ctgcctcaat gggaccgtgc acctctcctg ccaggagaaa   420

cagaacaccg tgtgcacctg ccatgcaggt ttctttctaa gagaaaacga gtgtgtctcc   480

tgtagtaact gtaagaaaag cctggagtgc acgaagttgt gcctacccca gattgagaat   540

gttaagggca ctgaggactc agggatcccc aaggtggaca agaaagttga gcccaaatct   600

tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca   660

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc   720

acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg   780

gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg   840

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac   900

aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc   960

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc  1020

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg  1080

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac  1140

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag  1200

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag  1260

agcctctccc tgtctccggg taaa                                         1284

<210>72

<211>428

<212>PRT

<213>Artificial sequence

<400>72

Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro His Leu Gly Asp Arg

1               5                   10                  15

Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His Pro Gln

            20                  25                  30

Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr

        35                  40                  45

Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu

    50                  55                  60

Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser

65                  70                  75                  80

Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys

                85                  90                  95

Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg

            100                 105                 110

His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys Ser Leu Cys

        115                 120                 125

Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu Lys Gln Asn Thr Val

    130                 135                 140

Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu Asn Glu Cys Val Ser

145                 150                 155                 160

Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu Pro

                165                 170                 175

Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser Gly Ile Pro Lys Val

            180                 185                 190

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

        195                 200                 205

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

    210                 215                 220

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

225                 230                 235                 240

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

                245                 250                 255

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

            260                 265                 270

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

        275                 280                 285

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

    290                 295                 300

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

305                 310                 315                 320

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

                325                 330                 335

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

            340                 345                 350

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

        355                 360                 365

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

    370                 375                 380

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

385                 390                 395                 400

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

                405                 410                 415

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

            420                 425

<210>73

<211>1290

<212>DNA

<213>Artificial sequence

<400>73

gtgggaatat acccctcagg ggttattgga ctggtccctc acctagggga cagggagaag    60

agagatagtg tgtgtcccca aggaaaatat atccaccctc aaaataattc gatttgctgt   120

accaagtgcc acaaaggaac ctacttgtac aatgactgtc caggcccggg gcaggatacg   180

gactgcaggg agtgtgagag cggctccttc accgcttcag aaaaccacct cagacactgc   240

ctcagctgct ccaaatgccg aaaggaaatg ggtcaggtgg agatctcttc ttgcacagtg   300

gaccgggaca ccgtgtgtgg ctgcaggaag aaccagtacc ggcattattg gagtgaaaac   360

cttttccagt gcttcaattg cagcctctgc ctcaatggga ccgtgcacct ctcctgccag   420

gagaaacaga acaccgtgtg cacctgccat gcaggtttct ttctaagaga aaacgagtgt   480

gtctcctgta gtaactgtaa gaaaagcctg gagtgcacga agttgtgcct accccagatt   540

gagaatgtta agggcactga ggacteaggg atccccaagg tggacaagaa agttgagccc   600

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga   660

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct   720

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg   780

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac   840

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag   900

gagtacaagt gcagggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc   960

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag  1020

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc  1080

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg  1140

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg  1200

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg    1260

cagaagagcc tctccctgtc tecgggtaaa                                     1290

<210>74

<211>430

<212>PRT

<213>Artificial sequence

<400>74

Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro His Leu Gly

1               5                   10                  15

Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His

            20                  25                  30

Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr

        35                  40                  45

Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Glu

    50                  55                  60

Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys

65                  70                  75                  80

Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser

                85                  90                  95

Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln

            100                  105                  110

Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys Ser

        115                  120                  125

Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu Lys Gln Asn

    130                 135                 140

Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu Asn Glu Cys

145                 150                 155                 160

Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr Lys Leu Cys

                165                 170                 175

Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser Gly Ile Pro

            180                 185                 190

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

        195                 200                 205

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

    210                 215                 220

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

225                 230                 235                 240

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

                245                 250                 255

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

            260                 265                 270

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

        275                 280                 285

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

    290                 295                 300

Arg Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

305                 310                 315                 320

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

                325                 330                 335

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

            340                 345                 350

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

        355                 360                 365

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

    370                 375                 380

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

385                 390                 395              400

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

                405                 410             415

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

            420                 425             430

<210>75

<211>1284

<212>DNA

<213>Artificial sequence

<400>75

atatacccct caggggttat tggactggtc cctcacctag gggacaggga gaagagagat     60

agtgtgtgtc cccaaggaaa atatatccac cctcaaaata attcgatttg ctgtaccaag    120

tgccacaaag gaacctactt gtacaatgac tgtccaggcc cggggcagga tacggactgc    180

agggagtgtg agagcggctc cttcaccgct tcagaaaacc acctcagaca ctgcctcagc    240

tgctccaaat gccgaaagga aatgggtcag gtggagatct cttcttgcac agtggaccgg    300

gacaccgtgt gtggctgcag gaagaaccag taccggcatt attggagtga aaaccttttc    360

cagtgcttca attgcagcct ctgcctcaat gggaccgtgc acctctcctg ccaggagaaa    420

cagaacaccg tgtgcacctg ccatgcaggt ttctttctaa gagaaaacga gtgtgtctcc    480

tgtagtaact gtaagaaaag cctggagtgc acgaagttgt gcctacccca gattgagaat    540

gttaagggca ctgaggactc agggatcccc aaggtggaca agaaagttga gcccaaatct    600

tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca    660

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc    720

acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg   780

gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg   840

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac   900

aagtgcaggg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc   960

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc  1020

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg  1080

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac  1140

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag  1200

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag  1260

agcctctccc tgtctccggg taaa                                         1284

<210>76

<211>428

<212>PRT

<213>Artificial sequence

<400>76

Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro His Leu Gly Asp Arg

1               5                   10                  15

Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His Pro Gln

            20                  25                  30

Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr

        35                  40                  45

Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu

    50                  55                  60

Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser

65                  70                  75                  80

Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys

                85                  90                  95

Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg

            100                 105                 110

His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys Ser Leu Cys

        115                 120                 125

Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu Lys Gln Asn Thr Val

    130                 135                 140

Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu Asn Glu Cys Val Ser

145                 150                 155                 160

Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu Pro

                165                 170                 175

Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser Gly Ile Pro Lys Val

            180                 185                 190

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

        195                 200                 205

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

    210                 215                 220

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

225                 230                 235                 240

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

                245                 250                 255

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

            260                 265                 270

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

        275                 280                 285

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Arg Val

    290                 295                 300

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

305                 310                 315                 320

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

                325                 330                 335

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

            340                 345                 350

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

        355                 360                 365

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

    370                 375                 380

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

385                 390                 395                 400

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

                405                 410                 415

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

            420                 425

<210>77

<211>1299

<212>DNA

<213>Artificial sequence

<400>77

gtgggaatat acccctcagg ggttattgga ctggtccctc acctagggga cagggagaag    60

agagatagtg tgtgtcccca aggaaaatat atccaccctc aaaataattc gatttgctgt   120

accaagtgcc acaaaggaac ctacttgtac aatgactgtc caggcccggg gcaggatacg   180

gactgcaggg agtgtgagag cggctccttc accgcttcag aaaaccacct cagacactgc   240

ctcagctgct ccaaatgccg aaaggaaatg ggtcaggtgg agatctcttc ttgcacagtg   300

gaccgggaca ccgtgtgtgg ctgcaggaag aaccagtacc ggcattattg gagtgaaaac   360

cttttccagt gcttcaattg cagcctctgc ctcaatggga ccgtgcacct ctcctgccag   420

gagaaacaga acaccgtgtg cacctgccat gcaggtttct ttctaagaga aaacgagtgt   480

gtctcctgta gtaactgtaa gaaaagcctg gagtgcacga agttgtgcct accccagatt   540

gagaatgtta agggcactga ggactcaggg ggatccagca acaccaaggt ggacaagaaa   600

gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc   660

ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc   720

cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag   780

ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag   840

cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg   900

aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa   960

accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc  1020

cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc  1080

agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg  1140

cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag  1200

agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac  1260

cactacacgc agaagagcct ctccctgtct ccgggtaaa                         1299

<210>78

<211>433

<212>PRT

<213>Artificial sequence

<400>78

Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro His Leu Gly

1               5                   10                  15

Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His

            20                  25                  30

Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr

        35                  40                  45

Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Glu

    50                  55                  60

Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys

65                  70                  75                  80

Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser

                85                  90                  95

Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln

            100                 105                 110

Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys Ser

        115                 120                 125

Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu Lys Gln Asn

    130                 135                 140

Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu Asn Glu Cys

145                 150                 155                 160

Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr Lys Leu Cys

                165                 170                 175

Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser Gly Gly Ser

            180                 185                 190

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

        195                 200                 205

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

    210                 215                 220

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

225                 230                 235                 240

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

                245                 250                 255

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

            260                 265                 270

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

        275                 280                 285

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

    290                 295                 300

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

305                 310                 315                 320

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

                325                 330                 335

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

            340                 345                 350

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

        355                 360                 365

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

    370                 375                 380

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

385                 390                 395                 400

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

                405                 410                 415

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

            420                 425                 430

Lys

<210>79

<211>1293

<212>DNA

<213>Artificial sequence

<400>79

atatacccct caggggttat tggactggtc cctcacctag gggacaggga gaagagagat    60

agtgtgtgtc cccaaggaaa atatatccac cctcaaaata attcgatttg ctgtaccaag   120

tgccacaaag gaacctactt gtacaatgac tgtccaggcc cggggcagga tacggactgc   180

agggagtgtg agagcggctc cttcaccgct tcagaaaacc acctcagaca ctgcctcagc   240

tgctccaaat gccgaaagga aatgggtcag gtggagatct cttcttgcac agtggaccgg   300

gacaccgtgt gtggctgcag gaagaaccag taccggcatt attggagtga aaaccttttc   360

cagtgcttca attgcagcct ctgcctcaat gggaccgtgc acctctcctg ccaggagaaa   420

cagaacaccg tgtgcacctg ccatgcaggt ttctttctaa gagaaaacga gtgtgtctcc   480

tgtagtaact gtaagaaaag cctggagtgc acgaagttgt gcctacccca gattgagaat   540

gttaagggca ctgaggactc agggggatcc agcaacacca aggtggacaa gaaagttgag   600

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg   660

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc   720

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac   780

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac   840

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc   900

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc   960

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat  1020

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac  1080

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc  1140

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg    1200

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac    1260

acgcagaaga gcctctccct gtctccgggt aaa                                 1293

<210>80

<211>431

<212>PRT

<213>Artificial sequence

<400>80

Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro His Leu Gly Asp Arg

1               5                   10                  15

Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His Pro Gln

            20                  25                  30

Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr

        35                  40                  45

Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu

    50                  55                  60

Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser

65                  70                  75                  80

Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys

                85                  90                  95

Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg

            100                 105                 110

His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys Ser Leu Cys

        115                 120                 125

Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu Lys Gln Asn Thr Val

    130                 135                 140

Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu Asn Glu Cys Val Ser

145                 150                 155                 160

Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu Pro

                165                 170                 175

Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser Gly Gly Ser Ser Asn

            180                 185                 190

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

        195                 200                 205

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

    210                 215                 220

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

225                 230                 235                 240

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

                245                 250                 255

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

            260                 265                 270

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

        275                 280                 285

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

    290                 295                 300

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

305                 310                 315                 320

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

                325                 330                 335

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

            340                 345                 350

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

        355                 360                 365

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

    370                 375                 380

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

385                 390                 395                 400

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

                405                 410                 415

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

            420                 425                 430

<210>81

<211>1347

<212>DNA

<213>Artificial sequence

<400>81

atgggcctct ccaccgtgcc tgacctgctg ctgccgctgg tgctcctgga gctgttggtg     60

ggaatatacc cctcaggggt tattggactg gtccctcacc taggggacag ggagaagaga    120

gatagtgtgt gtccccaagg aaaatatatc caccctcaaa ataattcgat ttgctgtacc    180

aagtgccaca aaggaaccta cttgtacaat gactgtccag gcccggggca ggatacggac    240

tgcagggagt gtgagagcgg ctccttcacc gcttcagaaa accacctcag acactgcctc    300

agctgctcca aatgccgaaa ggaaatgggt caggtggaga tctcttcttg cacagtggac    360

cgggacaccg tgtgtggctg caggaagaac cagtaccggc attattggag tgaaaacctt    420

ttccagtgct tcaattgcag cctctgcctc aatgggaccg tgcacctctc ctgccaggag    480

aaacagaaca ccgtgtgcac ctgccatgca ggtttctttc taagagaaaa cgagtgtgtc    540

tcctgtagta actgtaagaa aagcctggag tgcacgaagt tgtgcctacc ccagattgag    600

aatgttaagg gcactgagga ctcagggatc cccaaggtgg acaagaaagt tgagcccaaa    660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg   720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag   780

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac   840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc   900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag   960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa  1020

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg  1080

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc  1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg  1200

gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag  1260

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag  1320

aagagcctct ccctgtctcc gggtaaa                                      1347

<210>82

<211>449

<212>PRT

<213>Artificial sequence

<400>82

Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu

1               5                   10                  15

Glu Leu Leu Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro

            20                  25                  30

His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys

        35                  40                  45

Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys

    50                  55                  60

Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp

65                  70                  75                  80

Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu

                85                  90                  95

Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val

            100                 105                 110

Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg

        115                 120                 125

Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe

    130                 135                 140

Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu

145                 150                 155                 160

Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu

                165                 170                 175

Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr

            180                 185                 190

Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser

        195                 200                 205

Gly Ile Pro Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

    210                 215                 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225                 230                 235                 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

                245                 250                 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

            260                 265                 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

        275                 280                 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

    290                 295                 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305                 310                 315                 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

                325                 330                 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

            340                 345                 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

        355                 360                 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

    370                 375                 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385                 390                 395                 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

                405                 410                 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

            420                 425                 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

        435                 440                 445

Lys

<210>83

<211>1347

<212>DNA

<213>Artificial sequence

<400>83

atgggcctct ccaccgtgcc tgacctgctg ctgccgctgg tgctcctgga gctgttggtg     60

ggaatatacc cctcaggggt tattggactg gtccctcacc taggggacag ggagaagaga    120

gatagtgtgt gtccccaagg aaaatatatc caccctcaaa ataattcgat ttgctgtacc    180

aagtgccaca aaggaaccta cttgtacaat gactgtccag gcccggggca ggatacggac    240

tgcagggagt gtgagagcgg ctccttcacc gcttcagaaa accacctcag acactgcctc    300

agctgctcca aatgccgaaa ggaaatgggt caggtggaga tctcttcttg cacagtggac    360

cgggacaccg tgtgtggctg caggaagaac cagtaccggc attattggag tgaaaacctt    420

ttccagtgct tcaattgcag cctctgcctc aatgggaccg tgcacctctc ctgccaggag    480

aaacagaaca ccgtgtgcac ctgccatgca ggtttctttc taagagaaaa cgagtgtgtc    540

tcctgtagta actgtaagaa aagcctggag tgcacgaagt tgtgcctacc ccagattgag    600

aatgttaagg gcactgagga ctcagggatc cccaaggtgg acaagaaagt tgagcccaaa    660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg    720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag    780

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac    840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc    900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag    960

tacaagtgca gggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa   1020

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg   1080

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc   1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg   1200

gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag   1260

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag   1320

aagagcctct ccctgtctcc gggtaaa                                       1347

<210>84

<211>449

<212>PRT

<213>Artificial sequence

<400>84

Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu

1               5                   10                  15

Glu Leu Leu Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro

            20                  25                  30

His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys

        35                  40                  45

Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys

    50                  55                  60

Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp

65                  70                  75                  80

Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu

                85                  90                  95

Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val

            100                 105                 110

Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg

        115                 120                 125

Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe

    130                 135                 140

Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu

145                 150                 155                 160

Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu

                165                 170                 175

Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr

            180                 185                 190

Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser

        195                 200                 205

Gly Ile Pro Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

    210                 215                 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225                 230                 235                 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

                245                 250                 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

            260                 265                 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

        275                 280                 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

    290                 295                 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305                 310                 315                 320

Tyr Lys Cys Arg Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

                325                 330                 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

            340                 345                 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

        355                 360                 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

    370                 375                 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385                 390                 395                 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

                405                 410                 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

            420                 425                 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

        435                 440                 445

Lys

<210>85

<211>1356

<212>DNA

<213>Artificial sequence

<400>85

atgggcctct ccaccgtgcc tgacctgctg ctgccgctgg tgctcctgga gctgttggtg     60

ggaatatacc cctcaggggt tattggactg gtccctcacc taggggacag ggagaagaga    120

gatagtgtgt gtccccaagg aaaatatatc caccctcaaa ataattcgat ttgctgtacc    180

aagtgccaca aaggaaccta cttgtacaat gactgtccag gcccggggca ggatacggac    240

tgcagggagt gtgagagcgg ctccttcacc gcttcagaaa accacctcag acactgcctc    300

agctgctcca aatgccgaaa ggaaatgggt caggtggaga tctcttcttg cacagtggac    360

cgggacaccg tgtgtggctg caggaagaac cagtaccggc attattggag tgaaaacctt    420

ttccagtgct tcaattgcag cctctgcctc aatgggaccg tgcacctctc ctgccaggag    480

aaacagaaca ccgtgtgcac ctgccatgca ggtttctttc taagagaaaa cgagtgtgtc    540

tcctgtagta actgtaagaa aagcctggag tgcacgaagt tgtgcctacc ccagattgag    600

aatgttaagg gcactgagga ctcaggggga tccagcaaca ccaaggtgga caagaaagtt   660

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg   720

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg   780

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc   840

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag   900

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat   960

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc  1020

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg  1080

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc  1140

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct  1200

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc  1260

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac  1320

tacacgcaga agagcctctc cctgtctccg ggtaaa                            1356

<210>86

<211>452

<212>PRT

<213>Artificial sequence

<400>86

Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu

1               5                   10                  15

Glu Leu Leu Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro

            20                  25                  30

His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys

        35                  40                  45

Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys

    50                  55                  60

Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp

65                  70                  75                  80

Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu

                85                  90                  95

Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val

            100                 105                 110

Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg

        115                 120                 125

Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe

    130                 135                 140

Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu

145                 150                 155                 160

Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu

                165                 170                 175

Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr

            180                 185                 190

Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser

        195                 200                 205

Gly Gly Ser Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

    210                 215                 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225                 230                 235                 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

                245                 250                 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

            260                 265                 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

        275                 280                 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

    290                 295                 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305                 310                 315                 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

                325                 330                 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

            340                 345                 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

        355                 360                 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

    370                 375                 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385                 390                 395                 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

                405                 410                 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

            420                 425                 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

        435                 440                 445

Ser Pro Gly Lys

    450

<210>87

<211>27

<212>DNA

<213>Artificial sequence

<400>87

ggcgcgatat cagggggcaa ccggacc                                        27

<210>88

<211>30

<212>DNA

<213>Human sequence

<400>88

gagcgaagtc gccagggatc ccttcagctg                                     30

<210>89

<211>66

<212>DNA

<213>People

<400>89

atggcgcccg tcgccgtctg ggccgcgctg gccgtcggac tggagctctg ggctgcggcg    60

cacgcc                                                               66

<210>90

<211>22

<212>PRT

<213>People

<400>90

Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu

1               5                   10                  15

Trp Ala Ala Ala His Ala

            20

<210>91

<211>693

<212>DNA

<213>People

<400>91

ttgcccgccc aggtggcatt tacaccctac gccccggagc ccgggagcac atgccggctc     60

agagaatact atgaccagac agctcagatg tgctgcagca aatgctcgcc gggccaacat    120

gcaaaagtct tctgtaccaa gacctcggac accgtgtgtg actcetgtga ggacagcaca    180

tacacccagc tctggaactg ggttcccgag tgcttgagct gtggctcccg ctgtagctct    240

gaccaggtgg aaactcaagc ctgcactcgg gaacagaacc gcatctgcac ctgeaggccc    300

ggctggtact gcgcgctgag caagcaggag gggtgccggc tgtgcgcgcc gctgcgcaag    360

tgccgcccgg gcttcggcgt ggccagacca ggaactgaaa catcagacgt ggtgtgcaag    420

ccctgtgccc cggggacgtt ctccaacacg acttcatcca cggatatttg caggccccac    480

cagatctgta acgtggtggc catccctggg aatgcaagca tggatgcagt ctgcacgtcc    540

acgtccccca cccggagtat ggccccaggg gcagtacact taccccagcc agtgtccaca    600

cgatcccaac acacgcagcc aactccagaa cccagcactg ctceaagcac ctccttcctg    660

ctcccaatgg gccccagccc cccagctgaa ggg                                 693

<210>92

<211>231

<212>PRT

<213>People

<400>92

Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser

1               5                   10                  15

Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys

            20                  25                  30

Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr

        35                  40                  45

Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu

    50                  55                  60

Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser

65                  70                  75                  80

Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys

                85                  90                  95

Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys

            100                 105                 110

Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala

        115                 120                 125

Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro

    130                 135                 140

Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His

145                 150                 155                 160

Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala

                165                 170                 175

Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val

            180                 185                 190

His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr

        195                 200                 205

Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly

    210                 215                 220

Pro Ser Pro Pro Ala Glu Gly

225                 230

<210>93

<211>693

<212>DNA

<213>People

<400>93

ttgcccgccc aggtggcatt tacaccctac gccccggagc ccgggagcac atgccggctc     60

agagaatact atgaccagac agctcagatg tgctgcagca agtgctcgcc gggccaacat    120

gcaaaagtct tctgtaccaa gacctcggac accgtgtgtg actcctgtga ggacagcaca    180

tacacccagc tctggaactg ggttcccgag tgcttgagct gtggctcccg ctgtagctct    240

gaccaggtgg aaactcaagc ctgcactcgg gaacagaacc gcatctgcac ctgcaggccc    300

ggctggtact gcgcgctgag caagcaggag gggtgccggc tgtgcgcgcc gctgcgcaag    360

tgccgcccgg gcttcggcgt ggccagacca ggaactgaaa catcagacgt ggtgtgcaag    420

ccctgtgccc cggggacgtt ctccaacacg acttcatcca cggatatttg caggccccac    480

cagatctgta acgtggtggc catccctggg aatgcaagca gggatgcagt  ctgcacgtcc   540

acgtccccca cccggagtat ggccccaggg gcagtacact taccccagcc agtgtccaca    600

cgatcccaac acacgcagcc aactccagaa cccagcactg ctccaagcac ctccttcctg    660

ctcccaatgg gccccagccc cccagctgaa ggg                                 693

<210>94

<211>231

<212>PRT

<213>People

<400>94

Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser

1               5                   10                  15

Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys

            20                  25                  30

Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr

        35                  40                  45

Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu

    50                  55                  60

Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser

65                  70                  75                  80

Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys

                85                  90                  95

Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys

            100                 105                 110

Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala

        115                 120                 125

Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro

    130                 135                 140

Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His

145                 150                 155                 160

Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Arg Asp Ala

                165                 170                 175

Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val

            180                 185                 190

His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr

        195                 200                 205

Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly

    210                 215                 220

Pro Ser Pro Pro Ala Glu Gly

225                 230

<210>95

<211>759

<212>DNA

<213>Artificial sequence

<400>95

atggcgcccg tcgccgtctg ggccgcgctg gccgtcggac tggagctctg ggctgcggcg     60

cacgccttgc ccgcccaggt ggcatttaca ccctacgccc cggagcccgg gagcacatgc    120

cggctcagag aatactatga ccagacagct cagatgtgct gcagcaaatg ctcgccgggc    180

caacatgcaa aagtcttctg taccaagacc tcggacaccg tgtgtgactc ctgtgaggac    240

agcacataca cccagctctg gaactgggtt cccgagtgct tgagctgtgg ctcccgctgt    300

agctctgacc aggtggaaac tcaagcctgc actcgggaac agaaccgcat ctgcacctgc    360

aggcccggct ggtactgcgc gctgagcaag caggaggggt gccggctgtg cgcgccgctg    420

cgcaagtgcc gcccgggctt cggcgtggcc agaccaggaa ctgaaacatc agacgtggtg    480

tgcaagccct gtgccccggg gacgttctcc aacacgactt catccacgga tatttgcagg    540

ccccaccaga tctgtaacgt ggtggccatc cctgggaatg caagcatgga tgcagtctgc    600

acgtccacgt cccccacccg gagtatggcc ccaggggcag tacacttacc ccagccagtg    660

tccacacgat cccaacacac gcagccaact ccagaaccca gcactgctcc aagcacctcc    720

ttcctgctcc caatgggccc cagcccccca gctgaaggg                           759

<210>96

<211>253

<212>PRT

<213>Artificial sequence

<400>96

Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu

1               5                   10                  15

Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr

            20                  25                  30

Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln

        35                  40                  45

Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys

    50                  55                  60

Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp

65                  70                  75                  80

Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys

                85                  90                  95

Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg

            100                 105                 110

Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu

        115                 120                 125

Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg

    130                 135                 140

Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val

145                 150                 155                 160

Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr

                165                 170                 175

Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val AlaIle Pro Gly

            180                 185                 190

Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser

        195                 200                 205

Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser

    210                 215                 220

Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser

225                 230                 235                 240

Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly

                245                 250

<210>97

<211>759

<212>DNA

<213>Artificial sequence

<400>97

atggcgcccg tcgccgtctg ggccgcgctg gccgtcggac tggagctctg ggctgcggcg     60

cacgccttgc ccgcccaggt ggcatttaca ccctacgccc cggagcccgg gagcacatgc    120

cggctcagag aatactatga ccagacagct cagatgtgct gcagcaagtg ctcgccgggc    180

caacatgcaa aagtcttctg taccaagacc tcggacaccg tgtgtgactc ctgtgaggac    240

agcacataca cccagctctg gaactgggtt cccgagtgct tgagctgtgg ctcccgctgt    300

agctctgacc aggtggaaac tcaagcctgc actcgggaac agaaccgcat ctgcacctgc    360

aggcccggct ggtactgcgc gctgagcaag caggaggggt gccggctgtg cgcgccgctg    420

cgcaagtgcc gcccgggctt cggcgtggcc agaccaggaa ctgaaacatc agacgtggtg    480

tgcaagccct gtgccccggg gacgttctcc aacacgactt catccacgga tatttgcagg    540

ccccaccaga tctgtaacgt ggtggccatc cctgggaatg caagcaggga tgcagtctgc    600

acgtccacgt cccccacccg gagtatggcc ccaggggcag tacacttacc ccagccagtg    660

tccacacgat cccaacacac gcagccaact ccagaaccca gcactgctcc aagcacctcc    720

ttcctgctcc caatgggccc cagcccccca gctgaaggg    759

<210>98

<211>253

<212>PRT

<213>Artificial sequence

<400>98

Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu

1               5                   10                  15

Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr

            20                  25                  30

Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln

        35                  40                  45

Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys

    50                  55                  60

Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp

65                  70                  75                  80

Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys

                85                  90                  95

Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg

            100                 105                 110

Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu

        115                 120                 125

Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg

    130                 135                 140

Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val

145                 150                 155                 160

Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr

                165                 170                 175

Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly

            180                 185                 190

Asn Ala Ser Arg Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser

        195                 200                 205

Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser

    210                 215                 220

Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser

225                 230                 235                 240

Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly

                245                 250

<210>99

<211>1413

<212>DNA

<213>Artificial sequence

<400>99

ttgcccgccc aggtggcatt tacaccctac gccccggagc ccgggagcac atgccggctc     60

agagaatact atgaccagac agctcagatg tgctgcagca aatgctcgcc gggccaacat    120

gcaaaagtct tctgtaccaa gacctcggac accgtgtgtg actcctgtga ggacagcaca    180

tacacccagc tctggaactg ggttcccgag tgcttgagct gtggctcccg ctgtagctct    240

gaccaggtgg aaactcaagc ctgcactcgg gaacagaacc gcatctgcac ctgcaggccc    300

ggctggtact gcgcgctgag caagcaggag gggtgccggc tgtgcgcgcc gctgcgcaag    360

tgccgcccgg gcttcggcgt ggccagacca ggaactgaaa catcagacgt ggtgtgcaag    420

ccctgtgccc cggggacgtt ctccaacacg acttcatcca cggatatttg caggccccac    480

cagatctgta acgtggtggc catccctggg aatgcaagca tggatgcagt ctgcacgtcc     540

acgtccccca cccggagtat ggccccaggg gcagtacact taccccagcc agtgtccaca     600

cgatcccaac acacgcagcc aactccagaa cccagcactg ctccaagcac ctccttcctg     660

ctcccaatgg gccccagccc cccagctgaa gggatcccca aggtggacaa gaaagttgag     720

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg     780

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc     840

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac     900

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac     960

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc    1020

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc    1080

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat    1140

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac    1200

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc    1260

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg    1320

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac    1380

acgcagaaga gcctctccct gtctccgggt aaa                                 1413

<210>100

<211>471

<212>PRT

<213>Artificial sequence

<400>100

Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser

1               5                   10                  15

Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys

            20                  25                  30

Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr

        35                  40                  45

Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu

    50                  55                  60

Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser

65                  70                  75                  80

Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys

                85                  90                  95

Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys

            100                 105                 110

Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala

        115                 120                 125

Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro

    130                 135                 140

Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His

145                 150                 155                 160

Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala

                165                 170                 175

Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val

            180                 185                 190

His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr

        195                 200                 205

Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly

    210                 215                 220

Pro Ser Pro Pro Ala Glu Gly Ile Pro Lys Val Asp Lys Lys Val Glu

225                 230                 235                 240

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

                245                 250                 255

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

            260                 265                 270

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

        275                 280                 285

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

    290                 295                 300

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

305                 310                 315                 320

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

                325                 330                 335

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

            340                 345                 350

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

        355                 360                 365

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

    370                 375                 380

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

385                 390                 395                 400

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

                405                 410                 415

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

            420                 425                 430

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

        435                 440                 445

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

    450                 455                 460

Leu Ser Leu Ser Pro Gly Lys

465                 470

<210>101

<211>1413

<212>DNA

<213>Artificial sequence

<400>101

ttgcccgccc aggtggcatt tacaccctac gccccggagc ccgggagcac atgccggctc     60

agagaatact atgaccagac agctcagatg tgctgcagca agtgctcgcc gggccaacat    120

gcaaaagtct tctgtaccaa gacctcggac accgtgtgtg actcctgtga ggacagcaca    180

tacacccagc tctggaactg ggttcccgag tgcttgagct gtggctcccg ctgtagctct    240

gaccaggtgg aaactcaagc ctgcactcgg gaacagaacc gcatctgcac ctgcaggccc    300

ggctggtact gcgcgctgag caagcaggag gggtgccggc tgtgcgcgcc gctgcgcaag    360

tgccgcccgg gcttcggcgt ggccagacca ggaactgaaa catcagacgt ggtgtgcaag    420

ccctgtgccc cggggacgtt ctccaacacg acttcatcca cggatatttg caggccccac    480

cagatctgta acgtggtggc catccctggg aatgcaagca gggatgcagt ctgcacgtcc    540

acgtccccca cccggagtat ggccccaggg gcagtacact taccccagcc agtgtccaca    600

cgatcccaac acacgcagcc aactccagaa cccagcactg ctccaagcac ctccttcctg    660

ctcccaatgg gccccagccc cccagctgaa gggatcccca aggtggacaa gaaagttgag    720

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg    780

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc    840

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac    900

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac    960

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc   1020

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc   1080

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat    1140

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac    1200

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc    1260

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg    1320

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac    1380

acgcagaaga gcctctccct gtctccgggt aaa                                 1413

<210>102

<211>471

<212>PRT

<213>Artificial sequence

<400>102

Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser

1               5                   10                  15

Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys

            20                  25                  30

Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr

        35                  40                  45

Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu

    50                  55                  60

Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser

65                  70                  75                  80

Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys

                85                  90                  95

Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys

            100                 105                 110

Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala

        115                 120                 125

Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro

    130                 135                 140

Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His

145                 150                 155                 160

Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Arg Asp Ala

                165                 170                 175

Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val

            180                 185                 190

His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr

        195                 200                 205

Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly

    210                 215                 220

Pro Ser Pro Pro Ala Glu Gly Ile Pro Lys Val Asp Lys Lys Val Glu

225                 230                 235                 240

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

                245                 250                 255

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

            260                 265                 270

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

        275                 280                 285

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

    290                 295                 300

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

305                 310                 315                 320

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

                325                 330                 335

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

            340                 345                 350

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

        355                 360                 365

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

    370                 375                 380

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

385                 390                 395                 400

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

                405                 410                 415

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

            420                 425                 430

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

        435                 440                 445

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

    450                 455                 460

Leu Ser Leu Ser Pro Gly Lys

465                 470

<210>103

<211>1413

<212>DNA

<213>Artificial sequence

<400>103

ttgcccgccc aggtggcatt tacaccctac gccccggagc ccgggagcac atgccggctc    60

agagaatact atgaccagac agctcagatg tgctgcagca aatgctcgcc gggccaacat    120

gcaaaagtct tctgtaccaa gacctcggac accgtgtgtg actcctgtga ggacagcaca    180

tacacccagc tctggaactg ggttcccgag tgcttgagct gtggctcccg ctgtagctct    240

gaccaggtgg aaactcaagc ctgcactcgg gaacagaacc gcatctgcac ctgcaggccc    300

ggctggtact gcgcgctgag caagcaggag gggtgccggc tgtgcgcgcc gctgcgcaag    360

tgccgcccgg gcttcggcgt ggccagacca ggaactgaaa catcagacgt ggtgtgcaag    420

ccctgtgccc cggggacgtt ctccaacacg acttcatcca cggatatttg caggccccac    480

cagatctgta acgtggtggc catccctggg aatgcaagca tggatgcagt ctgcacgtcc    540

acgtccccca cccggagtat ggccccaggg gcagtacact taccccagcc agtgtccaca    600

cgatcccaac acacgcagcc aactccagaa cccagcactg ctccaagcac ctccttcctg    660

ctcccaatgg gccccagccc cccagctgaa gggatcccca aggtggacaa gaaagttgag    720

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg    780

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc    840

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac    900

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac    960

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc   1020

aaggagtaca agtgcagggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc   1080

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat   1140

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac   1200

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc   1260

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg   1320

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac   1380

acgcagaaga gcctctccct gtctccgggt aaa                                1413

<210>104

<211>471

<212>PRT

<213>Artificial sequence

<400>104

Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser

1               5                   10                  15

Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys

            20                  25                  30

Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr

        35                  40                  45

Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu

    50                  55                  60

Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser

65                  70                  75                  80

Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys

                85                  90                  95

Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys

            100                 105                 110

Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala

        115                 120                 125

Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro

    130                 135                 140

Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His

145                 150                 155                 160

Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala

                165                 170                 175

Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val

            180                 185                 190

His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr

        195                 200                 205

Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly

    210                 215                 220

Pro Ser Pro Pro Ala Glu Gly Ile Pro Lys Val Asp Lys Lys Val Glu

225                 230                 235                 240

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

                245                 250                 255

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

            260                 265                 270

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

        275                 280                 285

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

    290                 295                 300

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

305                 310                 315                 320

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

                325                 330                 335

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Arg Val Ser Asn Lys Ala Leu

            340                 345                 350

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

        355                 360                 365

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

    370                 375                 380

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

385                 390                 395                 400

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

                405                 410                 415

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

            420                 425                 430

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

        435                 440                 445

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

    450                 455                 460

Leu Ser Leu Ser Pro Gly Lys

465                 470

<210>105

<211>1413

<212>DNA

<213>Artificial sequence

<400>105

ttgcccgccc aggtggcatt tacaccctac gccccggagc ccgggagcac atgccggctc     60

agagaatact atgaccagac agctcagatg tgctgcagca aatgctcgcc gggccaacat    120

gcaaaagtct tctgtaccaa gacctcggac accgtgtgtg actcctgtga ggacagcaca    180

tacacccagc tctggaactg ggttcccgag tgcttgagct gtggctcccg ctgtagctct    240

gaccaggtgg aaactcaagc ctgcactcgg gaacagaacc gcatctgcac ctgcaggccc    300

ggctggtact gcgcgctgag caagcaggag gggtgccggc tgtgcgcgcc gctgcgcaag    360

tgccgcccgg gcttcggcgt ggccagacca ggaactgaaa catcagacgt ggtgtgcaag    420

ccctgtgccc cggggacgtt ctccaacacg acttcatcca cggatatttg caggccccac    480

cagatctgta acgtggtggc catccctggg aatgcaagca gggatgcagt ctgcacgtcc    540

acgtccccca cccggagtat ggccccaggg gcagtacact taccccagcc agtgtccaca    600

cgatcccaac acacgcagcc aactccagaa cccagcactg ctccaagcac ctccttcctg    660

ctcccaatgg gccccagccc cccagctgaa gggatcccca aggtggacaa gaaagttgag   720

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg   780

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc   840

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac   900

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac   960

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc  1020

aaggagtaca agtgcagggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc  1080

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat  1140

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac  1200

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc  1260

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg  1320

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac  1380

acgcagaaga gcctctccct gtctccgggt aaa                               1413

<210>106

<211>471

<212>PRT

<213>Artificial sequence

<400>106

Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser

1               5                   10                  15

Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys

            20                  25                  30

Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr

        35                  40                  45

Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu

    50                  55                  60

Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser

65                  70                  75                  80

Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys

                 85                  90                  95

Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys

            100                 105                 110

Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala

        115                 120                 125

Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro

    130                 135                 140

Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His

145                 150                 155                 160

Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Arg Asp Ala

                165                 170                 175

Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val

            180                 185                 190

His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr

        195                 200                 205

Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly

    210                 215                 220

Pro Ser Pro Pro Ala Glu Gly Ile Pro Lys Val Asp Lys Lys Val Glu

225                 230                 235                 240

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

                245                 250                 255

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

            260                 265                 270

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

        275                 280                 285

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

    290                 295                 300

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

305                 310                 315                 320

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

                325                 330                 335

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Arg Val Ser Asn Lys Ala Leu

             340                 345                 350

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

        355                 360                 365

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

    370                 375                 380

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

385                 390                 395                 400

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

                405                 410                 415

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

            420                 425                 430

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

        435                 440                 445

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

    450                 455                 460

Leu Ser Leu Ser Pro Gly Lys

465                 470

<210>107

<211>1422

<212>DNA

<213>Artificial sequence

<400>107

ttgcccgccc aggtggcatt tacaccctac gccccggagc ccgggagcac atgccggctc     60

agagaatact atgaccagac agctcagatg tgctgcagca aatgctcgcc gggccaacat    120

gcaaaagtct tctgtaccaa gacctcggac accgtgtgtg actcctgtga ggacagcaca    180

tacacccagc tctggaactg ggttcccgag tgcttgagct gtggctcccg ctgtagctct    240

gaccaggtgg aaactcaagc ctgcactcgg gaacagaacc gcatctgcac ctgcaggccc    300

ggctggtact gcgcgctgag caagcaggag gggtgccggc tgtgcgcgcc gctgcgcaag    360

tgccgcccgg gcttcggcgt ggccagacca ggaactgaaa catcagacgt ggtgtgcaag    420

ccctgtgccc cggggacgtt ctccaacacg acttcatcca cggatatttg caggccccac    480

cagatctgta acgtggtggc catccctggg aatgcaagca tggatgcagt ctgcacgtcc    540

acgtccccca cccggagtat ggccccaggg gcagtacact taccccagcc agtgtccaca    600

cgatcccaac acacgcagcc aactccagaa cccagcactg ctccaagcac ctccttcctg    660

ctcccaatgg gccccagccc cccagctgaa gggggatcca gcaacaccaa ggtggacaag    720

aaagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa    780

ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc    840

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc    900

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag    960

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg   1020

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag   1080

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca   1140

tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat   1200

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc   1260

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac    1320

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac    1380

aaccactaca cgcagaagag cctctccctg tctccgggta aa                       1422

<210>108

<211>474

<212>PRT

<213>Artificial sequence

<400>108

Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser

1               5                   10                  15

Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys

            20                  25                  30

Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr

        35                  40                  45

Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu

    50                  55                  60

Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser

65                  70                  75                  80

Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys

                85                  90                  95

Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys

            100                 105                 110

Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala

        115                 120                 125

Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro

    130                 135                 140

Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His

145                 150                 155                 160

Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala

                165                 170                 175

Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val

            180                 185                 190

His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr

        195                 200                 205

Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly

    210                 215                 220

Pro Ser Pro Pro Ala Glu Gly Gly Ser Ser Asn Thr Lys Val Asp Lys

225                 230                 235                 240

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

                245                 250                 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

            260                 265                 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

        275                 280                 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

    290                 295                 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305                 310                 315                 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

                325                 330                 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

            340                 345                 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

        355                 360                 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

    370                 375                 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385                 390                 395                 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

                405                 410                 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

            420                 425                 430

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

        435                 440                 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

    450                 455                 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465                 470

<210>109

<211>1422

<212>DNA

<213>Artificial sequence

<400>109

ttgcccgccc aggtggcatt tacaccctac gccccggagc ccgggagcac atgccggctc     60

agagaatact atgaccagac agctcagatg tgctgcagca agtgctcgcc gggccaacat    120

gcaaaagtct tctgtaccaa gacctcggac accgtgtgtg actcctgtga ggacagcaca    180

tacacccagc tctggaactg ggttcccgag tgcttgagct gtggctcccg ctgtagctct    240

gaccaggtgg aaactcaagc ctgcactcgg gaacagaacc gcatctgcac ctgcaggccc    300

ggctggtact gcgcgctgag caagcaggag gggtgccggc tgtgcgcgcc gctgcgcaag    360

tgccgcccgg gcttcggcgt ggccagacca ggaactgaaa catcagacgt ggtgtgcaag    420

ccctgtgccc cggggacgtt ctccaacacg acttcatcca cggatatttg caggccccac    480

cagatctgta acgtggtggc catccctggg aatgcaagca gggatgcagt ctgcacgtcc    540

acgtccccca cccggagtat ggccccaggg gcagtacact taccccagcc agtgtccaca    600

cgatcccaac acacgcagcc aactccagaa cccagcactg ctccaagcac ctccttcctg    660

ctcccaatgg gccccagccc cccagctgaa gggggatcca gcaacaccaa ggtggacaag    720

aaagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa    780

ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc    840

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc    900

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag    960

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg   1020

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag   1080

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca   1140

tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat   1200

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc   1260

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac   1320

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac   1380

aaccactaca cgcagaagag cctctccctg tctccgggta aa                      1422

<210>110

<211>474

<212>PRT

<213>Artificial sequence

<400>110

Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser

1               5                   10                  15

Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys

            20                  25                  30

Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr

        35                  40                  45

Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu

    50                  55                  60

Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser

65                  70                  75                  80

Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys

                85                  90                  95

Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys

            100                 105                 110

Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala

        115                 120                 125

Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro

    130                 135                 140

Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His

145                 150                 155                 160

Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Arg Asp Ala

                165                 170                 175

Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val

            180                 185                 190

His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr

        195                 200                 205

Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly

     210                 215                 220

Pro Ser Pro Pro Ala Glu Gly Gly Ser Ser Asn Thr Lys Val Asp Lys

225                 230                 235                 240

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

                245                 250                 255

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

            260                 265                 270

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

        275                 280                 285

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

    290                 295                 300

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

305                 310                 315                 320

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

                325                 330                 335

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

            340                 345                 350

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

        355                 360                 365

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

   370                 375                 380

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

385                 390                 395                 400

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

                405                 410                 415

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

            420                 425                 430

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

        435                 440                 445

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

    450                 455                 460

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465                 470

<210>111

<211>1479

<212>DNA

<213>Artificial sequence

<400>111

atggcgcccg tcgccgtctg ggccgcgctg gccgtcggac tggagctctg ggctgcggcg   60

cacgccttgc ccgcccaggt ggcatttaca ccctacgccc cggagcccgg gagcacatgc  120

cggctcagag aatactatga ccagacagct cagatgtgct gcagcaaatg ctcgccgggc  180

caacatgcaa aagtcttctg taccaagacc tcggacaccg tgtgtgactc ctgtgaggac  240

agcacataca cccagctctg gaactgggtt cccgagtgct tgagctgtgg ctcccgctgt  300

agctctgacc aggtggaaac tcaagcctgc actcgggaac agaaccgcat ctgcacctgc  360

aggcccggct ggtactgcgc gctgagcaag caggaggggt gccggctgtg cgcgccgctg  420

cgcaagtgcc gcccgggctt cggcgtggcc agaccaggaa ctgaaacatc agacgtggtg  480

tgcaagccct gtgccccggg gacgttctcc aacacgactt catccacgga tatttgcagg  540

ccccaccaga tctgtaacgt ggtggccatc cctgggaatg caagcatgga tgcagtctgc  600

acgtccacgt cccccacccg gagtatggcc ccaggggcag tacacttacc ccagccagtg  660

tccacacgat cccaacacac gcagccaact ccagaaccca gcactgctcc aagcacctcc  720

ttcctgctcc caatgggccc cagcccccca gctgaaggga tccccaaggt ggacaagaaa  780

gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc  840

ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc  900

cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag   960

ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag  1020

cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg  1080

aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa  1140

accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc  1200

cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc  1260

agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg  1320

cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag  1380

agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac  1440

cactacacgc agaagagcct ctccctgtct ccgggtaaa                         1479

<210>112

<211>493

<212>PRT

<213>Artificial sequence

<400>112

Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu

1               5                   10                  15

Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr

            20                  25                  30

Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln

        35                  40                  45

Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys

    50                  55                  60

Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp

65                  70                  75                  80

Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys

                85                  90                  95

Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg

            100                 105                 110

Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu

        115                 120                 125

Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg

    130                 135                 140

Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val

145                 150                 155                 160

Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr

                165                 170                 175

Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly

            180                 185                 190

Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser

        195                 200                 205

Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser

    210                 215                 220

Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser

225                 230                 235                 240

Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ile Pro Lys

                245                 250                 255

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

            260                 265                 270

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

        275                 280                 285

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

    290                 295                 300

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

305                 310                 315                 320

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

                325                 330                 335

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

            340                 345                 350

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

        355                 360                 365

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

    370                 375                 380

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

385                 390                 395                 400

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

                405                 410                 415

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

            420                 425                 430

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

        435                 440                 445

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

    450                 455                 460

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

465                 470                 475                 480

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

                485                 490

<210>113

<211>1479

<212>DNA

<213>Artificial sequence

<400>113

atggcgcccg tcgccgtctg ggccgcgctg gccgtcggac tggagctctg ggctgcggcg     60

cacgccttgc ccgcccaggt ggcatttaca ccctacgccc cggagcccgg gagcacatgc    120

cggctcagag aatactatga ccagacagct cagatgtgct gcagcaagtg ctcgccgggc    180

caacatgcaa aagtcttctg taccaagacc tcggacaccg tgtgtgactc ctgtgaggac    240

agcacataca cccagctctg gaactgggtt cccgagtgct tgagctgtgg ctcccgctgt    300

agctctgacc aggtggaaac tcaagcctgc actcgggaac agaaccgcat ctgcacctgc    360

aggcccggct ggtactgcgc gctgagcaag caggaggggt gccggctgtg cgcgccgctg    420

cgcaagtgcc gcccgggctt cggcgtggcc agaccaggaa ctgaaacatc agacgtggtg    480

tgcaagccct gtgccccggg gacgttctcc aacacgactt catccacgga tatttgcagg    540

ccccaccaga tctgtaacgt ggtggccatc cctgggaatg caagcaggga tgcagtctgc    600

acgtccacgt cccccacccg gagtatggcc ccaggggcag tacacttacc ccagccagtg    660

tccacacgat cccaacacac gcagccaact ccagaaccca gcactgctcc aagcacctcc    720

ttcctgctcc caatgggccc cagcccccca gctgaaggga tccccaaggt ggacaagaaa    780

gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc    840

ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc    900

cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag    960

ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag   1020

cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg   1080

aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa   1140

accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc   1200

cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc   1260

agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg   1320

cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag    1380

agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac    1440

cactacacgc agaagagcct ctccctgtct ccgggtaaa                           1479

<210>114

<211>493

<212>PRT

<213>Artificial sequence

<400>114

Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu

1               5                   10                  15

Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr

            20                  25                  30

Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln

         35                  40                  45

Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys

    50                  55                  60

Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp

65                  70                  75                  80

Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys

                85                  90                  95

Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg

            100                 105                 110

Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu

        115                 120                 125

Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg

    130                 135                 140

Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val

145                 150                 155                 160

Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr

                165                 170                 175

Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly

            180                 185                 190

Asn Ala Ser Arg Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser

        195                 200                 205

Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser

    210                 215                 220

Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser

225                 230                 235                 240

Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ile Pro Lys

                245                 250                 255

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

            260                 265                 270

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

        275                 280                 285

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

    290                 295                 300

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

305                 310                 315                 320

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

                325                 330                 335

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

            340                 345                 350

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

        355                 360                 365

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

    370                 375                 380

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

385                 390                 395                 400

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

                405                 410                 415

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

            420                 425                 430

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

        435                 440                 445

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

    450                 455                 460

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

465                 470                 475                 480

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

                485                 490

<210>115

<211>1479

<212>DNA

<213>Artificial sequence

<400>115

atggcgcccg tcgccgtctg ggccgcgctg gccgtcggac tggagctctg ggctgcggcg   60

cacgccttgc ccgcccaggt ggcatttaca ccctacgccc cggagcccgg gagcacatgc  120

cggctcagag aatactatga ccagacagct cagatgtgct gcagcaaatg ctcgccgggc    180

caacatgcaa aagtcttctg taccaagacc tcggacaccg tgtgtgactc ctgtgaggac    240

agcacataca cccagctctg gaactgggtt cccgagtgct tgagctgtgg ctcccgctgt    300

agctctgacc aggtggaaac tcaagcctgc actcgggaac agaaccgcat ctgcacctgc    360

aggcccggct ggtactgcgc gctgagcaag caggaggggt gccggctgtg cgcgccgctg    420

cgcaagtgcc gcccgggctt cggcgtggcc agaccaggaa ctgaaacatc agacgtggtg    480

tgcaagccct gtgccccggg gacgttctcc aacacgactt catccacgga tatttgcagg    540

ccccaccaga tctgtaacgt ggtggccatc cctgggaatg caagcatgga tgcagtctgc    600

acgtccacgt cccccacccg gagtatggcc ccaggggcag tacacttacc ccagccagtg    660

tccacacgat cccaacacac gcagccaact ccagaaccca gcactgctcc aagcacctcc    720

ttcctgctcc caatgggccc cagcccccca gctgaaggga tccccaaggt  ggacaagaaa   780

gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc    840

ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc    900

cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag    960

ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag   1020

cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg   1080

aatggcaagg agtacaagtg cagggtctcc aacaaagccc tcccagcccc catcgagaaa   1140

accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct  gcccccatcc  1200

cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc   1260

agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg   1320

cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag   1380

agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac   1440

cactacacgc agaagagcct ctccctgtct ccgggtaaa                          1479

<210>116

<211>493

<212>PRT

<213>Artificial sequence

<400>116

Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu

1               5                   10                  15

Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr

            20                  25                  30

Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln

        35                  40                  45

Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys

    50                  55                  60

Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp

65                  70                  75                  80

Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys

                85                  90                  95

Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg

            100                 105                 110

Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu

        115                 120                 125

Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg

    130                 135                 140

Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val

145                 150                 155                 160

Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr

                165                 170                 175

Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly

            180                 185                 190

Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser

        195                 200                 205

Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser

    210                 215                 220

Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser

225                 230                 235                 240

Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ile Pro Lys

                245                 250                 255

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

            260                 265                 270

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

        275                 280                 285

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

    290                 295                 300

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

305                 310                 315                 320

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

                325                 330                 335

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

            340                 345                 350

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Arg

        355                 360                 365

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

    370                 375                 380

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

385                 390                 395                 400

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

                405                 410                 415

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

            420                 425                 430

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

        435                 440                 445

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

    450                 455                 460

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

465                 470                 475                 480

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

                485                 490

<210>117

<21l>1479

<212>DNA

<213>Artificial sequence

<400>117

atggcgcccg tcgccgtctg ggccgcgctg gccgtcggac tggagctctg ggctgcggcg     60

cacgccttgc ccgcccaggt ggcatttaca ccctacgccc cggagcccgg gagcacatgc    120

cggctcagag aatactatga ccagacagct cagatgtgct gcagcaaatg ctcgccgggc    180

caacatgcaa aagtcttctg taccaagacc tcggacaccg tgtgtgactc ctgtgaggac    240

agcacataca cccagctctg gaactgggtt cccgagtgct tgagctgtgg ctcccgctgt    300

agctctgacc aggtggaaac tcaagcctgc actcgggaac agaaccgcat ctgcacctgc    360

aggcccggct ggtactgcgc gctgagcaag caggaggggt gccggctgtg cgcgccgctg    420

cgcaagtgcc gcccgggctt cggcgtggcc agaccaggaa ctgaaacatc agacgtggtg    480

tgcaagccct gtgccccggg gacgttctcc aacacgactt catccacgga tatttgcagg    540

ccccaccaga tctgtaacgt ggtggccatc cctgggaatg caagcaggga tgcagtctgc   600

acgtccacgt cccccacccg gagtatggcc ccaggggcag tacacttacc ccagccagtg   660

tccacacgat cccaacacac gcagccaact ccagaaccca gcactgctcc aagcacctcc   720

ttcctgctcc caatgggccc cagcccccca gctgaaggga tccccaaggt ggacaagaaa   780

gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc   840

ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc   900

cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag   960

ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag  1020

cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg  1080

aatggcaagg agtacaagtg cagggtctcc aacaaagccc tcccagcccc catcgagaaa  1140

accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc  1200

cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc  1260

agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg  1320

cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag  1380

agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac  1440

cactacacgc agaagagcct ctccctgtct ccgggtaaa                         1479

<210>118

<211>493

<212>PRT

<213>Artificial sequence

<400>118

Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu

1               5                   10                  15

Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr

            20                  25                  30

Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln

        35                  40                  45

Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys

    50                  55                  60

Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp

65                  70                  75                  80

Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys

                85                  90                  95

Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg

            100                 105                 110

Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu

        115                 120                 125

Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg

    130                 135                 140

Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val

145                 150                 155                 160

Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr

                165                 170                 175

Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly

            180                 185                 190

Asn Ala Ser Arg Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser

        195                 200                 205

Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser

    210                 215                 220

Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser

225                 230                 235                 240

Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ile Pro Lys

                245                 250                 255

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

            260                 265                 270

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

        275                 280                 285

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

    290                 295                 300

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

305                 310                 315                 320

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

                325                 330                 335

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

            340                 345                 350

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Arg

        355                 360                 365

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

    370                 375                 380

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

385                 390                 395                 400

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

                405                 410                 415

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

            420                 425                 430

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

        435                 440                 445

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

    450                 455                 460

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

465                 470                 475                 480

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

                485                 490

<210>119

<211>1488

<212>DNA

<213>Artificial sequence

<400>119

atggcgcccg tcgccgtctg ggccgcgctg gccgtcggac tggagctctg ggctgcggcg   60

cacgccttgc ccgcccaggt ggcatttaca ccctacgccc cggagcccgg gagcacatgc  120

cggctcagag aatactatga ccagacagct cagatgtgct gcagcaaatg ctcgccgggc  180

caacatgcaa aagtcttctg taccaagacc tcggacaccg tgtgtgactc ctgtgaggac  240

agcacataca cccagctctg gaactgggtt cccgagtgct tgagctgtgg ctcccgctgt  300

agctctgacc aggtggaaac tcaagcctgc actcgggaac agaaccgcat ctgcacctgc  360

aggcccggct ggtactgcgc gctgagcaag caggaggggt gccggctgtg cgcgccgctg  420

cgcaagtgcc gcccgggctt cggcgtggcc agaccaggaa ctgaaacatc agacgtggtg  480

tgcaagccct gtgccccggg gacgttctcc aacacgactt catccacgga tatttgcagg  540

ccccaccaga tctgtaacgt ggtggccatc cctgggaatg caagcatgga tgcagtctgc  600

acgtccacgt cccccacccg gagtatggcc ccaggggcag tacacttacc ccagccagtg  660

tccacacgat cccaacacac gcagccaact ccagaaccca gcactgctcc aagcacctcc  720

ttcctgctcc caatgggccc cagcccccca gctgaagggg gatccagcaa caccaaggtg  780

gacaagaaag ttgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca  840

cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc  900

atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct  960

gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg  1020

cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag  1080

gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc  1140

atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg  1200

cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc  1260

ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac  1320

aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc  1380

gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct  1440

ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaa               1488

<210>120

<211>496

<212>PRT

<213>Artificial sequence

<400>120

Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu

1               5                   10                  15

Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr

            20                  25                  30

Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln

         35                  40                  45

Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys

    50                  55                  60

Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp

65                  70                  75                  80

Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys

                 85                  90                  95

Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg

             100                 105                 110

Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu

        115                 120                 125

Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg

    130                 135                 140

Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val

145                 150                 155                 160

Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr

                165                 170                 175

Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly

            180                 185                 190

Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser

        195                 200                 205

Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser

    210                 215                 220

Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser

225                 230                 235                 240

Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Gly Ser Ser

                245                 250                 255

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

            260                 265                 270

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

        275                 280                 285

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

    290                 295                 300

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

305                 310                 315                 320

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

                325                 330                 335

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

            340                 345                 350

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

        355                 360                 365

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

    370                 375                 380

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

385                 390                 395                 400

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

                405                 410                 415

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

            420                 425                 430

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

        435                 440                 445

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

    450                 455                 460

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

465                 470                 475                 480

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

                485                 490                 495

<210>121

<211>1488

<212>DNA

<213>Artificial sequence

<400>121

atggcgcccg tcgccgtctg ggccgcgctg gccgtcggac tggagctctg ggctgcggcg     60

cacgccttgc ccgcccaggt ggcatttaca ccctacgccc cggagcccgg gagcacatgc    120

cggctcagag aatactatga ceagacagct cagatgtgct gcagcaagtg ctcgccgggc    180

caacatgcaa aagtcttctg taccaagacc tcggacaccg tgtgtgactc ctgtgaggac    240

agcacataca cccagctctg gaactgggtt cccgagtgct tgagctgtgg ctcccgctgt    300

agctctgacc aggtggaaac tcaagcctgc actcgggaac agaaccgcat ctgcacctgc    360

aggcccggct ggtactgcgc gctgagcaag caggaggggt gccggctgtg cgcgccgctg    420

cgcaagtgcc gcccgggctt cggcgtggcc agaccaggaa ctgaaacatc agacgtggtg    480

tgcaagccct gtgccccggg gacgttctcc aacacgactt catccacgga tatttgcagg    540

ccccaccaga tctgtaacgt ggtggccatc cctgggaatg caagcaggga tgcagtctgc    600

acgtccacgt cccccacccg gagtatggcc ccaggggcag tacacttacc ccagccagtg    660

tccacacgat cccaacacac gcagccaact ccagaaccca gcactgctcc aagcacctcc    720

ttcctgctcc caatgggccc cagcccccca gctgaagggg gatccagcaa caccaaggtg    780

gacaagaaag ttgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca    840

cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc    900

atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct    960

gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg   1020

cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag   1080

gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc   1140

atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg   1200

cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct  ggtcaaaggc  1260

ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac   1320

aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc   1380

gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct  1440

ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaa               1488

<210>122

<211>496

<212>PRT

<213>Artificial sequence

<400>122

Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu

1               5                   10                  15

Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr

            20                  25                  30

Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln

        35                  40                  45

Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys

    50                  55                  60

Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp

65                  70                  75                  80

Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys

                85                  90                  95

Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg

            100                 105                 110

Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu

        115                 120                 125

Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg

    130                 135                 140

Pro Gly Phe Gly yal Ala Arg Pro Gly Thr Glu Thr Ser Asp yal Val

145                 150                 155                 160

Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr

                165                 170                 175

Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly

            180                 185                 190

Asn Ala Ser Arg Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser

        195                 200                 205

Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser

    210                 215                 220

Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser

225                 230                 235                 240

Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Gly Ser Ser

                245                 250                 255

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

            260                 265                 270

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

        275                 280                 285

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

    290                 295                 300

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

305                 310                 315                 320

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

                325                 330                 335

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

            340                 345                 350

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

        355                 360                 365

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

    370                 375                 380

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

385                 390                 395                 400

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

                405                 410                 415

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

            420                 425                 430

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

        435                 440                 445

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

    450                 455                 460

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

465                 470                 475                 480

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

                485                 490                 495

<210>123

<211>31

<212>DNA

<213>Artificial sequence

<400>123

aaggatatcg aggatgtgcg tgggggctcg g                                31

<210>124

<211>36

<212>DNA

<213>Artificial sequence

<400>124

tcggatccac ttctgaggtt acaccacgtg cagggc                              36

<210>125

<211>27

<212>DNA

<213>Artificial sequence

<400>125

gtggatccga gcggaagcag ctgtgca                                        27

<210>126

<211>28

<212>DNA

<213>Artificial sequence

<400>126

aaaaagggga tccccacggg ccgggtgg                                       28

<210>127

<211>84

<212>DNA

<213>People

<400>127

atgtgcgtgg gggctcggcg gctgggccgc gggccgtgtg cggctctgct cctcctgggc    60

ctggggctga gcaccgtgac gggg                                           84

<210>128

<211>28

<212>PRT

<213>People

<400>128

Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu

1               5                   10                  15

Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly

            20                  25

<210>129

<211>540

<212>DNA

<213>People

<400>129

ctccactgtg tcggggacac ctaccccagc aacgaccggt gctgccacga gtgcaggcca     60

ggcaacggga tggtgagccg ctgcagccgc tcccagaaca cggtgtgccg tccgtgcggg    120

ccgggcttct acaacgacgt ggtcagctcc aagccgtgca agccctgcac gtggtgtaac    180

ctcagaagtg gatccgagcg gaagcagctg tgcacggcca cacaggacac agtctgccgc    240

tgccgggcgg gcacccagcc cctggacagc tacaagcctg gagttgactg tgccccctgc    300

cctccagggc acttctcccc aggcgacaac caggcctgca agccctggac caactgcacc    360

ttggctggga agcacaccct gcagccggcc agcaatagct cggacgcaat ctgtgaggac    420

agggaccccc cagccacgca gccccaggag acccagggcc ccccggccag gcccatcact    480

gtccagccca ctgaagcctg gcccagaacc tcacagggac cctccacccg gcccgtgggg    540

<210>130

<211>180

<212>PRT

<213>People

<400>130

Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His

1               5                   10                  15

Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln

            20                  25                  30

Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val

        35                  40                  45

Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly

    50                  55                  60

Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg

65                  70                  75                  80

Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp

                85                  90                  95

Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala

            100                 105                 110

Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln

        115                 120                 125

Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro

    130                 135                 140

Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr

145                 150                 155                 160

Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr

                165                 170                 175

Arg Pro Val Gly

            180

<210>131

<211>624

<212>DNA

<213>Artificial sequence

<400>131

atgtgcgtgg gggctcggcg gctgggccgc gggccgtgtg cggctctgct cctcctgggc   60

ctggggctga gcaccgtgac ggggctccac tgtgtcgggg acacctaccc cagcaacgac  120

cggtgctgcc acgagtgcag gccaggcaac gggatggtga gccgctgcag ccgctcccag  180

aacacggtgt gccgtccgtg cgggccgggc ttctacaacg acgtggtcag ctccaagccg  240

tgcaagccct gcacgtggtg taacctcaga agtggatccg agcggaagca gctgtgcacg  300

gccacacagg acacagtctg ccgctgccgg gcgggcaccc agcccctgga cagctacaag  360

cctggagttg actgtgcccc ctgccctcca gggcacttct ccccaggcga caaccaggcc  420

tgcaagccct ggaccaactg caccttggct gggaagcaca ccctgcagcc ggccagcaat  480

agctcggacg caatctgtga ggacagggac cccccagcca cgcagcccca ggagacccag  540

ggccccccgg ccaggcccat cactgtccag cccactgaag cctggcccag aacctcacag  600

ggaccctcca cccggcccgt gggg                                         624

<210>132

<211>208

<212>PRT

<213>Artificial sequence

<400>132

Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu

1               5                   10                  15

Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val

            20                  25                  30

Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro

        35                  40                  45

Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys

    50                  55                  60

Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro

65                  70                  75                  80

Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys

                85                  90                  95

Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly

            100                 105                 110

Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys

        115                 120                 125

Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp

    130                 135                 140

Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn

145                 150                 155                 160

Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro

                165                 170                 175

Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr

            180                 185                 190

Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Gly

        195                 200                 205

<210>133

<211>1260

<212>DNA

<213>Artificial sequence

<400>133

ctccactgtg tcggggacac ctaccccagc aacgaccggt gctgccacga gtgcaggcca     60

ggcaacggga tggtgagccg ctgcagccgc tcccagaaca cggtgtgccg tccgtgcggg    120

ccgggcttct acaacgacgt ggtcagctcc aagccgtgca agccctgcac gtggtgtaac    180

ctcagaagtg ggagtgagcg gaagcagctg tgcacggcca cacaggacac agtctgccgc    240

tgccgggcgg gcacccagcc cctggacagc tacaagcctg gagttgactg tgccccctgc    300

cctccagggc acttctcccc aggcgacaac caggcctgca agccctggac caactgcacc    360

ttggctggga agcacaccct gcagccggcc agcaatagct cggacgcaat ctgtgaggac    420

agggaccccc cagccacgca gccccaggag acccagggcc ccccggccag gcccatcact    480

gtccagccca ctgaagcctg gcccagaacc tcacagggac cctccacccg gcccgtgggg    540

atccccaagg tggacaagaa agttgagccc aaatcttgtg acaaaactca cacatgccca    600

ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc    660

aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc    720

cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc    780

aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc    840

gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc    900

ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag    960

gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc   1020

ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg   1080

gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac   1140

agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg   1200

atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa   1260

<210>134

<211>420

<212>PRT

<213>Artificial sequence

<400>134

Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His

1               5                   10                  15

Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln

            20                  25                  30

Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val

        35                  40                  45

Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly

    50                  55                  60

Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg

65                  70                  75                  80

Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp

                85                  90                  95

Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala

            100                 105                 110

Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln

        115                 120                 125

Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro

    130                 135                 140

Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr

145                 150                 155                 160

Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr

                165                 170                 175

Arg Pro Val Gly Ile Pro Lys Val Asp Lys Lys Val Glu Pro Lys Ser

            180                 185                 190

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

        195                 200                 205

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

    210                 215                 220

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

225                 230                 235                 240

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

                245                 250                 255

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

            260                 265                 270

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

        275                 280                 285

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

    290                 295                 300

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

305                 310                 315                 320

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

                325                 330                 335

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

            340                 345                 350

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

        355                 360                 365

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

    370                 375                 380

Val Asp Lys Aer Arg Trp Gln 6ln Gly Asn Val Phe 5er Cys 5er Val

385                 390                 395                 400

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

                405                 410                 415

Ser Pro Gly Lys

            420

<210>135

<211>1260

<212>DNA

<213>Artificial sequence

<400>135

ctccactgtg tcggggacac ctaccccagc aacgaccggt gctgccacga gtgcaggcca   60

ggcaacggga tggtgagccg ctgcagccgc tcccagaaca cggtgtgccg tccgtgcggg  120

ccgggcttct acaacgacgt ggtcagctcc aagccgtgca agccctgcac gtggtgtaac  180

ctcagaagtg gatccgagcg gaagcagctg tgcacggcca cacaggacac agtctgccgc  240

tgccgggcgg gcacccagcc cctggacagc tacaagcctg gagttgactg tgccccctgc  300

cctccagggc acttctcccc aggcgacaac caggcctgca agccctggac caactgcacc  360

ttggctggga agcacaccct gcagccggcc agcaatagct cggacgcaat ctgtgaggac  420

agggaccccc cagccacgca gccccaggag acccagggcc ccccggccag gcccatcact  480

gtccagccca ctgaagcctg gcccagaacc tcacagggac cctccacccg gcccgtgggg  540

atccccaagg tggacaagaa agttgagccc aaatcttgtg acaaaactca cacatgccca  600

ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc  660

aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc  720

cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc  780

aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc  840

gtcctgcacc aggactggct gaatggcaag gagtacaagt gcagggtctc caacaaagcc  900

ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag   960

gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc  1020

ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg  1080

gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac  1140

agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg  1200

atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa  1260

<210>136

<211>420

<212>PRT

<213>Artificial sequence

<400>136

Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His

1               5                   10                  15

Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln

            20                  25                  30

Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val

        35                  40                  45

Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly

    50                  55                  60

Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg

65                  70                  75                  80

Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp

                85                  90                  95

Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala

            100                 105                 110

Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln

        115                 120                 125

Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro

    130                 135                 140

Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr

145                 150                 155                 160

Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr

                165                 170                 175

Arg Pro Val Gly Ile Pro Lys Val Asp Lys Lys Val Glu Pro Lys Ser

            180                 185                 190

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

        195                 200                 205

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

    210                 215                 220

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

225                 230                 235                 240

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

                245                 250                 255

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

            260                 265                 270

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

        275                 280                 285

Gly Lys Glu Tyr Lys Cys Arg Val Ser Asn Lys Ala Leu Pro Ala Pro

    290                 295                 300

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

305                 310                 315                 320

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

                325                 330                 335

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

            340                 345                 350

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

        355                 360                 365

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

    370                 375                 380

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

385                 390                 395                 400

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

                405                 410                 415

Ser Pro Gly Lys

            420

<210>137

<211>1269

<212>DNA

<213>Artificial sequence

<400>137

ctccactgtg tcggggacac ctaccccagc aacgaccggt gctgccacga gtgcaggcca   60

ggcaacggga tggtgagccg ctgcagccgc tcccagaaca cggtgtgccg tccgtgcggg  120

ccgggcttct acaacgacgt ggtcagctcc aagccgtgca agccctgcac gtggtgtaac  180

ctcagaagtg gatccgagcg gaagcagctg tgcacggcca cacaggacac agtctgccgc  240

tgccgggcgg gcacccagcc cctggacagc tacaagcctg gagttgactg tgccccctgc  300

cctccagggc acttctcccc aggcgacaac caggcctgca agccctggac caactgcacc  360

ttggctggga agcacaccct gcagccggcc agcaatagct cggacgcaat ctgtgaggac  420

agggaccccc cagccacgca gccccaggag acccagggcc ccccggccag gcccatcact  480

gtccagccca ctgaagcctg gcccagaacc tcacagggac cctccacccg gcccgtgggg   540

ggatccagca acaccaaggt ggacaagaaa gttgagccca aatcttgtga caaaactcac   600

acatgcccac cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc   660

ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg   720

gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg   780

cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc   840

gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc   900

aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga   960

gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc  1020

ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat  1080

gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc  1140

ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca  1200

tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct  1260

ccgggtaaa                                                          1269

<210>138

<211>423

<212>PRT

<213>Artificial sequence

<400>138

Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His

1               5                   10                  15

Glu Cys Arg Pro Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gin

            20                  25                  30

Asn Thr Val Cys Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val

        35                  40                  45

Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly

    50                  55                  60

Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg

65                  70                  75                  80

Cys Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp

                85                  90                  95

Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala

            100                 105                 110

Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln

        115                 120                 125

Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro

    130                 135                 140

Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr

145                 150                 155                 160

Val Gln Pro Thr Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr

                165                 170                 175

Arg Pro Val Gly Gly Ser Ser Asn Thr Lys Val Asp Lys Lys Val Glu

            180                 185                 190

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

        195                 200                 205

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

    210                 215                 220

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

225                 230                 235                 240

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

                245                 250                 255

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

            260                 265                 270

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

        275                 280                 285

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

    290                 295                 300

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

305                 310                 315                 320

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

                325                 330                 335

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

            340                 345                 350

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

        355                 360                 365

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

    370                 375                 380

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

385                 390                 395                 400

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

                405                 410                 415

Leu Ser Leu Ser Pro Gly Lys

            420

<210>139

<211>1344

<212>DNA

<213>Artificial sequence

<400>139

atgtgcgtgg gggctcggcg gctgggccgc gggccgtgtg cggctctgct cctcctgggc     60

ctggggctga gcaccgtgac ggggctccac tgtgtcgggg acacctaccc cagcaacgac    120

cggtgctgcc acgagtgcag gccaggcaac gggatggtga gccgctgcag ccgctcccag    180

aacacggtgt gccgtccgtg cgggccgggc ttctacaacg acgtggtcag ctccaagccg    240

tgcaagccct gcacgtggtg taacctcaga agtgggagtg agcggaagca gctgtgcacg    300

gccacacagg acacagtctg ccgctgccgg gcgggcaccc agcccctgga cagctacaag    360

cctggagttg actgtgcccc ctgccctcca gggcacttct ccccaggcga caaccaggcc    420

tgcaagccct ggaccaactg caccttggct gggaagcaca ccctgcagcc ggccagcaat    480

agctcggacg caatctgtga ggacagggac cccccagcca cgcagcccca ggagacccag    540

ggccccccgg ccaggcccat cactgtccag cccactgaag cctggcccag aacctcacag    600

ggaccctcca cccggcccgt ggggatcccc aaggtggaca agaaagttga gcccaaatct    660

tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca    720

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc    780

acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg    840

gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg    900

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac    960

aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat  ctccaaagc   1020

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc   1080

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg   1140

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac   1200

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag   1260

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag   1320

agcctctccc tgtctccggg taaa                                          1344

<210>140

<211>448

<212>PRT

<213>Artificial sequence

<400>140

Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu

1               5                   10                  15

Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val

            20                  25                  30

Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro

        35                  40                  45

Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys

    50                  55                  60

Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro

65                  70                  75                  80

Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys

                85                  90                  95

Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly

            100                 105                 110

Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys

        115                 120                 125

Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp

    130                 135                 140

Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn

145                 150                 155                 160

Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro

                165                 170                 175

Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr

            180                 185                 190

Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Gly

        195                 200                 205

Ile Pro Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

    210                 215                 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225                 230                 235                 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

                245                 250                 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

            260                 265                 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

        275                 280                 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

    290                 295                 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305                 310                 315                 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

                325                 330                 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

            340                 345                 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

        355                 360                 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

    370                 375                 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385                 390                 395                 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

                405                 410                 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

            420                 425                 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

        435                 440                 445

<210>141

<211>1344

<212>DNA

<213>Artificial sequence

<400>14l

atgtgcgtgg gggctcggcg gctgggccgc gggccgtgtg cggctctgct cctcctgggc   60

ctggggctga gcaccgtgac ggggctccac tgtgtcgggg acacctaccc cagcaacgac  120

cggtgctgcc acgagtgcag gccaggcaac gggatggtga gccgctgcag ccgctcccag  180

aacacggtgt gccgtccgtg cgggccgggc ttctacaacg acgtggtcag ctccaagccg  240

tgcaagccct gcacgtggtg taacctcaga agtggatccg agcggaagca gctgtgcacg  300

gccacacagg acacagtctg ccgctgccgg gcgggcaccc agcccctgga cagctacaag  360

cctggagttg actgtgcccc ctgccctcca gggcacttct ccccaggcga caaccaggcc  420

tgcaagccct ggaccaactg caccttggct gggaagcaca ccctgcagcc ggccagcaat  480

agctcggacg caatctgtga ggacagggac cccccagcca cgcagcccca ggagacccag  540

ggccccccgg ccaggcccat cactgtccag cccactgaag cctggcccag aacctcacag  600

ggaccctcca cccggcccgt ggggatcccc aaggtggaca agaaagttga gcccaaatct  660

tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca  720

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc  780

acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg  840

gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg   900

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac   960

aagtgcaggg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc  1020

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc  1080

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg  1140

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac  1200

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag  1260

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag  1320

agcctctccc tgtctccggg taaa                                         1344

<210>142

<211>448

<212>PRT

<213>Artificial sequence

<400>142

Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu

1               5                   10                  15

Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val

            20                  25                  30

Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro

        35                  40                  45

Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys

    50                  55                  60

Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro

65                  70                  75                  80

Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys

                85                  90                  95

Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly

            100                 105                 110

Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys

        115                 120                 125

Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp

    130                 135                 140

Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn

145                 150                 155                 160

Ser Ser Asp Ala Tle Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro

                165                 170                 175

Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr

            180                 185                 190

Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Gly

        195                 200                 205

Ile Pro Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

    210                 215                 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225                 230                 235                 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

                245                 250                 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

            260                 265                 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

        275                 280                 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

    290                 295                 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305                 310                 315                 320

Lys Cys Arg Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

                325                 330                 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

            340                 345                 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

        355                 360                 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

    370                 375                 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385                 390                 395                 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

                405                 410                 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

            420                 425                 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

        435                 440                 445

<210>143

<211>1353

<212>DNA

<213>Artificial sequence

<400>143

atgtgcgtgg gggctcggcg gctgggccgc gggccgtgtg cggctctgct cctcctgggc   60

ctggggctga gcaccgtgac ggggctccac tgtgtcgggg acacctaccc cagcaacgac  120

cggtgctgcc acgagtgcag gccaggcaac gggatggtga gccgctgcag ccgctcccag  180

aacacggtgt gccgtccgtg cgggccgggc ttctacaacg acgtggtcag ctccaagccg    240

tgcaagccct gcacgtggtg taacctcaga agtggatccg agcggaagca gctgtgcacg    300

gccacacagg acacagtctg ccgctgccgg gcgggcaccc agcccctgga cagctacaag    360

cctggagttg actgtgcccc ctgccctcca gggcacttct ccccaggcga caaccaggcc    420

tgcaagccct ggaccaactg caccttggct gggaagcaca ccctgcagcc ggccagcaat    480

agctcggacg caatctgtga ggacagggac cccccagcca cgcagcccca ggagacccag    540

ggccccccgg ccaggcccat cactgtccag cccactgaag cctggcccag aacctcacag    600

ggaccctcca cccggcccgt ggggggatcc agcaacacca aggtggacaa gaaagttgag    660

cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg    720

ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc    780

cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac    840

tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac    900

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc    960

aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc   1020

tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat   1080

gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac   1140

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc   1200

gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg   1260

tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac   1320

acgcagaaga gcctctccct gtctccgggt aaa                                1353

<210>144

<211>451

<212>PRT

<213>Artificial sequence

<400>144

Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu

1               5                   10                  15

Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val

            20                  25                  30

Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro

        35                  40                  45

Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys

    50                  55                  60

Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro

65                  70                  75                  80

Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys

                85                  90                  95

Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly

            100                 105                 110

Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys

        115                 120                 125

Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp

    130                 135                 140

Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn

145                 150                 155                 160

Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro

                165                 170                 175

Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr

            180                 185                 190

Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Gly

        195                 200                 205

Gly Ser Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

    210                 215                 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225                 230                 235                 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

                245                 250                 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

            260                 265                 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

        275                 280                 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

    290                 295                 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305                 310                 315                 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

                325                 330                 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

            340                 345                 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

        355                 360                 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

    370                 375                 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385                 390                 395                 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

                405                 410                 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

            420                 425                 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

        435                 440                 445

Pro Gly Lys

    450

<210>145

<211>28

<212>DNA

<213>Artificial sequence

<400>145

cttgatatct caagtagtga tatggatg                                        28

<210>146

<211>28

<212>DNA

<213>Artificial sequence

<400>146

agacatggat ccagtaggtc acagcagt                                        28

<210>147

<211>399

<212>DNA

<213>People

<400>147

atggatgact ccacagaaag ggagcagtca cgccttactt cttgccttaa gaaaagagaa     60

gaaatgaaac tgaaggagtg tgtttccatc ctcccacgga aggaaagccc ctctgtccga    120

tcctccaaag acggaaagct gctggctgca accttgctgc tggcactgct gtcttgctgc    180

ctcacggtgg tgtctttcta ccaggtggcc gccctgcaag gggacctggc cagcctccgg    240

gcagagctgc agggccacca cgcggagaag ctgccagcag gagcaggagc ccccaaggcc    300

ggcctggagg aagctccagc tgtcaccgcg ggactgaaaa tctttgaacc accagctcca    360

ggagaaggca actccagtca gaacagcaga aataagcgt                           399

<210>148

<211>133

<212>PRT

<213>People

<400>148

Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu

1               5                   10                  15

Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro

            20                  25                  30

Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu

        35                  40                  45

Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val

    50                  55                  60

Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg

65                  70                  75                  80

Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly

                85                  90                  95

Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu

            100                 105                 110

Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn

        115                 120                 125

Ser Arg Asn Lys Arg

    130

<210>149

<211>399

<212>DNA

<213>People

<400>149

atggatgact ccacagaaag ggagcagtca cgccttactt cttgccttaa gaaaagagaa     60

gaaatgaaac tgaaggagtg tgtttccatc ctcccacgga aggaaatccc ctctgtccga    120

tcctccaaag acggaaagct gctggctgca accttgctgc tggcactgct gtcttgctgc    180

ctcacggtgg tgtctttcta ccaggtggcc gccctgcaag gggacctggc cagcctccgg    240

gcagagctgc agggccacca cgcggagaag ctgccagcag gagcaggagc ccccaaggcc    300

ggcctggagg aagctccagc tgtcaccgcg ggactgaaaa tctttgaacc accagctcca    360

ggagaaggca actccagtca gaacagcaga aataagcgt                           399

<210>150

<211>133

<212>PRT

<213>People

<400>150

Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu

1               5                   10                  15

Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro

            20                  25                  30

Arg Lys Glu Ile Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu

        35                  40                  45

Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val

    50                  55                  60

Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg

65                  70                  75                  80

Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly

                85                  90                  95

Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu

            100                 105                 110

Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn

        115                 120                 125

Ser Arg Asn Lys Arg

    130

<210>151

<211>456

<212>DNA

<213>People

<400>151

gccgttcagg gtccagaaga aacagtcact caagactgct tgcaactgat tgcagacagt     60

gaaacaccaa ctatacaaaa aggatcttac acatttgttc catggcttct cagctttaaa    120

aggggaagtg ccctagaaga aaaagagaat aaaatattgg tcaaagaaac tggttacttt    180

tttatatatg gtcaggtttt atatactgat aagacctacg ccatgggaca tctaattcag    240

aggaagaagg tccatgtctt tggggatgaa ttgagtctgg tgactttgtt tcgatgtatt    300

caaaatatgc ctgaaacact acccaataat tcctgctatt cagctggcat tgcaaaactg    360

gaagaaggag atgaactcca acttgcaata ccaagagaaa atgcacaaat atcactggat    420

ggagatgtca cattttttgg tgcattgaaa ctgctg                           456

<210>152

<211>152

<212>PRT

<213>People

<400>152

Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln Asp Cys Leu Gln Leu

1               5                   10                  15

Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe

            20                  25                  30

Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys

        35                  40                  45

Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile Tyr Gly

    50                  55                  60

Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu Ile Gln

65                  70                  75                  80

Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val Thr Leu

                85                  90                  95

Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn Ser Cys

            100                 105                 110

Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu

        115                 120                 125

Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp Val Thr

    130                 135                 140

Phe Phe Gly Ala Leu Lys Leu Leu

145                 150

<210>153

<211>855

<212>DNA

<213>Artificial sequence

<400>153

atggatgact ccacagaaag ggagcagtca cgccttactt cttgccttaa gaaaagagaa     60

gaaatgaaac tgaaggagtg tgtttccatc ctcccacgga aggaaagccc ctctgtccga    120

tcctccaaag acggaaagct gctggctgca accttgctgc tggcactgct gtcttgctgc    180

ctcacggtgg tgtctttcta ccaggtggcc gccctgcaag gggacctggc cagcctccgg    240

gcagagctgc agggccacca cgcggagaag ctgccagcag gagcaggagc ccccaaggcc    300

ggcctggagg aagctccagc tgtcaccgcg ggactgaaaa tctttgaacc accagctcca    360

ggagaaggca actccagtca gaacagcaga aataagcgtg ccgttcaggg tccagaagaa    420

acagtcactc aagactgctt gcaactgatt gcagacagtg aaacaccaac tatacaaaaa    480

ggatcttaca catttgttcc atggcttctc agctttaaaa ggggaagtgc cctagaagaa    540

aaagagaata aaatattggt caaagaaact ggttactttt ttatatatgg tcaggtttta    600

tatactgata agacctacgc catgggacat ctaattcaga ggaagaaggt ccatgtcttt    660

ggggatgaat tgagtctggt gactttgttt cgatgtattc aaaatatgcc tgaaacacta    720

cccaataatt cctgctattc agctggcatt gcaaaactgg aagaaggaga tgaactccaa    780

cttgcaatac caagagaaaa tgcacaaata tcactggatg gagatgtcac attttttggt    840

gcattgaaac tgctg                                                     855

<210>154

<211>285

<212>PRT

<213>Artificial sequence

<400>154

Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu

1               5                   10                  15

Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro

            20                  25                  30

Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu

        35                  40                  45

Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val

    50                  55                  60

Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg

65                  70                  75                  80

Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly

                85                  90                  95

Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu

            100                 105                 110

Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn

        115                 120                 125

Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln

    130                 135                 140

Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys

145                 150                 155                 160

Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser

                165                 170                 175

Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr

            180                 185                 190

Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met

        195                 200                 205

Gly His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu

    210                 215                 220

Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu

225                 230                 235                 240

Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly

                245                 250                 255

Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu

            260                 265                 270

Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu

        275                 280                 285

<210>155

<211>855

<212>DNA

<213>Artificial sequence

<400>155

atggatgact ccacagaaag ggagcagtca cgccttactt cttgccttaa gaaaagagaa     60

gaaatgaaac tgaaggagtg tgtttccatc ctcccacgga aggaaatccc ctctgtccga    120

tcctccaaag acggaaagct gctggctgca accttgctgc tggcactgct gtcttgctgc    180

ctcacggtgg tgtctttcta ccaggtggcc gccctgcaag gggacctggc cagcctccgg    240

gcagagctgc agggccacca cgcggagaag ctgccagcag gagcaggagc ccccaaggcc    300

ggcctggagg aagctccagc tgtcaccgcg ggactgaaaa tctttgaacc accagctcca    360

ggagaaggca actccagtca gaacagcaga aataagcgtg ccgttcaggg tccagaagaa    420

acagtcactc aagactgctt gcaactgatt gcagacagtg aaacaccaac tatacaaaaa    480

ggatcttaca catttgttcc atggcttctc agctttaaaa ggggaagtgc cctagaagaa    540

aaagagaata aaatattggt caaagaaact ggttactttt ttatatatgg tcaggtttta    600

tatactgata agacctacgc catgggacat ctaattcaga ggaagaaggt ccatgtcttt    660

ggggatgaat tgagtctggt gactttgttt cgatgtattc aaaatatgcc tgaaacacta    720

cccaataatt cctgctattc agctggcatt gcaaaactgg aagaaggaga tgaactccaa    780

cttgcaatac caagagaaaa tgcacaaata tcactggatg gagatgtcac attttttggt    840

gcattgaaac tgctg                                                     855

<210>156

<211>285

<212>PRT

<213>Artificial sequence

<400>156

Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu

1               5                   10                  15

Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro

            20                  25                  30

Arg Lys Glu Ile Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu

        35                  40                  45

Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val

    50                  55                  60

Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg

65                  70                  75                  80

Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly

                85                  90                  95

Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu

            100                 105                 110

Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn

        115                 120                 125

 Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln

     130                 135                 140

Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys

145                 150                 155                 160

Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser

                165                 170                 175

Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr

            180                 185                 190

Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met

        195                 200                 205

Gly His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu

    210                 215                 220

Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu

225                 230                 235                 240

Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly

                245                 250                 255

Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu

            260                 265                 270

Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu

        275                 280                 285

<210>157

<211>1584

<212>DNA

<213>Artificial sequence

<400>157

atggatgact ccacagaaag ggagcagtca cgccttactt cttgccttaa gaaaagagaa      60

gaaatgaaac tgaaggagtg tgtttccatc ctcccacgga aggaaagccc ctctgtccga     120

tcctccaaag acggaaagct gctggctgca accttgctgc tggcactgct gtcttgctgc     180

ctcacggtgg tgtctttcta ccaggtggcc gccctgcaag gggacctggc cagcctccgg     240

gcagagctgc agggccacca cgcggagaag ctgccagcag gagcaggagc ccccaaggcc     300

ggcctggagg aagctccagc tgtcaccgcg ggactgaaaa tctttgaacc accagctcca     360

ggagaaggca actccagtca gaacagcaga aataagcgtg ccgttcaggg tccagaagaa     420

acagtcactc aagactgctt gcaactgatt gcagacagtg aaacaccaac tatacaaaaa     480

ggatcttaca catttgttcc atggcttctc agctttaaaa ggggaagtgc cctagaagaa     540

aaagagaata aaatattggt caaagaaact ggttactttt ttatatatgg tcaggtttta     600

tatactgata agacctacgc catgggacat ctaattcaga ggaagaaggt ccatgtcttt     660

ggggatgaat tgagtctggt gactttgttt cgatgtattc aaaatatgcc tgaaacacta     720

cccaataatt cctgctattc agctggcatt gcaaaactgg aagaaggaga tgaactccaa     780

cttgcaatac caagagaaaa tgcacaaata tcactggatg gagatgtcac attttttggt     840

gcattgaaac tgctgggatc cagcaacacc aaggtggaca agaaagttga gcccaaatct     900

tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca     960

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc    1020

acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg    1080

gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg    1140

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac    1200

aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc    1260

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc    1320

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg    1380

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac    1440

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag    1500

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag    1560

agcctctccc tgtctccggg taaa                                           1584

<210>158

<211>528

<212>PRT

<213>Artificial sequence

<400>158

Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu

1               5                   10                  15

Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro

            20                  25                  30

Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu

        35                  40                  45

Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val

    50                  55                  60

Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg

65                  70                  75                  80

Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly

                85                  90                  95

Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu

            100                 105                 110

Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn

        115                 120                 125

Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln

    130                 135                 140

Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys

145                 150                 155                 160

Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser

                165                 170                 175

Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr

            180                 185                 190

Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met

        195                 200                 205

Gly His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu

    210                 215                 220

Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu

225                 230                 235                 240

Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly

                245                 250                 255

Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu

            260                 265                 270

Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu Gly Ser Ser

        275                 280                 285

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

    290                 295                 300

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

305                 310                 315                 320

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

                325                 330                 335

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

            340                 345                 350

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

        355                 360                 365

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

    370                 375                 380

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

385                 390                 395                 400

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

                405                 410                 415

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

            420                 425                 430

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

        435                 440                 445

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

    450                 455                 460

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

465                 470                 475                 480

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

                485                 490                 495

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

            500                 505                 510

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

        515                 520                 525

<210>159

<211>1584

<212>DNA

<213>Artificial sequence

<400>159

atggatgact ccacagaaag ggagcagtca cgccttactt cttgccttaa gaaaagagaa    60

gaaatgaaac tgaaggagtg tgtttccatc ctcccacgga aggaaatccc ctctgtccga     120

tcctccaaag acggaaagct gctggctgca accttgctgc tggcactgct gtcttgctgc     180

ctcacggtgg tgtctttcta ccaggtggcc gccctgcaag gggacctggc cagcctccgg     240

gcagagctgc agggccacca cgcggagaag ctgccagcag gagcaggagc ccccaaggcc     300

ggcctggagg aagctccagc tgtcaccgcg ggactgaaaa tctttgaacc accagctcca     360

ggagaaggca actccagtca gaacagcaga aataagcgtg ccgttcaggg tccagaagaa     420

acagtcactc aagactgctt gcaactgatt gcagacagtg aaacaccaac tatacaaaaa     480

ggatcttaca catttgttcc atggcttctc agctttaaaa ggggaagtgc cctagaagaa     540

aaagagaata aaatattggt caaagaaact ggttactttt ttatatatgg tcaggtttta     600

tatactgata agacctacgc catgggacat ctaattcaga ggaagaaggt ccatgtcttt     660

ggggatgaat tgagtctggt gactttgttt cgatgtattc aaaatatgcc tgaaacacta     720

cccaataatt cctgctattc agctggcatt gcaaaactgg aagaaggaga tgaactccaa     780

cttgcaatac caagagaaaa tgcacaaata tcactggatg gagatgtcac attttttggt     840

gcattgaaac tgctgggatc cagcaacacc aaggtggaca agaaagttga gcccaaatct     900

tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca     960

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc    1020

acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg    1080

gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg    1140

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac    1200

aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc    1260

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc    1320

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg    1380

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac    1440

tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag    1500

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag    1560

agcctctccc tgtctccggg taaa                                           1584

<210>160

<211>528

<212>PRT

<213>Artificial sequence

<400>160

Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu

1               5                   10                  15

Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro

            20                  25                  30

Arg Lys Glu Ile Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu

        35                  40                  45

Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val

    50                  55                  60

Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg

65                  70                  75                  80

Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly

                85                  90                  95

Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu

            100                 105                 110

Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn

        115                 120                 125

Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln

    130                 135                 140

Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys

145                 150                 155                 160

Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser

                165                 170                 175

Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr

            180                 185                 190

Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met

        195                 200                 205

Gly His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu

    210                 215                 220

Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu

225                 230                 235                 240

Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly

                245                 250                 255

Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu

            260                 265                 270

Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu Gly Ser Ser

        275                 280                 285

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

    290                 295                 300

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

305                 310                 315                 320

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

                325                 330                 335

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

            340                 345                 350

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

        355                 360                 365

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

    370                 375                 380

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

385                 390                 395                 400

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

                405                 410                 415

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

            420                 425                 430

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

        435                 440                 445

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

    450                 455                 460

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

465                 470                 475                 480

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

                485                 490                 495

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

            500                 505                 510

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

        515                 520                 525

<210>161

<211>30

<212>DNA

<213>Artificial sequence

<400>161

aaggatatcc gggcgatggg ggcaggtgcc                                    30

<210>162

<211>30

<212>DNA

<213>Artificial sequence

<400>162

tggggatccc catcactgtg gtcaccacac                                     30

<210>163

<211>84

<212>DNA

<213>People

<400>163

atgggggcag gtgccaccgg ccgcgccatg gacgggccgc gcctgctgct gttgctgctt    60

ctgggggtgt cccttggagg tgcc                                           84

<210>164

<211>28

<212>PRT

<213>People

<400>164

Met Gly Ala Gly Ala Thr Gly Arg Ala Met Asp Gly Pro Arg Leu Leu

1               5                   10                  15

Leu Leu Leu Leu Leu Gly Val Ser Leu Gly Gly Ala

            20                  25

<210>165

<211>627

<212>DNA

<213>People

<400>165

aaggaggcat gccccacagg cctgtacaca cacagcggtg agtgctgcaa agcctgcaac     60

ctgggcgagg gtgtggccca gccttgtgga gccaaccaga ccgtgtgtga gccctgcctg    120

gacagcgtga cgttctccga cgtggtgagc gcgaccgagc cgtgcaagcc gtgcaccgag    180

tgcgtggggc tccagagcat gtcggcgcca tgcgtggagg ccgacgacgc cgtgtgccgc    240

tgcgcctacg gctactacca ggatgagacg actgggcgct gcgaggcgtg ccgcgtgtgc    300

gaggcgggct cgggcctcgt gttctcctgc caggacaagc agaacaccgt gtgcgaggag    360

tgccccgacg gcacgtattc cgacgaggcc aaccacgtgg acccgtgcct gccctgcacc    420

gtgtgcgagg acaccgagcg ccagctccgc gagtgcacac gctgggccga cgccgagtgc    480

gaggagatcc ctggccgttg gattacacgg tccacacccc cagagggctc ggacagcaca    540

gcccccagca cccaggagcc tgaggcacct ccagaacaag acctcatagc cagcacggtg    600

gcaggtgtgg tgaccacagt gatgggg                                        627

<210>166

<211>209

<212>PRT

<213>People

<400>166

Lys Glu Ala Cys Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys

1               5                   10                  15

Lys Ala Cys Asn Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn

            20                  25                  30

Gln Thr Val Cys Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val

        35                  40                  45

Val Ser Ala Thr Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu

    50                  55                  60

Gln Ser Met Ser Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg

65                  70                  75                  80

Cys Ala Tyr Gly Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala

                85                  90                  95

Cys Arg Val Cys Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp

            100                 105                 110

Lys Gln Asn Thr Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp

        115                 120                 125

Glu Ala Asn His Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp

    130                 135                 140

Thr Glu Arg Gln Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys

145                 150                 155                 160

Glu Glu Ile Pro Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly

                165                 170                 175

Ser Asp Ser Thr Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu

            180                 185                 190

Gln Asp Leu Ile Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met

        195                 200                 205

Gly

<210>167

<211>711

<212>DNA

<213>Artificial sequence

<400>167

atgggggcag gtgccaccgg ccgcgccatg gacgggccgc gcctgctgct gttgctgctt     60

ctgggggtgt cccttggagg tgccaaggag gcatgcccca caggcctgta cacacacagc    120

ggtgagtgct gcaaagcctg caacctgggc gagggtgtgg cccagccttg tggagccaac    180

cagaccgtgt gtgagccctg cctggacagc gtgacgttct ccgacgtggt gagcgcgacc    240

gagccgtgca agccgtgcac cgagtgcgtg gggctccaga gcatgtcggc gccatgcgtg    300

gaggccgacg acgccgtgtg ccgctgcgcc tacggctact accaggatga gacgactggg    360

cgctgcgagg cgtgccgcgt gtgcgaggcg ggctcgggcc tcgtgttctc ctgccaggac    420

aagcagaaca ccgtgtgcga ggagtgcccc gacggcacgt attccgacga ggccaaccac    480

gtggacccgt gcctgccctg caccgtgtgc gaggacaccg agcgccagct ccgcgagtgc    540

acacgctggg ccgacgccga gtgcgaggag atccctggcc gttggattac acggtccaca    600

cccccagagg gctcggacag cacagccccc agcacccagg agcctgaggc acctccagaa    660

caagacctca tagccagcac ggtggcaggt gtggtgacca cagtgatggg g             711

<210>168

<211>237

<212>PRT

<213>Artificial sequence

<400>168

Met Gly Ala Gly Ala Thr Gly Arg Ala Met Asp Gly Pro Arg Leu Leu

1               5                   10                  15

Leu Leu Leu Leu Leu Gly Val Ser Leu Gly Gly Ala Lys Glu Ala Cys

            20                  25                  30

Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys Lys Ala Cys Asn

            35                  40                  45

Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn Gln Thr Val Cys

    50                  55                  60

Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val Val Ser Ala Thr

65                  70                  75                  80

Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu Gln Ser Met Ser

                85                  90                  95

Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg Cys Ala Tyr Gly

            100                 105                 110

Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala Cys Arg Val Cys

        115                 120                 125

Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp Lys Gln Asn Thr

    130                 135                 140

Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp Glu Ala Asn His

145                 150                 155                 160

Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp Thr Glu Arg Gln

                165                 170                 175

Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys Glu Glu Ile Pro

            180                 185                 190

Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly Ser Asp Ser Thr

        195                 200                 205

Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu Gln Asp Leu Ile

    210                 215                 220

Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met Gly

225                 230                 235

<210>169

<211>1347

<212>DNA

<213>Artificial sequence

<400>169

aaggaggcat gccccacagg cctgtacaca cacagcggtg agtgctgcaa agcctgcaac    60

ctgggcgagg gtgtggccca gccttgtgga gccaaccaga ccgtgtgtga gccctgcctg   120

gacagcgtga cgttctccga cgtggtgagc gcgaccgagc cgtgcaagcc gtgcaccgag   180

tgcgtggggc tccagagcat gtcggcgcca tgcgtggagg ccgacgacgc cgtgtgccgc   240

tgcgcctacg gctactacca ggatgagacg actgggcgct gcgaggcgtg ccgcgtgtgc   300

gaggcgggct cgggcctcgt gttctcctgc caggacaagc agaacaccgt gtgcgaggag   360

tgccccgacg gcacgtattc cgacgaggcc aaccacgtgg acccgtgcct gccctgcacc   420

gtgtgcgagg acaccgagcg ccagctccgc gagtgcacac gctgggccga cgccgagtgc   480

gaggagatcc ctggccgttg gattacacgg tccacacccc cagagggctc ggacagcaca   540

gcccccagca cccaggagcc tgaggcacct ccagaacaag acctcatagc cagcacggtg   600

gcaggtgtgg tgaccacagt gatggggatc cccaaggtgg acaagaaagt tgagcccaaa   660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg   720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag   780

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac   840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc   900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag   960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa  1020

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg  1080

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc  1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg  1200

gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag  1260

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag  1320

aagagcctct ccctgtctcc gggtaaa                                      1347

<210>170

<211>449

<212>PRT

<213>Artificial sequence

<400>170

Lys Glu Ala Cys Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys

1               5                   10                  15

Lys Ala Cys Asn Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn

            20                  25                  30

Gln Thr Val Cys Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val

        35                  40                  45

Val Ser Ala Thr Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu

    50                  55                  60

Gln Ser Met Ser Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg

65                  70                  75                  80

Cys Ala Tyr Gly Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala

                85                  90                  95

Cys Arg Val Cys Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp

            100                 105                 110

Lys Gln Asn Thr Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp

        115                 120                 125

Glu Ala Asn His Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp

    130                 135                 140

Thr Glu Arg Gln Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys

145                 150                 155                 160

Glu Glu Ile Pro Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly

                165                 170                 175

Ser Asp Ser Thr Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu

            180                 185                 190

Gln Asp Leu Ile Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met

        195                 200                 205

Gly Ile Pro Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

    210                 215                 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225                 230                 235                 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

                245                 250                 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

            260                 265                 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

        275                 280                 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

    290                 295                 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305                 310                 315                 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

                325                 330                 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

            340                 345                 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

        355                 360                 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

    370                 375                 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385                 390                 395                 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

                405                 410                 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

            420                 425                 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

        435                 440                 445

Lys

<210>171

<211>1347

<212>DNA

<213>Artificial sequence

<400>171

aaggaggcat gccccacagg cctgtacaca cacagcggtg agtgctgcaa agcctgcaac   60

ctgggcgagg gtgtggccca gccttgtgga gccaaccaga ccgtgtgtga gccctgcctg  120

gacagcgtga cgttctccga cgtggtgagc gcgaccgagc cgtgcaagcc gtgcaccgag  180

tgcgtggggc tccagagcat gtcggcgeca tgcgtggagg ccgacgacgc cgtgtgccgc  240

tgcgcctacg gctactacca ggatgagacg actgggcgct gcgaggcgtg ccgcgtgtgc  300

gaggcgggct cgggcctcgt gttctcctgc caggacaagc agaacaccgt gtgcgaggag  360

tgccccgacg gcacgtattc cgacgaggcc aaccacgtgg acccgtgcct gccctgcacc  420

gtgtgcgagg acaccgagcg ccagctccgc gagtgcacac gctgggccga cgccgagtgc  480

gaggagatcc ctggccgttg gattacacgg tccacacccc cagagggctc ggacagcaca  540

gcccccagca cccaggagcc tgaggcacct ccagaacaag acctcatagc cagcacggtg  600

gcaggtgtgg tgaccacagt gatggggatc cccaaggtgg acaagaaagt tgagcccaaa  660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg   720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag   780

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac   840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc   900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag   960

tacaagtgca gggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa  1020

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg  1080

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc  1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg  1200

gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag  1260

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag  1320

aagagcctct ccctgtctcc gggtaaa                                      1347

<210>172

<211>449

<212>PRT

<213>Artificial sequence

<400>172

Lys Glu Ala Cys Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys

1               5                   10                  15

Lys Ala Cys Asn Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn

            20                  25                  30

Gln Thr Val Cys Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val

        35                  40                  45

Val Ser Ala Thr Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu

    50                  55                  60

Gln Ser Met Ser Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg

65                  70                  75                  80

Cys Ala Tyr Gly Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala

                85                  90                  95

Cys Arg Val Cys Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp

            100                 105                 110

Lys Gln Asn Thr Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp

        115                 120                 125

Glu Ala Asn His Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp

    130                 135                 140

Thr Glu Arg Gln Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys

145                 150                 155                 160

Glu Glu Ile Pro Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly

               165                 170                 175

Ser Asp Ser Thr Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu

            180                 185                 190

Gln Asp Leu Ile Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met

        195                 200                 205

Gly Ile Pro Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

    210                 215                 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225                 230                 235                 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

                245                 250                 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

        260                 265                 270

Pro Glu Val Lys Phe Asr Trp Tyr Val Asp Gly Val Glu Val His Asn

        275                 280                 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

    290                 295                 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asr Gly Lys Glu

305                 310                 315                 320

Tyr Lys Cys Arg Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

                325                 330                 335

Thr Ilc Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

            340                 345                 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

        355                 360                 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

    370                 375                 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385                 390                 395                 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

                405                 410                 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

            420                 425                 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

        435                 440                 445

Lys

<210>173

<211>1356

<212>DNA

<213>Artificial sequence

<400>173

aaggaggcat gccccacagg cctgtacaca cacagcggtg agtgctgcaa agcctgcaac   60

ctgggcgagg gtgtggccca gccttgtgga gccaaccaga ccgtgtgtga gccctgcctg  120

gacagcgtga cgttctccga cgtggtgagc gcgaccgagc cgtgcaagcc gtgcaccgag  180

tgcgtggggc tccagagcat gtcggcgcca tgcgtggagg ccgacgacgc cgtgtgccgc  240

tgcgcctacg gctactacca ggatgagacg actgggcgct gcgaggcgtg ccgcgtgtgc  300

gaggcgggct cgggcctcgt gttctcctgc caggacaagc agaacaccgt gtgcgaggag  360

tgccccgacg gcacgtattc cgacgaggcc aaccacgtgg acccgtgcct gccctgcacc  420

gtgtgcgagg acaccgagcg ccagctccgc gagtgcacac gctgggccga cgccgagtgc  480

gaggagatcc ctggccgttg gattacacgg tccacacccc cagagggctc ggacagcaca  540

gcccccagca cccaggagcc tgaggcacct ccagaacaag acctcatagc cagcacggtg  600

gcaggtgtgg tgaccacagt gatgggggga tccagcaaca ccaaggtgga caagaaagtt  660

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg  720

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg  780

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc  840

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag  900

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat  960

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1020

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1080

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1140

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1200

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1260

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1320

tacacgcaga agagcctctc cctgtctccg ggtaaa                           1356

<210>174

<211>452

<212>PRT

<213>Artificial sequence

<400>174

Lys Glu Ala Cys Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys

1               5                   10                  15

Lys Ala Cys Asn Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn

            20                  25                  30

Gln Thr Val Cys Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val

        35                  40                  45

Val Ser Ala Thr Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu

    50                  55                  60

Gln Ser Met Ser Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg

65                  70                  75                  80

Cys Ala Tyr Gly Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala

                85                  90                  95

Cys Arg Val Cys Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp

            100                 105                 110

Lys Gln Asn Thr Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp

        115                 120                 125

Glu Ala Asn His Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp

    130                 135                 140

Thr Glu Arg Gln Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys

145                 150                 155                 160

Glu Glu Ile Pro Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly

                165                 170                 175

Ser Asp Ser Thr Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu

            180                 185                 190

Gln Asp Leu Ile Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met

        195                 200                 205

Gly Gly Ser Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

    210                 215                 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225                 230                 235                 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

                245                 250                 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

            260                 265                 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

        275                 280                 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

    290                 295                 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305                 310                 315                 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

                325                 330                 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

            340                 345                 350

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

        355                 360                 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

    370                 375                 380

Glu Trp Glu Ser Asn Gly Gln Pro G1u Asn Asn Tyr Lys Thr Thr Pro

385                 390                 395                 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

                405                 410                 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

            420                 425                 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

        435                 440                 445

Ser Pro Gly Lys

    450

<210>175

<211>1431

<212>DNA

<213>Artificial sequence

<400>175

atgggggcag gtgccaccgg ccgcgccatg gacgggccgc gcctgctgct gttgctgctt   60

ctgggggtgt cccttggagg tgccaaggag gcatgcccca caggcctgta cacacacagc  120

ggtgagtgct gcaaagcctg caacctgggc gagggtgtgg cccagccttg tggagccaac  180

cagaccgtgt gtgagccctg cctggacagc gtgacgttct ccgacgtggt gagcgcgacc  240

gagccgtgca agccgtgcac cgagtgcgtg gggctccaga gcatgtcggc gccatgcgtg  300

gaggccgacg acgccgtgtg ccgctgcgcc tacggctact accaggatga gacgactggg  360

cgctgcgagg cgtgccgcgt gtgcgaggcg ggctcgggcc tcgtgttctc ctgccaggac  420

aagcagaaca ccgtgtgcga ggagtgcccc gacggcacgt attccgacga ggccaaccac  480

gtggacccgt gcctgccctg caccgtgtgc gaggacaccg agcgccagct ccgcgagtgc  540

acacgctggg ccgacgccga gtgcgaggag atccctggcc gttggattac acggtccaca  600

cccccagagg gctcggacag cacagccccc agcacccagg agcctgaggc acctccagaa   660

caagacctca tagccagcac ggtggcaggt gtggtgacca cagtgatggg gatccccaag   720

gtggacaaga aagttgagcc caaatcttgt gacaaaactc acacatgccc accgtgccca   780

gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc   840

ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac   900

cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag   960

ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac  1020

caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc  1080

cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc  1140

ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa  1200

ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac  1260

tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc  1320

accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag  1380

gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa a           143l

<210>176

<211>477

<212>PRT

<213>Artificial sequence

<400>176

Met Gly Ala Gly Ala Thr Gly Arg Ala Met Asp Gly Pro Arg Leu Leu

1               5                   10                  15

Leu Leu Leu Leu Leu Gly Val Ser Leu Gly Gly Ala Lys Glu Ala Cys

            20                  25                  30

Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys Lys Ala Cys Asn

        35                  40                  45

Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn Gln Thr Val Cys

    50                  55                  60

Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val Val Ser Ala Thr

65                  70                  75                  80

Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu Gln Ser Met Ser

                85                  90                  95

Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg Cys Ala Tyr Gly

            100                 105                 110

Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala Cys Arg Val Cys

        115                 120                 125

Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp Lys Gln Asn Thr

    130                 135                 140

Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp Glu Ala Asn His

145                 150                 155                 160

Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp Thr Glu Arg Gln

                165                 170                 175

Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys Glu Glu Ile Pro

            180                 185                 190

Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly Ser Asp Ser Thr

            195                 200                 205

Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu Gln Asp Leu Ile

    210                 215                 220

Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met Gly Ile Pro Lys

225                 230                 235                 240

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

                245                 250                 255

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

            260                 265                 270

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

        275                 280                 285

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

    290                 295                 300

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

305                 310                 315                 320

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

                325                 330                 335

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

            340                 345                 350

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

        355                 360                 365

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

    370                 375                 380

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

385                 390                 395                 400

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

                405                 410                 415

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

            420                 425                 430

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

        435                 440                 445

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

    450                 455                 460

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465                 470                 475

<210>177

<211>1431

<212>DNA

<213>Artificial sequence

<400>177

atgggggcag gtgccaccgg ccgcgccatg gacgggccgc gcctgctgct gttgctgctt     60

ctgggggtgt cccttggagg tgccaaggag gcatgcccca caggcctgta cacacacagc    120

ggtgagtgct gcaaagcctg caacctgggc gagggtgtgg cccagccttg tggagccaac    180

cagaccgtgt gtgagccctg cctggacagc gtgacgttct ccgacgtggt gagcgcgacc    240

gagccgtgca agccgtgcac cgagtgcgtg gggctccaga gcatgtcggc gccatgcgtg    300

gaggccgacg acgccgtgtg ccgctgcgcc tacggctact accaggatga gacgactggg    360

cgctgcgagg cgtgccgcgt gtgcgaggcg ggctcgggcc tcgtgttctc ctgccaggac    420

aagcagaaca ccgtgtgcga ggagtgcccc gacggcacgt attccgacga ggccaaccac    480

gtggacccgt gcctgccctg caccgtgtgc gaggacaccg agcgccagct ccgcgagtgc    540

acacgctggg ccgacgccga gtgcgaggag atccctggcc gttggattac acggtccaca    600

cccccagagg gctcggacag cacagccccc agcacccagg agcctgaggc acctccagaa    660

caagacctca tagccagcac ggtggcaggt gtggtgacca cagtgatggg gatccccaag    720

gtggacaaga aagttgagcc caaatcttgt gacaaaactc acacatgccc accgtgccca    780

gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc    840

ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac    900

cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag    960

ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac   1020

caggactggc tgaatggcaa ggagtacaag tgcagggtct ccaacaaagc cctcccagcc   1080

cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc   1140

ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa   1200

ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac  1260

tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc  1320

accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag  1380

gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa a           1431

<210>178

<211>477

<212>PRT

<213>Artificial sequence

<400>178

Met Gly Ala Gly Ala Thr Gly Arg Ala Met Asp Gly Pro Arg Leu Leu

1               5                   10                  15

Leu Leu Leu Leu Leu Gly Val Ser Leu Gly Gly Ala Lys Glu Ala Cys

            20                  25                  30

Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys Lys Ala Cys Asn

        35                  40                  45

Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn Gln Thr Val Cys

    50                  55                  60

Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val Val Ser Ala Thr

65                  70                  75                  80

Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu Gln Ser Met Ser

                85                  90                  95

Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg Cys Ala Tyr Gly

            100                 105                 110

Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala Cys Arg Val Cys

        115                 120                 125

Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp Lys Gln Asn Thr

    130                 135                 140

Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp Glu Ala Asn His

145                 150                 155                 160

Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp Thr Glu Arg Gln

                165                 170                 175

Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys Glu Glu Ile Pro

            180                 185                 190

Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly Ser Asp Ser Thr

        195                 200                 205

Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu Gln Asp Leu Ile

    210                 215                 220

Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met Gly Ile Pro Lys

225                 230                 235                 240

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

                245                 250                 255

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

            260                 265                 270

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

        275                 280                 285

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

    290                 295                 300

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

305                 310                 315                 320

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

                325                 330                 335

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Arg

            340                 345                 350

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

        355                 360                 365

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

    370                 375                 380

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

385                 390                 395                 400

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

                405                 410                 415

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

            420                 425                 430

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

        435                 440                 445

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

    450                 455                 460

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465                 470                 475

<210>179

<211>1440

<212>DNA

<213>Artificial sequence

<400>179

atgggggcag gtgccaccgg ccgcgccatg gacgggccgc gcctgctgct gttgctgctt     60

ctgggggtgt cccttggagg tgccaaggag gcatgcccca caggcctgta cacacacagc    120

ggtgagtgct gcaaagcctg caacctgggc gagggtgtgg cccagccttg tggagccaac    180

cagaccgtgt gtgagccctg cctggacagc gtgacgttct ccgacgtggt gagcgcgacc    240

gagccgtgca agccgtgcac cgagtgcgtg gggctccaga gcatgtcggc gccatgcgtg    300

gaggccgacg acgccgtgtg ccgctgcgcc tacggctact accaggatga gacgactggg    360

cgctgcgagg cgtgccgcgt gtgcgaggcg ggctcgggcc tcgtgttctc ctgccaggac    420

aagcagaaca ccgtgtgcga ggagtgcccc gacggcacgt attccgacga ggccaaccac    480

gtggacccgt gcctgccctg caccgtgtgc gaggacaccg agcgccagct ccgcgagtgc    540

acacgctggg ccgacgccga gtgcgaggag atccctggcc gttggattac acggtccaca    600

cccccagagg gctcggacag cacagccccc agcacccagg agcctgaggc acctccagaa    660

caagacctca tagccagcac ggtggcaggt gtggtgacca cagtgatggg gggatccagc    720

aacaccaagg tggacaagaa agttgagccc aaatcttgtg acaaaactca cacatgccca    780

ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc    840

aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc    900

cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc    960

aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc   1020

gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc   1080

ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag   1140

gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc   1200

ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg   1260

gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac   1320

agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg   1380

atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa   1440

<210>180

<211>480

<212>PRT

<213>Artificial sequence

<400>180

Met Gly Ala Gly Ala Thr Gly Arg Ala Met Asp Gly Pro Arg Leu Leu

1               5                   10                  15

Leu Leu Leu Leu Leu Gly Val Ser Leu Gly Gly Ala Lys Glu Ala Cys

            20                  25                  30

Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys Lys Ala Cys Asn

        35                  40                  45

Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn Gln Thr Val Cys

    50                  55                  60

Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val Val Ser Ala Thr

65                  70                  75                  80

Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu Gln Ser Met Ser

                85                  90                  95

Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg Cys Ala Tyr Gly

            100                 105                 110

Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala Cys Arg Val Cys

        115                 120                 125

Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp Lys Gln Asn Thr

    130                 135                 140

Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp Glu Ala Asn His

145                 150                 155                 160

Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp Thr Glu Arg Gln

                165                 170                 175

Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys Glu Glu Ile Pro

            180                 185                 190

Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly Ser Asp Ser Thr

        195                 200                 205

Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu Gln Asp Leu Ile

    210                 215                 220

Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met Gly Gly Ser Ser

225                 230                 235                 240

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

                245                 250                 255

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

            260                 265                 270

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

        275                 280                 285

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

    290                 295                 300

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

305                 310                 315                 320

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

                325                 330                 335

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

            340                 345                 350

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

        355                 360                 365

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

    370                 375                 380

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

385                 390                 395                 400

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

                405                 410                 415

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

            420                 425                 430

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

        435                 440                 445

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

    450                 455                 460

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

465                 470                 475                 480

<210>181

<211>28

<212>DNA

<213>Artificial sequence

<400>181

ccttgatatc tcagcctcta caggactg                                      28

<210>182

<211>27

<212>DNA

<213>Artificial sequence

<400>182

agcccagaat tctatgttct tccgtca                                       27

<210>183

<211>387

<212>DNA

<213>People

<400>183

atgcagcagc ccttcaatta cccatatccc cagatctact gggtggacag cagtgccagc     60

tctccctggg cccctccagg cacagttctt ccctgtccaa cctctgtgcc cagaaggcct    120

ggtcaaagga ggccaccacc accaccgcca ccgccaccac taccacctcc gccgccgccg    180

ccaccactgc ctccactacc gctgccaccc ctgaagaaga gagggaacca cagcacaggc    240

ctgtgtctcc ttgtgatgtt tttcatggtt ctggttgcct tggtaggatt gggcctgggg    300

atgtttcagc tcttccacct acagaaggag ctggcagaac tccgagagtc  taccagccag   360

atgcacacag catcatcttt ggagaag                                        387

<210>184

<211>129

<212>PRT

<213>People

<400>184

Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Tyr Trp Val Asp

1               5                   10                  15

Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro Cys

            20                  25                  30

Pro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro

        35                  40                  45

Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro

    50                  55                  60

Pro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly

65                  70                  75                  80

Leu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly

                85                  90                  95

Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala

            100                 105                 110

Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu Glu

        115                 120                 125

Lys

<210>185

<211>456

<212>DNA

<213>People

<400>185

caaataggcc accccagtcc accccctgaa aaaaaggagc tgaggaaagt ggcccattta     60

acaggcaagt ccaactcaag gtccatgcct ctggaatggg aagacaccta tggaattgtc    120

ctgctttctg gagtgaagta taagaagggt ggccttgtga tcaatgaaac tgggctgtac    180

tttgtatatt ccaaagtata cttccggggt caatcttgca acaacctgcc cctgagccac    240

aaggtctaca tgaggaactc taagtatccc caggatctgg tgatgatgga ggggaagatg    300

atgagctact gcactactgg gcagatgtgg gcccgcagca gctacctggg ggcagtgttc    360

aatcttacca gtgctgatca tttatatgtc aacgtatctg agctctctct ggtcaatttt    420

gaggaatctc agacgttttt cggcttatat aagctc                              456

<210>186

<211>152

<212>PRT

<213>People

<400>186

Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg Lys

1               5                   10                  15

Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu Glu

            20                  25                  30

Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys Tyr Lys

        35                  40                  45

Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr Ser

    50                  55                  60

Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser His

65                  70                  75                  80

Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met Met

                85                  90                  95

Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala Arg

            100                 105                 110

Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His Leu

        115                 120                 125

Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser Gln

    130                 135                 140

Thr Phe Phe Gly Leu Tyr Lys Leu

145                 150

<210>187

<211>843

<212>DNA

<213>Artificial sequence

<400>187

atgcagcagc ccttcaatta cccatatccc cagatctact gggtggacag cagtgccagc   60

tctccctggg cccctccagg cacagttctt ccctgtccaa cctctgtgcc cagaaggcct  120

ggtcaaagga ggccaccacc accaccgcca ccgccaccac taccacctcc gccgccgccg  180

ccaccactgc ctccactacc gctgccaccc ctgaagaaga gagggaacca cagcacaggc  240

ctgtgtctcc ttgtgatgtt tttcatggtt ctggttgcct tggtaggatt gggcctgggg  300

atgtttcagc tcttccacct acagaaggag ctggcagaac tccgagagtc taccagccag  360

atgcacacag catcatcttt ggagaagcaa ataggccacc ccagtccacc ccctgaaaaa  420

aaggagctga ggaaagtggc ccatttaaca ggcaagtcca actcaaggtc catgcctctg  480

gaatgggaag acacctatgg aattgtcctg ctttctggag tgaagtataa gaagggtggc  540

cttgtgatca atgaaactgg gctgtacttt gtatattcca aagtatactt ccggggtcaa  600

tcttgcaaca acctgcccct gagccacaag gtctacatga ggaactctaa gtatccccag  660

gatctggtga tgatggaggg gaagatgatg agctactgca ctactgggca gatgtgggcc  720

cgcagcagct acctgggggc agtgttcaat cttaccagtg ctgatcattt atatgtcaac  780

gtatctgagc tctctctggt caattttgag gaatctcaga cgtttttcgg cttatataag  840

ctc                                                                843

<210>188

<211>281

<212>PRT

<213>Artificial sequence

<400>188

Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Tyr Trp Val Asp

1               5                   10                  15

Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro Cys

            20                  25                  30

Pro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro

        35                  40                  45

Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro

    50                  55                  60

Pro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly

65                  70                  75                  80

Leu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly

                85                  90                  95

Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala

            100                 105                 110

Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu Glu

        115                 120                 125

Lys Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg

    130                 135                 140

Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu

145                 150                 155                 160

Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys Tyr

                165                 170                 175

Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr

            180                 185                 190

Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser

        195                 200                 205

His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met

    210                 215                 220

Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala

225                 230                 235                 240

Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His

                245                 250                 255

Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser

            260                 265                 270

Gln Thr Phe Phe Gly Leu Tyr Lys Leu

        275                 280

<210>189

<211>1572

<212>DNA

<213>Artificial sequence

<400>189

atgcagcagc ccttcaatta cccatatccc cagatctact gggtggacag cagtgccagc   60

tctccctggg cccctccagg cacagttctt ccctgtccaa cctctgtgcc cagaaggcct  120

ggtcaaagga ggccaccacc accaccgcca ccgccaccac taccacctcc gccgccgccg  180

ccaccactgc ctccactacc gctgccaccc ctgaagaaga gagggaacca cagcacaggc  240

ctgtgtctcc ttgtgatgtt tttcatggtt ctggttgcct tggtaggatt gggcctgggg  300

atgtttcagc tcttccacct acagaaggag ctggcagaac tccgagagtc taccagccag  360

atgcacacag catcatcttt ggagaagcaa ataggccacc ccagtccacc ccctgaaaaa  420

aaggagctga ggaaagtggc ccatttaaca ggcaagtcca actcaaggtc catgcctctg  480

gaatgggaag acacctatgg aattgtcctg ctttctggag tgaagtataa gaagggtggc  540

cttgtgatca atgaaactgg gctgtacttt gtatattcca aagtatactt ccggggtcaa  600

tcttgcaaca acctgcccct gagccacaag gtctacatga ggaactctaa gtatccccag  660

gatctggtga tgatggaggg gaagatgatg agctactgca ctactgggca gatgtgggcc  720

cgcagcagct acctgggggc agtgttcaat cttaccagtg ctgatcattt atatgtcaac  780

gtatctgagc tctctctggt caattttgag gaatctcaga cgtttttcgg cttatataag  840

ctcggatcca gcaacaccaa ggtggacaag aaagttgagc ccaaatcttg tgacaaaact  900

cacacatgcc caccgtgccc agcacctgaa ctcctggggg gaccgtcagt cttcctcttc  960

cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg 1020

gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag 1080

gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 1140

agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc    1200

tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc    1260

cgagaaccac aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc    1320

agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc    1380

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc    1440

ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc    1500

tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg    1560

tctccgggta aa                                                        1572

<210>190

<211>524

<212>PRT

<213>Artificial sequence

<400>190

Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Tyr Trp Val Asp

1               5                   10                  15

Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro Cys

            20                  25                  30

Pro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro

        35                  40                  45

Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro

    50                  55                  60

Pro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly

65                  70                  75                  80

Leu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly

                85                  90                  95

Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala

            100                 105                 110

Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu Glu

        115                 120                 125

Lys Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg

    130                 135                 140

Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu

145                 150                 155                 160

Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys Tyr

                165                 170                 175

Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr

            180                 185                 190

Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser

        195                 200                 205

His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met

    210                 215                 220

Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala

225                 230                 235                 240

Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His

                245                 250                 255

Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser

            260                 265                 270

Gln Thr Phe Phe Gly Leu Tyr Lys Leu Gly Ser Ser Asn Thr Lys Val

        275                 280                 285

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

    290                 295                 300

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

305                 310                 315                 320

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

                325                 330                 335

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

            340                 345                 350

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

        355                 360                 365

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

    370                 375                 380

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

385                 390                 395                 400

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

                405                 410                 415

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

            420                 425                 430

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

        435                 440                 445

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

    450                 455                 460

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

465                 470                 475                 480

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

            485                 490                 495

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

        500                 505                 510

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

        515                 520

<210>191

<211>1797

<212>DNA

<213>People

<400>191

atgagcactg aaagcatgat ccgggacgtg gagctggccg aggaggcgct ccccaagaag     60

acaggggggc cccagggctc caggcggtgc ttgttcctca gcctcttctc cttcctgatc    120

gtggcaggcg ccaccacgct cttctgcctg ctgcactttg gagtgatcgg cccccagagg    180

gaagaggtga gtgcctggcc agccttcatc cactctccca cccaagggga aatgagagac    240

gcaagagagg gagagagatg ggatgggtga aagatgtgcg ctgataggga gggatgagag    300

agaaaaaaac atggagaaag acggggatgc agaaagagat gtggcaagag atggggaaga    360

gagagagaga aagatggaga gacaggatgt ctggcacatg gaaggtgctc actaagtgtg    420

tatggagtga atgaatgaat gaatgaatga acaagcagat atataaataa gatatggaga    480

cagatgtggg gtgtgagaag agagatgggg gaagaaacaa gtgatatgaa taaagatggt    540

gagacagaaa gagcgggaaa tatgacagct aaggagagag atgggggaga taaggagaga    600

agaagatagg gtgtctggca cacagaagac actcagggaa agagctgttg aatgctggaa    660

ggtgaataca cagatgaatg gagagagaaa accagacacc tcagggctaa gagcgcaggc    720

cagacaggca gccagctgtt cctcctttaa gggtgactcc ctcgatgtta accattctcc    780

ttctccccaa cagttcccca gggacctctc tctaatcagc cctctggccc aggcagtcag    840

taagtgtctc caaacctctt tcctaattct gggtttgggt ttgggggtag ggttagtacc    900

ggtatggaag cagtggggga aatttaaagt tttggtcttg ggggaggatg gatggaggtg    960

aaagtagggg ggtattttct aggaagttta agggtctcag ctttttcttt tctctctcct   1020

cttcaggatc atcttctcga accccgagtg acaagcctgt agcccatgtt gtaggtaaga   1080

gctctgagga tgtgtcttgg aacttggagg gctaggattt ggggattgaa gcccggctga   1140

tggtaggcag aacttggaga caatgtgaga aggactcgct gagctcaagg gaagggtgga   1200

ggaacagcac aggccttagt gggatactca gaacgtcatg gccaggtggg atgtgggatg    1260

acagacagag aggacaggaa ccggatgtgg ggtgggcaga gctcgagggc caggatgtgg    1320

agagtgaacc gacatggcca cactgactct cctctccctc tctccctccc tccagcaaac    1380

cctcaagctg aggggcagct ccagtggctg aaccgccggg ccaatgccct cctggccaat    1440

ggcgtggagc tgagagataa ccagctggtg gtgccatcag agggcctgta cctcatctac    1500

tcccaggtcc tcttcaaggg ccaaggctgc ccctccaccc atgtgctcct cacccacacc    1560

atcagccgca tcgccgtctc ctaccagacc aaggtcaacc tcctctctgc catcaagagc    1620

ccctgccaga gggagacccc agagggggct gaggccaagc cctggtatga gcccatctat    1680

ctgggagggg tcttccagct ggagaagggt gaccgactca gcgctgagat caatcggccc    1740

gactatctcg actttgccga gtctgggcag gtctactttg ggatcattgc cctgtga       1797

<210>192

<211>951

<212>DNA

<213>People

<400>192

atgacaccac ctgaacgtct cttcctccca agggtgtgtg gcaccaccct acacctcctc     60

cttctggggc tgctgctggt tctgctgcct ggggcccagg tgaggcagca ggagaatggg    120

ggctgctggg gtggctcagc caaaccttga gccctagagc ccccctcaac tctgttctcc    180

cctaggggct ccctggtgtt ggcctcacac cttcagctgc ccagactgcc cgtcagcacc    240

ccaagatgca tcttgcccac agcaccctca aacctgctgc tcacctcatt ggtaaacatc    300

cacctgacct cccagacatg tccccaccag ctctcctcct acccctgcct caggaaccca    360

agcatccacc cctctccccc aacttccccc acgctaaaaa aaacagaggg agcccactcc    420

tatgcctccc cctgccatcc cccaggaact cagttgttca gtgcccactt cctcagggat    480

tgagacctct gatccagacc cctgatctcc cacccccatc ccctatggct cttcctagga    540

gaccccagca agcagaactc actgctctgg agagcaaaca cggaccgtgc cttcctccag    600

gatggtttct ccttgagcaa caattctctc ctggtcccca ccagtggcat ctacttcgtc    660

tactcccagg tggtcttctc tgggaaagcc tactctccca aggccacctc ctccccactc    720

tacctggccc atgaggtcca gctcttctcc tcccagtacc ccttccatgt gcctctcctc    780

agctcccaga agatggtgta tccagggctg caggaaccct ggctgcactc gatgtaccac    840

ggggctgcgt tccagctcac ccagggagac cagctatcca cccacacaga tggcatcccc    900

cacctagtcc tcagccctag tactgtcttc tttggagcct tcgctctgta g             951

<210>193

<211>6815

<212>DNA

<213>People

<400>193

atgcagcagc ccttcaatta cccatatccc cagatctact gggtggacag cagtgccagc     60

tctccctggg cccctccagg cacagttctt ccctgtccaa cctctgtgcc cagaaggcct    120

ggtcaaagga ggccaccacc accaccgcca ccgccaccac taccacctcc gccgccgccg    180

ccaccactgc ctccactacc gctgccaccc ctgaagaaga gagggaacca cagcacaggc    240

ctgtgtctcc ttgtgatgtt tttcatggtt ctggttgcct tggtaggatt gggcctgggg    300

atgtttcagc tcttccacct acagaaggag ctggcagaac tccgagaggt aagcctgccg    360

gcagactgct gtgccctgga ggcaccaggc ataaggggat ggagggccca ctgcctggct    420

tgcaaagtgc ttttcaatcc tttttttttt tttcttagaa atgggttgtt ctagttattc    480

tattctatag acagagaaat ggaggctcat aggggtgaag ggaatatttt ttactttgta    540

aagaatcaga gcccaacagt ttgtctttcc tcagttgtca actcagtctg ttgaaccact    600

tactgcctca tacctattga tttaaggagt aaaaaacaaa caaacaaaac ttttaagttt    660

tgcttggaag taaatttgtt cagatgacca aactaaatct tgaaaattgg tacgattcca    720

aatcacaaaa ctgagggaca gtgaggcaaa attgcttggc caaagcccaa gttagcagaa    780

cttctgaggt atttggattc tctttccagg gcttggttta tttgacgatt ctgcctcttt    840

tgcttaaaga attttatttt tattatacat cttttctctt tctgttttac tagtctacca    900

gccagatgca cacagcatca tctttggaga agcaaatagg tgagtctttt ttcgcatgta     960

cattgagttc ccaaagatga tcctcagcac agaactatgt taatggaatg ccttaaattc    1020

tgtcccacac tttggtttct gtacactata agaggaattc tggctaattc agaatctctg    1080

gtctatgatt ccttgagctg ctttaaaaat gtgaagtgaa ttgaattgct cacaatcaat    1140

atagctgagc actgcacaca attcaaaatc acttccactt gactttggaa agagacacac    1200

atatacaacc ctggaccttt gccccctgag aagtggctcc aggcctgtcc ccttccacag    1260

acatcctggt cctggcacac acgccagtgg ctgtaactcc tgggaagaga aggcgaaatg    1320

aaggcagaga cagatgtttc tgagaaacgt cctttcctct tttaaatgcc ctaaagagat    1380

tacattgaag ctttatttac aatatgtttt ataggatgta agcttttaaa acatggataa    1440

tcaattcccc acattagaat ggtttttctt gaagtcactg agaagcttaa gggaagactt    1500

cagccatcat tcaaaggatg ttcagcttca cccacagagg cagtcatgag gtacccttga    1560

tcacaaagga aaatcctttg tatgggagct agggcaagaa gatgataggt tgaacactga    1620

tctcatgaaa tttcagggca tagttgtgca agctctgtac agatagattt tacagtgtgc    1680

ttcactagtc taagattcat tgaaatttta gatttagatt tacaatgaga ttcattgaat    1740

ttttagaggt ggaaggaagc ttagaatata tctagtccag ccatttcatt ttttggaaga    1800

gaaacaaaat accagatgga ttaggtgaat atgcccaaga ccactggtat gccattgggg    1860

ctagaaggaa cacaggctct tgatgccttg tcacctacag ggctttccag tactggactg    1920

gtgcctcatg aaggagggaa actcctgtct ctctgtagtg agcatggaat aagaaataga    1980

tgaaaagcag gatacctctg caaagactat aggtagatct catccttaga aactcagaat    2040

aaagaagaag gcagcactgg ccatctatta agtgaagcaa gttccagttt ctccggatgt    2100

ggctactgca catgacccag tcgcctgtct actatcctag actgttaagt tcttatatga    2160

gccactgttg ggatgctctg gcccctgctt ctgtgtccta atccttaaat gttagaaagt    2220

ccatatgtac acaagcagag tggaaatatt tgatagaaaa gtaaagcctc tggaggacgt    2280

ctgaaataag atcattttga aaatctgtgg aatgtgcctg gtctgatagg tatctaatta    2340

ttcatctttc acccactctc tggagtcaat ttagtgatta aaagtcaaat gatcaggtga    2400

gtcagatcca tctaccccag taagcaatta tgttctctag atcaacccaa ataaaccaga    2460

aattggtaaa tcatcacatg gaaatcaaat cagtaacttt actaataaga aaagagatgc    2520

aaattaaaaa tagggaatac caattttcat caaattaata aagattaaaa aaaaacccag    2580

actgacaaag atttgagaga aaagtgcctg tgcaaacatt agtttcagcc agtatacatg    2640

taaattgcta taactttttt tgaagagcgc tttagaaata catagcaaaa gcttgtaact    2700

agtagtttac gttgtaatcc tatcgctaag aatttatctt aagaaaataa tcagagatgt    2760

gcccgaagat ttatatataa ggatgttcat tacttcctta tgagcctatt tataataaat    2820

aattcagaag aaacttgaat gcctcatacg agattagtta tatacattat ggtgtattca    2880

gacaagcaca taataagcag caactaaaag tgatgtatca gaaggacatt taatgctttg    2940

gggattgtgc actctgtatt gttagataaa aagacaagtt acaaaagggt atgtttagtt    3000

tcatactgac tatgtaacca tgtatgcaca tttttatatt cttaggaact ggaaagtttt    3060

acacactgac atactaacag tgattttctc tgggagatac tattatagat tatttatact    3120

ttctaatttg ctccttttta cattttccat ttttttccgg ttaactaatt aaatctttta    3180

ttcattcaca tgtgtgaggc tagatgttgg gtgttggatg ctgattgagg atgagccagc    3240

ccagggggcc tgtccttatg gagcttttag cagggggaag aagctaaaca aaagcaagca    3300

aacagaccat gcaattccaa agttgtacta agtacaacct gagaatcttc tttaaggagg    3360

tgttgtttac gctaacacct tgggggtcag aagcccgatg agtgaacggc tgtgtgcatg    3420

tgctgtggag tggggtgggg ctgggggcgg gcatttcagc gctgggaacg gcacatgcaa    3480

aaaccctggg gcaggaagca ctcataacca gtgtctagga tatccttgca atttatttat    3540

tccagtatag gtacagcaga tattcttagt gagcatgttc tgtaagaata acgttgtact    3600

aggctctgtg agggatataa agacatgcaa catgtggttc tctgttccgt ttcctgccgg    3660

tgcgtggctg agtgccagat tagttgctcg ggtggtttgc ggtgtaggaa tgcagaggag    3720

actcagtttt gcctttcctg ttcgctaaca cctcctacat ggtgggttgt cttggatgac    3780

tagagaggtc atgattgctc agctaatgtt ctctgtggat tgggccattc tcataccata    3840

tgtgagtttt acaggctaat cacgaacctg acatagacaa gcatctcctg tgaatattta    3900

ttgaagtctc agttagctga attcattagc taagtggcag ctgtctatct tgtctgaaac    3960

agttgaagtc agagaaatag tgtctgtttt gagaactatt gtctatcctt ccatttaatt    4020

tgtccaacat ttgctagtaa catgccctgt gttagaaact gggcttctct ggtgaactca    4080

acgtatcgta aggaacttgt cgttatttga ggggaataag ataatttatg tagttacccc    4140

agagacaaga ggagtcagaa atgacaagag aggggaagac agagggatcc tagatttcaa    4200

aagaggggca gatcatattt gatcaaaaaa ggcttgagag ggtttcccaa agaaagcggt    4260

aataggtagt ccttatggga tgtttaggaa tttgacacag aaatctgctt gaaagaacat    4320

tacgggcaga agacatggca tgaacaaagg cacagaggaa ggaaagtggg ggtatgtgta    4380

ggccacagaa ctcccgttgt gtggcaatgc tcagtgggag taaggagata aggctggaca    4440

gatgggctgg gattgtgtga tggggatact tgtatacgac ggcatcttcg gggaggagca    4500

attaaaggtg tttgaggact cagtgatatg aggtgattgt cagaggtctt aagataatta    4560

atttggggca gattgtaaaa tgtcactggg ggaagatctg gtgacacgca gacagatcag    4620

tagtcttctt cagcctctgt cctgtaagtc tttggaacaa gagaatcaga gtggacagag    4680

ccctggaatt ggcgtcagga gagcagggtg tggtcccatc tgtgatgctg actagctggg    4740

tcttcttgga ttagtcaccc aacttctaag ctcattttgc aatcgataag gtgggacaat    4800

aatacctacc tcaaagggtt gtggtgagtt ctaaatgcaa taactgacat gacagctctt    4860

tatgaataaa tgtttatatt aattatgttt tattgtaaca ttataattta tacatatgtc    4920

aatcttaatt tggtttattt ttttacctct ttgtttctga aatatagact actatgaatc    4980

ctgcagttca gacctacatg attagtatat gttagactgt tgccatttac ggttttaaaa    5040

tctttttttt aaaatgattg gatttaaatt cccaccaaaa taatagttgc tatttcattt    5100

taacatatat ttttcctctc tctatgatac aggccacccc agtccacccc ctgaaaaaaa    5160

ggagctgagg aaagtggccc atttaacagg tctgtatctg gaaggtacag gtgagatttg    5220

ggaaagcttt tggcaaaagg caaaatggct gcctggtttc cattaccagg ctctagagag    5280

ttgttacttc ctggagacag tatttgaacc caataatact aacacttttc ttgtcgagtc    5340

agtcttatga acacactttg atcccctatt ctctgggcaa ggccctcttc taaattggag    5400

acctggagct gattatgaca tgacccctgc ccataagaag ctcattcttt tcagtcattc    5460

caaagtccct tgggggattg ggggcacaga tccaagaggt gagagatgag aaggtgagga    5520

atgaggactt attttagtca gtgctttctt ggttttctgg aagagcttca ctctgctttg    5580

tccaagagta taaataggag tgagaaggga gactcaatcc tggcacttgt agagtttcaa    5640

tatgtgcctg taaagtggag tgaaatgata tggtatgggc actcccatta taaactacat  5700

aaataatgga ataaatatca cagaaagtgg taggctattg tccctggaat tatttaggca  5760

gaaattatag aatgatccgg tcacatcagc agacacttac tgaatacctg ctgtgtgcca  5820

atttggggat gtgaagatga gtagcacatg gctcttgccc ccagggagct tgacatctaa  5880

tagggaaaga caggcactta tgataatttc agtacttcag agatttaggt tatgttcatg  5940

atgttgtggg gatacaaaga aaagggaact gtaccccaaa ttgaggggct gaataattct  6000

tactgcaggt gagaagatgg accagatggt ccctaagatc cttcccaact ttagaacttt  6060

agagttcctt ggatttggct ttttccttca ggaaaggact tcaaagccta gcagatttgg  6120

tgctagttct gaagatagta aaatctttgt tccagagagc aaatattttc tcaataattt  6180

cttactgcaa tggattacgg gtatatacta ttgttccaat tgtgtggatg acaaaatagg  6240

acaacgttgt tgaggaaatt ctgtgatgga tcaagttctg acccctcagc cagttctata  6300

ccagctgtca ttctgggtga aacatttgtt gaaggaaggg cccacagttt tgccttagaa  6360

acttagtttg ttggatgcat gactattcct tgttgaaagc tccttttgga tttatttcag  6420

gcaagtccaa ctcaaggtcc atgcctctgg aatgggaaga cacctatgga attgtcctgc  6480

tttctggagt gaagtataag aagggtggcc ttgtgatcaa tgaaactggg ctgtactttg  6540

tatattccaa agtatacttc cggggtcaat cttgcaacaa cctgcccctg agccacaagg  6600

tctacatgag gaactctaag tatccccagg atctggtgat gatggagggg aagatgatga  6660

gctactgcac tactgggcag atgtgggccc gcagcagcta cctgggggca gtgttcaatc  6720

ttaccagtgc tgatcattta tatgtcaacg tatctgagct ctctctggtc aattttgagg  6780

aatctcagac gtttttcggc ttatataagc tctaa                             6815

<210>194

<211>28

<212>DNA

<213>Artificial sequence

<400>194

gggattaggc ggccgcaggc ttgttttc                                     28

<210>195

<211>33

<212>DNA

<213>Artificial sequence

<400>195

gatggagaag agtgaggatc ctgtgcttag cag    33

<210>196

<211>28

<212>DNA

<213>Artificial sequence

<400>196

gggattagcc ggatccaggc ttgttttc          28

<210>197

<211>28

<212>DNA

<213>Artificial sequence

<400>197

agaagagtgc ggccgctgtg cttagcag          28

<210>198

<211>34

<212>DNA

<213>Artificial sequence

<400>198

aaaaactcga gaaccggacc ccgcccgcac ccat    34

<210>199

<211>32

<212>DNA

<213>Artificial sequence

<400>199

aaaaaggatc ctcatttacc cggagacagg ga      32

Claims (21)

1. the protein of separation, it has measurable physical and chemical parameter feature, wherein described character representation, the pharmacological characteristics for being associated with one or more characteristics or formed one or more characteristics pharmacological characteristics basis, wherein the protein of described separation, which has, includes multiple measurable physical and chemical parameter { [P_x]₁、[P_x]₂、...[P_x]_n, physicochemical characteristicses, wherein P_xRepresent measurable physical and chemical parameter and " n " be >=1 integer, wherein [P_x]₁To [P_x]_nIn each be respectively different measurable physical and chemical parameters, a series of value of the value of the measurable physicochemical property of any of which or more than one measurable physicochemical property represents, is associated with the pharmacological characteristics T of a characteristic_yBasis or series of features pharmacological characteristics { [T_y]₁、[T_y]₂、....[T_y]_mOr formed a characteristic pharmacological characteristics T_yBasis or series of features pharmacological characteristics { [T_y]₁、[T_y]₂、....[T_y]_mBasis, wherein T_yThe pharmacological characteristics and m for representing a characteristic are >=1 integers, wherein [T_y]₁To [T_y]_mIn each be respectively different pharmacological characteristics, wherein the protein separated is selected from TNF-α, LT- α, TNFRI-Fc, TNFRII-Fc, 0X40-Fc, BAFF, NGFR-Fc and FasL.

2. the protein of the separation of claim 1, wherein described protein has measurable physical and chemical parameter shown in one or more tables 2.

3. the protein of the separation of claim 1, wherein described protein has the pharmacological characteristics of the characteristic shown in one or more tables 3.

4. chimeric molecule, it includes TNF-α, LT- α, BAFF or the FasL or its segment for being fused to one or more peptides, polypeptide or the claim 1 on protein portion.

5. the chimeric molecule of claim 4, wherein, peptide, polypeptide or protein portion include the constant region (Fc) or framework region of human immunoglobulin(HIg).

6. the chimeric molecule of claim 4, wherein, chimeric molecule is selected from TNF-α-Fc, LT- α-Fc, BAFF-Fc or FasL-Fc.

7. pharmaceutical composition, the protein or chimeric molecule of the separation containing any one of claim 1 to 6.

8. the pharmaceutical composition of claim 7, the wherein pharmaceutical composition further include pharmaceutically acceptable topical vehicle.

9. the pharmaceutical composition of claim 8, wherein pharmaceutically acceptable topical vehicle is creme or lotion.

10. the pharmaceutical composition of claim 7 to 9, wherein chimeric molecule are TNFRI-Fc or TNFRII-Fc.

11. the method for disease is treated or prevented in mammalian subject, wherein described disease can be improved by increasing the amount or activity of protein, and described method includes the pharmaceutical composition for giving the chimeric molecule or claim 7 to 9 of any one of protein, the claim 4 to 6 of the separation according to any one of claims 1 to 3 of the mammalian subject effective dose.

12. nucleotide sequence, selected from SEQ ID No：27, 29, 31, 33, 35, 37, 39, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 127, 129, 131, 133, 135, 137, 139, 141, 143, 147, 149, 151, 153, 155, 157, 159, 163, 165, 167, 169, 171, 173, 175, 177, 179, 183, 185, 187, 189, or there is the nucleotide sequence of at least about 90% homogeneity with the above-mentioned any sequence enumerated, or the nucleotide sequence that can hybridize with any above-mentioned sequence or their complementary type under high stringency conditions.

13. the protein or chimeric molecule of separation, by selected from SEQ ID NO：27, 29, 31, 33, 35, 37, 39, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 127, 129, 131, 133, 135, 137, 139, 141, 143, 147, 149, 151, 153, 155, 157, 159, 163, 165, 167, 169, 171, 173, 175, 177, 179, 183, 185, 187, coded by 189 nucleotide sequence, or as coded by there is at least about nucleotide sequence of 90% homogeneity or the nucleotide sequence that can hybridize with any one above-mentioned sequence or they any complementary type under high stringency conditions with the above-mentioned any sequence enumerated.

14. the nucleic acid molecule of separation, encoding proteins matter or chimeric molecule or its funtion part, including with SEQ ID NO：27, 29, 31, 33, 35, 37, 39, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 127, 129, 131, 133, 135, 137, 139, 141, 143, 147, 149, 151, 153, 155, 157, 159, 163, 165, 167, 169, 171, 173, 175, 177, 179, 183, 185, 187, 189 have the nucleotide sequence of at least 90% similitude, or after optimal comparison and/or can be with SEQ ID NO：27, 29, 31, 33, 35, 37, 39, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 127, 129, 131, 133, 135, 137, 139, 141, 143, 147, 149, 151, 153, 155, 157, 159, 163, 165, 167, 169, 171, 173, 175, 177, 179, 183, 185, 187, 189 or one or more nucleotide sequences hybridized under high stringency conditions of their complementary type.

15. the nucleic acid molecule of separation, including coding have substantially such as SEQ ID NO：28, 30, 32, 34, 36, 38, 40, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 128, 130, 132, 134, 136, 138, 140, 142, 144, 148, 150, 152, 154, 156, 158, 160, 164, 166, 168, 170, 172, 174, 176, 178, 180, 184, 186, 188, the protein or the nucleotide sequence of chimeric molecule of 190 one or more shown amino acid sequences, or coding and SEQ ID NO：28, 30, 32, 34, 36, 38, 40, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 128, 130, 132, 134, 136, 138, 140, 142, 144, 148, 150, 152, 154, 156, 158, 160, 164, 166, 168, 170, 172, 174, 176, 178, 180, 184, 186, 188, one or more nucleotide sequences with least about protein of the amino acid sequence of 90% similitude or chimeric molecule after 190 optimal comparisons.

16. a kind of kit, for determining the human protein of the expression of people's cell present in biological products or the level of chimeric molecule, including (a) solid phase support matrix；(b) it is one or more anti-according to the human protein of any one of claims 1 to 3 or the antibody of the chimeric molecule according to any one of claim 4 to 6；(c) lock solution；(d) one or more substrate reservoirs；(e) substrate buffer solution；(f) standard human protein or chimeric molecule sample and (g) operation instructions.

17. the kit of claim 16, wherein standard human protein or chimeric molecule sample be the separation of any one of claim 2 to 3 protein or claim 4 chimeric molecule product.

18. the kit of claim 16 or 17, mammal is immunized the product that each of which antibody is derived from the chimeric molecule with the protein of the separation comprising any one of Claims 2 or 3 or claim 4.

19. the human protein of the kit of claim 16 to 18, wherein people's cell expression is naturally occurring TNF-α, LT- α, TNFRI, TNFRII, OX40, BAFF, NGFR or FasL.

20. sanatory method, the illness is characterized in that the TNF-α level in individual is excessive, or the level of the TNF-α in the cause of disease individual is excessively aggravated, or it is relevant with TNF-α, methods described includes the pharmaceutical composition that the claim 10 of therapeutically effective amount is partly applied to individual.

21. the method for claim 20, wherein, disease disease is selected from：Psoriasis, BehcetShi diseases, bullous dermatitis, eczema, mycotic infection, leprosy, neutrophilic dermatitis, spot pityriasis (or pityriasis rosea), pityriasis nigra (or tinea manuum nigra), pityriasis rubra pilaris, systemic lupus erythematosus, primary small vessel vasculitis and toxic epidermal necrolysis, erythema, erosion, ulcer, peeling, slabbing, drying, into scar, crust, skin secretion or ooze out or using any side effect caused by the treatment such as Aladara cream.

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