CN101198621A - 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 such as EPO, Flt3-Ligand, Flt3, PDGF-B or VEGF-165 or chimeric molecules thereof comprising at least a portion of the protein molecule, wherein the protein or chimeric molecule has a profile of measurable physiochemical parameters which 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 the invention provides the protein molecule of separation, such as EPO, Flt3- part, Flt3, PDGF-B or VEGF-165 or at least one of chimeric molecule comprising the protein molecule, wherein protein or chimeric molecule have the feature of measurable biochemical parameter, its expression, the basis for associating or being formed 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.

Growth factor and their acceptor are important during many relevant with differentiation from the propagation of the different cell categories of regulation, regulation to hematopoietic cell is the most notable, and it can be used for producing red blood cell, blood platelet, NK, precursor B cells, Precursor T-cell, monocyte/macrophage and BMDC.In addition to adjusting cell differentiation, growth factor can also adjust process such as angiogenesis, wound healing and neurological process such as Remyelination.Such growth factor and their acceptor include EPO, Flt3- part, F1t3, PDGF-B and VEGF-165.

EPO is glycoprotein, and it is relevant with RBC acceptor garland rate, for example, the survival of the red progenitor cells in marrow, propagation and differentiation.In addition, EPO stimulates the propagation of epithelial cell and induction B- cells propagation and megakaryocytopoiesis in angiogenesis.EPO can also be in localized cerebral ischemic; concussion property brain damage; neuroprotective factor (Brines etc., Proc Natl Acad Sci USA97 (19) are used as in the animal model of experimental autoimmune encephalomyelitis (EAE) and the epilepsy of kainic acid induction：10526-31,2000).

PDGF-B is the regulatory factor of mesenchymal cell such as fibroblast, smooth muscle cell and neuroectodermal cells such as oligodendrocyte proliferation.PDGF-B promotes vascular progenitor propagation to show PDGF-B to facilitating angiogenesis and because possible lymphatic metastasis caused by lymphatic vessel generation is a kind of important growth factor in tumour with it.In addition, PDGF-B plays an important role in wound healing, wherein PDGF is expressed by the blood platelet for adding to wound site.Research also shows that PDGF-B is relevant with CNS, and being included in developmental or ripe CNS has neuroprotection or neurotrophic effect, and angiogenesis reaction is promoted for the damage in CNS, and PDGF includes playing an important role in Remyelination in CNS.

VEGF-165 plays an important role in physiology and pathological angiogenesis and angiogenesis.It induces microvascular permeability, cell migration and the differentiation and survival of regulation HPC as the special mitogen and protective factors of vascular endothelial cell.VEGF also works in nerve to occur and blood-brain barrier effect.

Fms samples EGFR-TK 3 (Flt3) and its cognate ligand, that is Flt3- parts (Flt3L), it is the required growth factor relevant with differentiation of the survival with HPC, especially NK cell precursors B cell, Precursor T-cell, monocyte/macrophage and BMDC.Flt3 activation promotes survival and the propagation of early progenitor cells.However, Flt3 reactions are both being influenceed by other growth factors such as GM-CSF, G-CSF, IL-3 and EPO for cell category dependence again.In addition, Flt3 is also found with cell factor such as IL-3, IL-7, IL-11 with reference to relevant with lymph development.

Flt3 in the leukaemia hemogonia of marrow and lymph all expression and these cellular responses Flt3- parts and breed.Worked it can thus be appreciated that Flt3 and Flt3- parts are found in many people's white blood cell malignant tumours, including acute myelogenous leukemia (AML), acute promyelocytic leukemia (APL) B and T cell acute lymphoblastic leukemia (ALL), chronic granulocytic leukemia (CML) and myelodysplasia (MDS).It can also cause in the activation of acceptor constitutive character caused by mutation, cause hyperplasia and the blocking of bone marrow differentiation.

Potentiality of the Flt3- parts in clinical practice expand the ability and the ability (PNAS 17 of Fong etc. 2001 of the immune response of vaccine-induction of its increase anticancer and infectious agent of BMDC (DC) quantity dependent on it；98(15)：8809-14.Gregory et al 2001Cytokine 13 202).Further, since its effect in NK cell developments, Flt3- parts also promote anti-cell inner virus and non-viral pathogen it is immune in work.

In addition, Flt3- parts are used in vitro expanding hemopoietic cell and are found to expand the candidate stem cell from adult donor, and therefore it is likely to be used for stem cell preparation.

The biological effect factor acts through protein performance, through their own protein-bonded interaction, it is meant that protein discussed herein and their own part or acceptor have the significant potentiality of the treatment factor as regulation physiological processes.However, the small change of molecule such as one-level, two grades, three or four structure and common translation or posttranslational modification pattern may produce significant impact to the activity of albumen, secretion, antigenicity and removing.Accordingly, it is possible to which albumen can produce structure or assembling after special one-level, two grades, three or four structure, or common translation or rear translation, it assigns albumen uniqueness 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 1,4)-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.JBiol Chem 265：Although 7055-7061,1990) most of Chinese hamster ovary celI systems synthesize for recombinant protein, such as Dux-B11, (the α 1,3) galactosyl transferase of inactivation gene expression, 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 (Grameret al.Biotechnology 13 (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 biochemical and pharmacological characteristics such as half-life period, antigenicity, stability and the feature effects different from people's cell derived protein.

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 biochemical and pharmacological characteristic for diagnosis, prevention, treatment, nutrition and/or research application, and is used for clinical, business and research application the invention provides protein such as EPO, Flt3- part, Flt3, PDGF-B or VEGF-165 or all or part of chimeric molecule comprising these molecules.

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 the protein or its chimeric molecule of the separation with biochemical parameter feature, wherein described character representation, the basis for the pharmacological characteristics for associating or forming one or more characteristics.The protein or its chimeric molecule of the particularly separation that the present invention is provided are selected from EPO, EPO-Fc, Flt3- parts, Flt3- parts-Fc, Flt3, Flt3-Fc, PDGF-B, PDGF-B-Fc, VEGF-165 and VEGF-165-Fc, it, which has, includes biochemical character { [Px] 1 of multiple measurable biochemical parameters, [Px] 2, ... [Px] n, , wherein Px represent measurable biochemical parameter and " n " be >=1 integer, wherein between and the parameter that is included between [Px] 1 to [Px] n is respectively different measurable biochemical parameters, any of which or the value of more than one measurable biochemical characteristic are represented, the pharmacological characteristics Ty of one characteristic of association or formation basis or pharmacological characteristics { [Ty] 1 of series of features, [Ty] 2, ... [Ty] m } basis, wherein Ty represents that the pharmacological characteristics and m of a characteristic are >=1 integers, wherein [Ty] 1 to [Ty] m is 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 biochemical characteristic 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 biochemical 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 biochemical parameter (P_x), the parameter is used to intactly define protein molecule protein molecule or chimeric molecule.The biochemical parameter can be selected from：Apparent molecular weight (P₁), isoelectric point (PI) (P₂), constitutional isomer (P₃), the relative intensity (P of different isomer 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 ratio (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 (P₆O), 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 specific embodiment, with biochemical parameter (P_x) and pharmacological characteristics (T_x) feature come characterize the present invention EPO, include 8 arrive 60kD apparent molecular weight (P₁), such as 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, and be 23-43kD in a specific embodiment.EPO pI (P₂) scope is from about 2 to about 14, for example, 3,4,5,6,7,8,9,10,11,12,13,14, and be 3-10 in a specific embodiment, including at least about 2 to about 30 isoforms, 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 isoforms, and be 6-25 isoform (P in a specific embodiment₃).The EPO of present invention carbohydrate weight percentage (P₅) it is 10-90%, and be 21-58% in a specific embodiment.The EPO of present invention removal N- connection oligosaccharides apparent molecular weights (P₆) between 13 and 28kDa, and arrive 25kDa in a specific embodiment for 15.The EPO of present invention removal N- and O- connection oligosaccharides apparent molecular weight (P₇) between 13 and 26kDa, and arrive 23kDa in a specific embodiment for 15.The EPO of the present invention acid monose percentage composition (P₈) it is 10-50%, and be 19-50% in a specific embodiment.The EPO of present invention monose (P₉) and sialic acid (P₁₀) content, when normalizing to N- acetylgalactosamines (GalNAc), for 1: 0-3 trehalose, 1: 0.1-8GlcNAc, 1: 0.1-4 galactolipin, 1: 0-5 mannose, 1: 0-5N- n acetylneuraminic acid n (NeuNAc), and be 1: 0.1-2 trehalose, 1: 0.1-6GlcNAc, 1: 0.1-3 galactolipin, 1: 0.1-4 mannose, 1: 0.1-2NeuNAc in a specific embodiment；When normalizing to 3 times of mannoses, for 3: 0.1-6 trehalose, 3: 0-8GalNAc, 3: 2-17GlcNAc, 3: 1.0-6 galactolipin, 3: 0.1-9NeuNAc, and be in a specific embodiment, 3: 0.5-5 trehalose, 3: 0-6GalNAc, 3: 2-14GlcNAc, 3: 1-5 galactolipin, 3: 1-7NeuNAc.Neutral percentage (the P of the EPO of present invention N- connections oligosaccharides₁₃) it is 30 to 99% 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, it is 50 to 90% in a specific embodiment, it is 50 to 85% in a further embodiment.Acid percentage (the P of the EPO of present invention N- connections oligosaccharides₁₄) it is 1 to 70% such as 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, it is 10 to 50% in a specific embodiment, is 15 to 35% in a further embodiment.Neutral percentage (the P of the EPO of present invention O- connections oligosaccharides₁₅) be 50 to 100% such as 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 be 67 to 86%, be 71 to 81% in a further embodiment.Acid percentage (the P of the EPO of present invention O- connections oligosaccharides₁₆) be 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 be 14 to 33%, be 19 to 29% in a further embodiment.The EPO of present invention N- glycosylation sites (P₂₁) it is N-51, N-65, N-118 and N-110 (from the Base Serial Number of signal sequence).The EPO of present invention immunoreactivity feature (T₁₃) different with the people EPO that non-human cell's system is expressed, especially, when being detected using the people EPO expressed containing non-human cell's system ELISA kit, EPO of the invention protein concentration is underestimated.The EPO of present invention multiplication capacity (T₃₂) different with the people EPO that non-human cell's system is expressed, especially, the multiplication capacity (T of EPO of the invention to TF-1 cells₃₂) exceed the people EPO that non-human cell's system is expressed.

In a specific embodiment, with biochemical parameter (P_x) and pharmacological characteristics (T_x) feature come characterize the present invention Flt3 parts, include 1 to 250 apparent molecular weight (P₁), 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, and arrive 35kDa in a specific embodiment for 18.PI (the P of the Flt3 parts of the present invention₂) it is 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, and be 3 to 6 and about 2 to 50 in a specific embodiment, 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, and in a specific embodiment be 8-35 isoforms (P₃).Carbohydrate weight percentage (the P of the Flt3 parts of the present invention₅) 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, 99%, and be 5 to 48% in a specific embodiment.Contents of monosaccharides (the P of the Flt3 parts of the present invention₉) and sialic acid content (P₁₀), when normalizing to N- acetylgalactosamines (GalNAc), for 1: 0-1 trehalose, 1: 0.1-5GlcNAc, 1: 0.1-4 galactolipin, 1: 0.1-5 mannose, 1: 0-5N- n acetylneuraminic acid n (NeuNAc), and be 1: 0-0.5 trehalose, 1: 0.1-4GlcNAc, 1: 0.1-3 galactolipin, 1: 0.1-4 mannose, 1: 0-2NeuNAc in a specific embodiment；When normalizing to 3 times of mannoses, for 3: 0.1-6 trehalose, 3: 0.1-6GalNAc, 3: 0.1-12GlcNAc, 3: 0.1-7 galactolipin, 3: 0-5NeuNAc, and be in a specific embodiment, 3: 0-0.5 trehalose, 3: 0.1-5GalNAc, 3: 0.1-10Gl cNAc, 3: 0.1-5.5 galactolipin, 3: 0-2NeuNAc.The sialic acid of the Flt3 parts of the present invention accounts for the percentage composition (P of contents of monosaccharides₁₀) it is 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%, and be 0 to 25% in a specific embodiment.The sulfate of the Flt3 parts of the present invention accounts for the percentage composition (P of contents of monosaccharides₅₉) it is 0-75%, and be 0-70% in a specific embodiment.Neutral percentage (the P of the N- connections oligosaccharides of the Flt3 parts of the present invention₁₃) it is 50 to 100%, it is 80 to 100% in a specific embodiment, and be 90 to 100% in an other embodiments.Acid percentage (the P of the N- connections oligosaccharides of the Flt3 parts of the present invention₁₄) it is 0 to 50%, it is 0 to 20% in a specific embodiment, and be 0 to 10% in an other embodiments.Neutral percentage (the P of the O- connections oligosaccharides of the Flt3 parts of the present invention₁₅) it is 30 to 75%, it is 35 to 70% in a specific embodiment, and be 40 to 65% in an other embodiments.Acid percentage (the P of the O- connections oligosaccharides of the Flt3 parts of the present invention₁₆) it is 25 to 70%, it is 30 to 65% in a specific embodiment, and be 35 to 60% in an other embodiments.

In a specific embodiment, with biochemical parameter (P_x) and pharmacological characteristics (T_x) feature come characterize the present invention Flt3-Fc, include 1 to 350 apparent molecular weight (P₁), 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, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350kDa, and arrive 150kDa in a specific embodiment for 85.The Flt3-Fc of present invention pI (P₂) it is 2 to 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and be 4 to 8 and about 2 to 70 in a specific embodiment, 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 isoforms, and be 10-40 isoforms (P in a specific embodiment₃).The Flt3-Fc of present invention carbohydrate weight percentage (P₅) it is 0 to 99%, it is 0 to 45% in a specific embodiment.The F lt 3-Fc of present invention removal N- connection oligosaccharides apparent molecular weights (P₆) between 85 and 145kDa, and arrive 140kDa in a specific embodiment for 85.The Flt3-Fc of present invention removal N- and O- connection oligosaccharides apparent molecular weight (P₇) between 85 and 130kDa, and arrive 125kDa in a specific embodiment for 85.The Flt3-Fc of present invention contents of monosaccharides (P₉) and sialic acid content (P₁₀), when normalizing to N- acetylgalactosamines (GalNAc), for 1: 0-4 trehalose, 1: 0.1-27GlcNAc, 1: 0.1-10 galactolipin, 1: 0.1-8 mannose, 1: 0-5N- n acetylneuraminic acid n (NeuNAc), and be 1: 0-3 trehalose, 1: 0.5-20GlcNAc, 1: 0.5-7 galactolipin, 1: 0.5-6 mannose, 1: 0-1NeuNAc in a specific embodiment；When normalizing to 3 times of mannoses, for 3: 0-3 trehalose, 3: 0.1-5GalNAc, 3: 0.1-41GlcNAc, 3: 0.1-7 galactolipin, 3: 0-5NeuNAc, and be in a specific embodiment, 3: 0-2 trehalose, 3: 0.1-3GalNAc, 3: 0.5-30GlcNAc, 3: 0.5-6 galactolipin, 3: 0-1NeuNAc.The Flt3-Fc of present invention sialic acid accounts for the percentage composition (P of contents of monosaccharides₁₀) it is 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%, and be 1 to 25% in a specific embodiment.Neutral percentage (the P of the Flt3-Fc of present invention N- connections oligosaccharides₁₃) it is 50 to 95%, it is 70 to 90% in a specific embodiment, and be 75 to 85% in an other embodiments.Acid percentage (the P of the Flt3-Fc of present invention N- connections oligosaccharides₁₄) it is 5 to 50%, it is 10 to 30% in a specific embodiment, and be 15 to 25% in an other embodiments.Neutral percentage (the P of the Flt3-Fc of present invention O- connections oligosaccharides₁₅) it is 40 to 90%, it is 50 to 85% in a specific embodiment, and be 60 to 80% in an other embodiments.Acid percentage (the P of the Flt3-Fc of present invention O- connections oligosaccharides₁₆) it is 10 to 60%, it is 15 to 50% in a specific embodiment, and be 20 to 40% in an other embodiments.

In a specific embodiment, with biochemical parameter (P_x) and pharmacological characteristics (T_x) feature come characterize the present invention PDGF-B, include 1 to 350 apparent molecular weight (P₁), 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, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350kDa, it is 12-30kDa in a specific embodiment.The PDGF-B of present invention pI (P₂) it is 2 to 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14withabout 2 to 70, suchas 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 isoform (P₃).The PDGF-B of present invention carbohydrate weight percentage (P₅) 0 to 99%, it is 0 to 60% in a specific embodiment.

In a specific embodiment, with biochemical parameter (P_x) and pharmacological characteristics (T_x) feature come characterize the present invention VEGF-165, include 1 to 250 apparent molecular weight (P₁), 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 19.The VEGF-165 of present invention pI (P₂₎For 2 to 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, it is 4 to 9 and about 2 to 75 in a specific embodiment, 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 isoforms, it is 5 to 40 isoform (P in a specific embodiment₃).The VEGF-165 of present invention carbohydrate weight percentage (P₅) it is 0 to 99%, it is 0 to 46% in a specific embodiment.The VEGF-165 of present invention contents of monosaccharides (P₉) and sialic acid content (P₁₀), when normalizing to N- acetylgalactosamines (GalNAc), for 1: 0.1-6 trehalose, 1: 0.1-11GlcNAc, 1: 0.1-10 galactolipin, 1: 1: 0.1-6 mannose, 1: 0-5N- n acetylneuraminic acid n (NeuNAc), and be 1: 0.1-5 trehalose, 1: 1-8GlcNAc, 1: 0.1-4 galactolipin, 1: 0.1-5 mannose, 1: 0-3NeuNAc in a specific embodiment；When normalizing to 3 times of mannoses, for 3: 0.1-8 trehalose, 3: 0.1-5GalNAc, 3: 1-14GlcNAc, 3: 0.1-6 galactolipin, 3: 0-5NeuNAc, and be in a specific embodiment, 3: 0.1-6 trehalose, 3: 0.1-3GalNAc, 3: 2-10GlcNAc, 3: 0.5-4 galactolipin, 3: 0-3NeuNAc.The VEGF-165 of present invention sialic acid accounts for the percentage composition (P of contents of monosaccharides₁₀) it is 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%, it is 5-25% in a specific embodiment.Neutral percentage (the P of the VEGF-165 of present invention N- connections oligosaccharides₁₃) it is 50-90%, it is 60-85% in a specific embodiment, and be 65-80% in an other embodiments.Acid percentage (the P of the VEGF-165 of present invention N- connections oligosaccharides₁₄) it is 10-50%, it is 15-40% in a specific embodiment, and be 20-35% in an other embodiments.Neutral percentage (the P of the VEGF-165 of present invention O- connections oligosaccharides₁₅) it is 50-100%, it is 90-100% in a specific embodiment, and be 95-100% in an other embodiments.Acid percentage (the P of the VEGF-165 of present invention O- connections oligosaccharides₁₆) it is 0-50%, it is 0-10% in a specific embodiment, and be 0-5% in an other embodiments.

The VEGF-165 of present invention immunoreactivity feature (T₁₃) different with the people VEGF-165 that non-human cell's system is expressed, especially, when being detected using the people VEGF-165 expressed containing non-human cell's system ELISA kit, VEGF-165 of the invention protein concentration is overestimated.The VEGF-165 of present invention multiplication capacity (T₃₂) different with the people VEGF-165 that non-human cell's system is expressed, especially, the multiplication capacity (T of VEGF-165 of the invention to HUVEC cells₃₂) exceed the people VEGF-165 that non-human cell's system is expressed.

In another embodiment, it is contemplated by the invention that for selected from EPO, EPO-Fc, Flt3- part, Flt3- parts-Fc, the protein of Flt3, Flt3-Fc, PDGF-B, PDGF-B-Fc, VEGF-165 and VEGF-165-Fc unpack format or its chimeric molecule.The protein or chimeric molecule of the separation of the present invention have the pharmacological characteristics selected from following characteristics：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), administering mode (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), 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 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 linker combination human immunoglobulin(HIg)s.Such chimeric molecule also illustrates that into albumen-Fc herein.Include EPO-Fc, Flt3- parts-Fc, Flt3-Fc, PDGF-B-Fc and VEGF-165-Fc present invention contemplates the example of such albumen-Fc chimeras.

Such albumen-Fc chimeras have the characteristic of measurable biochemical 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 linker combination human immunoglobulin(HIg)s.In other respects, the Fc or framework region of mammalian immunoglobulin are derived from and are included 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 specific 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 EPO-Fc, Flt3- part-Fc, Flt3-Fc, PDGF-B-Fc and VEGF-165-Fc, it has the characteristic of measurable biochemical parameter, these personality presentations, the albumen-Fc of association separation one or more specific pharmacological characteristics.

Therefore, the invention provides by nucleotide sequence coded isolated polypeptide, the sequence is selected from SEQ ID NO：27th, 29,31,33,35,37,39,43,45,47,49,51,53.55,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,109,111,113,115,117,119,123,125,126,128,129,131 and 132, and have the nucleotide sequence of at least 65% similitude, or the sequence that can hybridize with any one in above-mentioned sequence or its complementary type under not high stringency conditions with above-mentioned sequence any of which one.

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 selected from SEQID NO：134,135,136 and 137.

Another aspect provides the polypeptide of separation, its amino acid sequence included is selected from SEQ ID NO：28th, 30,32,34,36,38,40,44,46,48,50,52,54,56,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,110,112,114,116,118,120,124,127,130 and 133, and there is with above-mentioned sequence any of which one amino acid sequence of at least 65% similitude.

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

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：27th, 29,31,33,35,37,39,43,45,47,49,51,53.55,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,109,111,113,115,117,119,123,125,126,128,129,131 and 132, and have the nucleotide sequence of at least 60% similitude, or the sequence that can hybridize with any one in above-mentioned sequence or its complementary type in not high stringency conditions with above-mentioned sequence any of which one.

In addition, other aspects of the present invention provide the isolated nucleic acid molecule of encoding proteins matter or its chimeric molecule or its functional area, comprising with SEQ ID NO：27th, 29,31,33,35,37,39,43,45,47,49,51,53.55,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,109,111,113,115,117,119,123,125,126,128,129,131 and 132 have at least 60% similitude and best pairing after and/or can be with SEQ ID NO：27th, 29,31,33,35,37,39,43,45,47,49,51,53.55,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,109,111,113,115,117,119,123,125,126,128,129,131,132 or they under low high stringency conditions complementary type hybridization nucleotide sequence.

In a specific embodiment, present invention contemplates the nucleic acid molecules of separation, comprising coding, the albumen or the nucleotide sequence of chimeric molecule of the segment selected from EPO, EPO-Fc, Flt3- part, Flt3- parts-Fc, Flt3, Flt3-Fc, PDGF-B, PDGF-B-Fc, VEGF-165 and VEGF-165-Fc and previous molecular, basically comprise SEQ ID NO：28th, 30,32,34,36,38,40,44,46,48,50,52,54,56,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,110,112,114,116,118,120,124,127,130,133 one or more amino acid segments, or with SEQ ID NO：28th, one or more sequences after 30,32,34,36,38,40,44,46,48,50,52,54,56,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,110,112,114,116,118,120,124,127,130,133 best pairings have the amino acid segment of at least 60% similitude.

In other respects, the invention provides the nucleic acid molecules of separation, coding is selected from EPO, EPO-Fc, Flt3- part, Flt3- parts-Fc, Flt3, Flt3-Fc, PDGF-B, PDGF-B-Fc, VEGF-165 and VEGF-165-Fc protein or chimeric molecule, or its segment, comprising selected from there is SEQ ID NO：29th, 31,33,35,45,47,49,51,67,69,71,73,75,77,79,81,113,125,126,128,129 nucleotide sequence, direct or through the nucleotide sequence of one or more industrial well known encoding proteins matter joints, 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 nucleotide 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, protein linker includes IP, GSSNT, TRA or VDGIQWIP.

In other respects, the invention provides the protein of separation, selected from EPO, Flt3- part, Flt3, PDGF-B, VEGF-165 or its segment, comprising selected from there is SEQ ID NO：30th, 32,34,36,46,48,50,52,68,70,72,74,76,78,80,82,114,127 and 130 amino acid sequence, direct or through one or more industrial well known protein linker 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 molecules 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.

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 pharmaceutical formulation containing albumen or its chimeric molecule in diagnosis, prevent, treatment, the application in nutrition and/or research application.

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.

Table 1

Sequence identifier

Sequence identifier	Sequence
		SEQ ID NO：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 clones) (nucleotide sequence)
SEQ ID NO：22	Human IgG1 Fc reverse primers (being used for pIRESbleo clones) (nucleotide sequence)
		SEQ ID NO：23	Human IgG1 Fc forward primers (being used for pIRESbleo GSSNT clones) (nucleic acid sequence Row)
SEQ ID NO：24	Human IgG1 Fc reverse primers (being used for pIRESbleo GSSNT clones) (nucleic acid sequence Row)
		SEQ ID NO：25	EPO forward primers (nucleotide sequence)
SEQ ID NO：26	EPO reverse primers (nucleotide sequence)

Sequence identifier	Sequence
		SEQ ID NO：27	EPO nucleotide sequences are used as signal peptide
SEQ ID NO：28	EPO amino acid sequences are used as signal peptide
		SEQ ID NO：29	EPO nucleotide sequences are used as mature peptide
SEQ ID NO：30	EPO amino acid sequences are used as mature peptide
		SEQ ID NO：31	EPO nucleotide sequences are used as mature peptide (variant)
SEQ ID NO：32	EPO amino acid sequences are used as mature peptide (variant)
		SEQ ID NO：33	EPO nucleotide sequences are used as signal peptide+mature peptide
SEQ ID NO：34	EPO amino acid sequences are used as signal peptide+mature peptide
		SEQ ID NO：35	EPO nucleotide sequences are used as signal peptide+mature peptide (variant)
SEQ ID NO：36	EPO amino acid sequences are used as signal peptide+mature peptide (variant)
		SEQ ID NO：37	EPO nucleotide sequences are used as complete construct (signal peptide+mature peptide+GSSNT Connexon+IgG1Fc)
SEQ ID NO：38	EPO amino acid sequences be used as complete construct (signal peptide+mature peptide+ GSSNT connexons+IgG1Fc)
		SEQ ID NO：39	EPO nucleotide sequences be used as complete construct (signal peptide+mature peptide variant+ GSSNT connexons+IgG1Fc)
SEQ ID NO：40	EPO amino acid sequences are used as complete construct (signal peptide+mature peptide variant + GSSNT connexons+IgG1Fc)
		SEQ ID NO：41	Flt3- parts forward primer (nucleotide sequence)
SEQ ID NO：42	Flt3- parts reverse primer (nucleotide sequence)
		SEQ ID NO：43	Flt3- ligand nucleic acids sequence is used as signal peptide
SEQ ID NO：44	Flt3- ligand amino acid sequences are used as signal peptide
		SEQ ID NO：45	Flt3- ligand nucleic acids sequence is used as mature peptide (ECD)
SEQ ID NO：46	Flt3- ligand amino acid sequences are used as mature peptide (ECD)
		SEQ ID NO：47	Flt3- ligand nucleic acids sequence is used as mature peptide (ECD) (variant)
SEQ ID NO：48	Flt3- ligand amino acid sequences are used as mature peptide (ECD) (variant)
		SEQ ID NO：49	Flt3- ligand nucleic acids sequence is used as signal peptide+mature peptide (ECD)
SEQ ID NO：50	Flt3- ligand amino acid sequences are used as signal peptide+mature peptide (ECD)
		SEQ ID NO：51	Flt3- ligand nucleic acids sequence is used as signal peptide+mature peptide (ECD) (variant)
SEQ ID NO：52	Flt3- ligand amino acid sequences are used as signal peptide+mature peptide (ECD) (variant)
		SEQ ID NO：53	Flt3- part-Fc nucleotide sequences are used as complete construct (signal peptide+maturation Peptide (ECD)+GSSNT connexons+IgG1Fc)
SEQ ID NO：54	Flt3- part-Fc amino acid sequences be used as complete construct (signal peptide+into Ripe peptide (ECD)+GSSNT connexons+IgG1Fc)
		SEQ ID NO：55	Flt3- part-Fc nucleotide sequences are used as complete construct (signal peptide+maturation Peptide (ECD) (variant)+GSSNT connexon IgG1Fc)
SEQ ID NO：56	Flt3- part-Fc amino acid sequences be used as complete construct (signal peptide+into

Sequence identifier	Sequence
			Ripe peptide (ECD) (variant)+GSSNT connexon IgG1Fc)
SEQ ID NO：57	Flt3 forward primers (nucleotide sequence)
		SEQ ID NO：58	Flt3 reverse primers (nucleotide sequence)
SEQ ID NO：59	Flt3 nucleotide sequences are used as signal peptide
		SEQ ID NO：60	Flt3 amino acid sequences are used as signal peptide
SEQ ID NO：61	Flt3 nucleotide sequences are used as selectable signal peptide
		SEQ ID NO：62	Flt3 amino acid sequences are used as selectable signal peptide
SEQ ID NO：63	Flt3 nucleotide sequences are used as signal peptide (variant)
		SEQ ID NO：64	Flt3 amino acid sequences are used as signal peptide (variant)
SEQ ID NO：65	Flt3 nucleotide sequences are used as selectable signal peptide (variant)
		SEQ ID NO：66	Flt3 amino acid sequences are used as selectable signal peptide (variant)
SEQ ID NO：67	Flt3 nucleotide sequences are used as mature peptide (ECD)
		SEQ ID NO：68	Flt3 amino acid sequences are used as mature peptide (ECD)
SEQ ID NO：69	The selectable nucleotide sequences of Flt3 are used as mature peptide (ECD)
		SEQ ID NO：70	The selectable amino acid sequences of Flt3 are used as mature peptide (ECD)
SEQ ID NO：71	Flt3 nucleotide sequences are used as mature peptide (ECD) (variant)
		SEQ ID NO：72	Flt3 amino acid sequences are used as mature peptide (ECD) (variant)
SEQ ID NO：73	The selectable nucleotide sequences of Flt3 are used as mature peptide (ECD) (variant)
		SEQ ID NO：74	The selectable amino acid sequences of Flt3 are used as mature peptide (ECD) (variant)
SEQ ID NO：75	Flt3 nucleotide sequences are used as signal peptide+mature peptide (ECD)
		SEQ ID NO：76	Flt3 amino acid sequences are used as signal peptide+mature peptide (ECD)
SEQ ID NO：77	Flt3 nucleotide sequences are used as signal peptide (variant)+mature peptide (ECD)
		SEQ ID NO：78	Flt3 amino acid sequences are used as signal peptide (variant)+mature peptide (ECD)
SEQ ID NO：79	Flt3 nucleotide sequences are used as signal peptide+mature peptide (ECD) (variant)
		SEQ ID NO：80	Flt3 amino acid sequences are used as signal peptide+mature peptide (ECD) (variant)
SEQ ID NO：81	Flt3 nucleotide sequences are used as signal peptide (variant)+mature peptide (ECD) (variant)
		SEQ ID NO：82	Flt3 amino acid sequences are used as signal peptide (variant)+mature peptide (ECD) (variant)
SEQ ID NO：83	Flt3-Fc nucleotide sequences are used as complete construct (signal peptide+mature peptide (ECD)+IP connexon IgG1Fc)
		SEQ ID NO：84	Flt3-Fc amino acid sequences are used as complete construct (signal peptide+mature peptide (ECD)+IP connexons+IgG1Fc)
SEQ ID NO：85	Flt3-Fc nucleotide sequences be used as complete construct (signal peptide (variant)+into Ripe peptide (ECD)+IP connexons+IgG1Fc)
		SEQ ID NO：86	Flt3-Fc amino acid is used as complete construct (signal peptide (variant)+maturation Peptide (ECD)+IP connexons+IgG1Fc)
SEQ ID NO：87	Flt3-Fc nucleotide sequences are used as complete construct (signal peptide+mature peptide (ECD) (variant)+IP connexons+IgG1Fc)

Sequence identifier	Sequence
		SEQ ID NO：88	Flt3-Fc amino acid is used as complete construct (signal peptide+mature peptide (ECD) (variant)+IP connexons+IgG1Fc)
SEQ ID NO：89	Flt3-Fc nucleotide sequences be used as complete construct (signal peptide (variant)+ Mature peptide (ECD) (variant)+IP connexons+IgG1Fc)
		SEQ ID NO：90	Flt3-Fc amino acid sequences be used as complete construct (signal peptide (variant)+ Mature peptide (ECD) (variant)+IP connexons+IgG1Fc)
SEQ ID NO：91	Flt3-Fc nucleotide sequences are used as complete construct (signal peptide+mature peptide (ECD)+IP connexons+IgG1Fc (variant))
		SEQ ID NO：92	Flt3-Fc amino acid sequences are used as complete construct (signal peptide+mature peptide (ECD)+IP connexons+IgG1Fc (variant))
SEQ ID NO：93	Flt3-Fc nucleotide sequences be used as complete construct (signal peptide (variant)+ Mature peptide (ECD)+IP connexons+IgG1Fc (variant))
		SEQ ID NO：94	Flt3-Fc amino acid be used as complete construct (signal peptide (variant)+into Ripe peptide (ECD)+IP connexons+IgG1Fc (variant))
SEQ ID NO：95	Flt3-Fc nucleotide sequences are used as complete construct (signal peptide+mature peptide (ECD) (variant)+IP connexons+IgG1Fc (variant))
		SEQ ID NO：96	Flt3-Fc amino acid is used as complete construct (signal peptide+mature peptide (ECD) (variant)+IP connexons+IgG1Fc (variant))
SEQ ID NO：97	Flt3-Fc nucleotide sequences be used as complete construct (signal peptide (variant)+into Ripe peptide (ECD) (variant)+IP connexons+IgG1Fc (variant))
		SEQ ID NO：98	Flt3-Fc amino acid sequences be used as complete construct (signal peptide (variant)+ Mature peptide (ECD) (variant)+IP connexons+IgG1Fc (variant))
SEQ ID NO：99	Flt3-Fc nucleotide sequences are used as complete construct (signal peptide+mature peptide (ECD)+GSSNT connexon IgG1Fc)
		SEQ ID NO：100	Flt3-Fc amino acid sequences are used as complete construct (signal peptide+mature peptide (ECD)+GSSNT connexons+IgG1Fc)
SEQ ID NO：101	Flt3-Fc nucleotide sequences be used as complete construct (signal peptide (variant)+into Ripe peptide (ECD)+GSSNT connexons+IgG1Fc)
		SEQ ID NO：102	Flt3-Fc amino acid is used as complete construct (signal peptide (variant)+maturation Peptide (ECD)+GSSNT connexons+IgG1Fc)
SEQ ID NO：103	Flt3-Fc nucleotide sequences are used as complete construct (signal peptide+mature peptide (ECD) (variant)+GSSNT connexons+IgG1Fc)
		SEQ ID NO：104	Flt3-Fc amino acid is used as complete construct (signal peptide+mature peptide (ECD) (variant)+GSSNT connexons+IgG1Fc)
SEQ ID NO：105	Flt3-Fc nucleotide sequences be used as complete construct (signal peptide (variant)+ Mature peptide (ECD) (variant)+GSSNT connexons+IgG1Fc)
		SEQ ID NO：106	Flt3-Fc amino acid sequences be used as complete construct (signal peptide (variant)+

Sequence identifier	Sequence
			Mature peptide (ECD) (variant)+GSSNT connexons+IgG1Fc)
SEQ ID NO：107	PDGF-B forward primers (nucleotide sequence)
		SEQ ID NO：108	PDGF-B reverse primers (nucleotide sequence)
SEQ ID NO：109	PDGF-B nucleotide sequences are used as signal peptide
		SEQ ID NO：110	PDGF-B amino acid sequences are used as signal peptide
SEQ ID NO：111	PDGF-B nucleotide sequences are used as pro peptides 1 (5 ' end)
		SEQ ID NO：112	PDGF-B amino acid sequences are used as pro peptides 1 (N- ends)
SEQ IDNO：113	PDGF-B nucleotide sequences are used as mature peptide
		SEQ ID NO：114	PDGF-B amino acid sequences are used as mature peptide
SEQ ID NO：115	PDGF-B nucleotide sequences are used as pro peptides 2 (3 ' end)
		SEQ ID NO：116	PDGF-B amino acid sequences are used as pro peptides 2 (C- ends)
SEQ ID NO：117	PDGF-B nucleotide sequences are used as signal peptide+pro peptide 1+ mature peptide+pro peptides   2
		SEQ ID NO：118	PDGF-B amino acid sequences are used as signal peptide+pro peptide 1+ mature peptides+pro Peptide 2
SEQ ID NO：119	PDGF-B-Fc nucleotide sequences are used as complete construct (signal peptide+pro peptides + mature peptide+GSSNT connexons+IgG1Fc)
		SEQ ID NO：120	PDGF-B-Fc amino acid sequences are used as complete construct (signal peptide+pro Peptide+mature peptide+GSSNT connexons+IgG1Fc)
SEQ ID NO：121	VEGF-165 forward primers (nucleotide sequence)
		SEQ ID NO：122	VEGF-165 reverse primers (nucleotide sequence)
SEQ ID NO：123	VEGF-165 nucleotide sequences are used as signal peptide
		SEQ ID NO：124	VEGF-165 amino acid sequences are used as signal peptide
SEQ ID NO：125	VEGF-165 nucleotide sequences are used as mature peptide
		SEQ ID NO：126	VEGF-165 nucleotide sequences are used as mature peptide (variant)
SEQ ID NO：127	VEGF-165 amino acid sequences are used as mature peptide (variant)
		SEQ ID NO：128	VEGF-165 nucleotide sequences are used as signal peptide+mature peptide
SEQ ID NO：129	VEGF-165 nucleotide sequences are used as signal peptide+mature peptide (variant)
		SEQ ID NO：130	VEGF-165 amino acid sequences are used as signal peptide+mature peptide
SEQ ID NO：131	VEGF-165-Fc nucleotide sequences are used as complete construct (signal peptide+maturation Peptide+GSSNT connexon IgG1Fc)
		SEQ ID NO：132	VEGF-165-Fc nucleotide sequences are used as complete construct (signal peptide+maturation Peptide (variant)+GSSNT connexon IgG1Fc)
SEQ ID NO：133	VEGF-165-Fc amino acid sequences be used as complete construct (signal peptide+into Ripe peptide+GSSNT connexon IgG1Fc)
		SEQ ID NO：134	EPO genomic nucleic acid sequences

Sequence identifier	Sequence
		SEQ ID NO：135	PDGF-B genomic nucleic acid sequences
SEQ ID NO：136	VEGF-165 genomic nucleic acid sequences
		SEQ ID NO：137	Flt3- ligand gene group nucleotide sequences

Table 2：Biochemical parameter list

Px	Biochemical parameter	EPO	Flt3-Ligand	Flt3-Fc	PDGF-B	VEGF-165
							P1	Apparent molecular weight	23-43kDa	18-35kDa	85-150kDa	12-30kDa	19-35kDa
P2	Isoelectric point (PI)	3-10	3-6	4-8	2-14	4-9
							P3	Constitutional isomer	6-25	8-35	10-40	2-70	5-40
P4	Different isomer number it is relative Intensity
							P5	Carbohydrate percetage by weight	21-58%	5-48%	0-45%	0-60%	0-46%
P6	N- connection oligosaccharides deglycosylations Actual measurement molecular weight afterwards	15-25kDa		85-140kDa
							P7	N- connections and O- connections Oligosaccharides deglycosylation after Survey molecular weight	15-23kDa		85-125kDa
P8	The percentage of acid contents of monosaccharides Than	19-50%
							P9	Contents of monosaccharides	When normalizing to During GalNAc： 1 arrives 0-3 marine algas Sugar, 1 arrives 0.1-8   GlcNAc、 1 arrives 0.1-4 Galactolipin, 1 to 0-5 is sweet Dew sugar； When normalizing to 3 X mannoses：3 To 0.1-6 marine algas Sugar, 3 arrive 0-8   GalNAc、 3 arrive 2-17	When being normalized into During GalNAc： 1 arrives 0-1 marine algas Sugar, 1 arrives 0.1-5 GlcNAc, 1 to 0.1-4 half Lactose, 1 to 0.1-5 is sweet Dew sugar； When being normalized into 3 X mannoses： 3 arrive 0-1 marine algas Sugar, 3 arrive 0.1-6 GalNAc, 3 arrive 0.1-12	When being normalized into During GalNAc： 1 arrives 0-4 marine algas Sugar, 1 arrives 0.1-27 GlcNAc, 1 to 0.1-10 half Lactose, 1 to 0.1-8 is sweet Dew sugar； When normalizing to 3 X mannoses：3 To 0-3 trehaloses, 3 arrive 0.1-5   GalNAc、 3 arrive 0.1-41   GlcNAc、		When being normalized into During GalNAc： 1 to 0.1-6 sea Algae sugar； 1 arrives 0.1-11 GlcNAc, 1 to 0.1-10 half Lactose, 1 to 0.1-6 is sweet Dew sugar； When normalizing to 3 X mannoses： 3 to 0.1-8 seas Algae sugar, 3 arrive 0.1-5   GalNAc、 3 arrive 1-14

Px	Biochemical parameter	EPO	Flt3-Ligand	Flt3-Fc	PDGF-B	VEGF-165
									GlcNAc、 3 arrive 1-6 galas Sugar	GlcNAc, 3 to 0.1-7 half Lactose	3 to 0.1-7 half Lactose		GlcNAc、 3 to 0.1-6 half Lactose
P10	Sialic acid content	When being normalized into During GalNAc： 1 arrives 0-5   NeuNAc； When being normalized into 3 X mannoses： 3 arrive 0.1-9   NeuNAc	When being normalized into During GalNAc： 1 arrives 0-5   NeuNAc； When normalizing to 3 X mannoses：3 arrive   0-5NeuNAc； Contain when with monose The percentage of amount During expression, 0-50%	When being normalized into During GalNAc：1 To 0-5   NeuNAc； When normalizing to 3 X mannoses：3 arrive   0-5NeuNAc； When with contents of monosaccharides Percentage represent When, 0-50%		When being normalized into During GalNAc： And 1 arrives 0-5   NeuNAc； When normalizing to 3 X mannoses：3 arrive   0-3NeuNAc； When with contents of monosaccharides Percentage represent When, 0-50%
							P11	Sulfate and phosphate content
P12	Ser/Thr: GalNAc ratio
							P13	The neutrality hundred of N- connection oligosaccharides Divide ratio	30-99%	50-100%	50-95%		50-90%
P14	The acidity hundred of N- connection oligosaccharides Divide ratio	1-70%	0-50%	5-50%		10-50%
							P15	The neutrality hundred of O- connection oligosaccharides Divide ratio	50-100%	30-75%	40-90%		50-100%
P16	The acidity hundred of O- connection oligosaccharides Divide ratio	0-50%	25-70%	10-60%		0-50%
							P17	The ratio of N- connection oligosaccharides
P18	The ratio of O- connection oligosaccharides
							P19	The knot of N- connection oligosaccharide ingredients Structure
P20	The knot of O- connection oligosaccharide ingredients Structure
							P21	The position of N- connection oligosaccharides and Constitute	Including N-51, N-65, N-118 With N-110 (from Signal sequence Beginning starts Numbering)

Px	Biochemical parameter	EPO	Flt3-Ligand	Flt3-Fc	PDGF-B	VEGF-165
							P22	The position of O- connection oligosaccharides and Constitute
P23	Common translation is modified
							P24	Posttranslational modification
P25	It is acylated
							P26	Acetylation
P27	Amidatioon
							P28	Deamidation
P29	Biotinylation
							P30	Carbamylation
P31	Carboxylation
							P32	Decarboxylation
P33	Disulfide formation
							P34	Fatty-acylation
P35	Myristoylation
							P36	Palmitoylation
P37	Octadecane is acylated
							P38	Formylated
P39	Saccharification
							P40	Glycosylation
P41	Glycophosphatidyl inositol grappling
							P42	Hydroxylating
P43	The combination of selenocysteine
							P44	Lipid
P45	The addition of lipoic acid
							P46	Methylate
P47	N or C-terminal closing
							P48	N or C-terminal are removed
P49	Nitrification
							P50	It is methionine oxidized
P51	Phosphorylation
							P52	Protease digestion
P53	Prenylation
							P54	Farnesylation
P55	Mang ox base
							P56	Phosphopyridoxal pyridoxal phosphate addition
P57	It is sialylated

Px	Biochemical parameter	EPO	Flt3-Ligand	Flt3-Fc	PDGF-B	VPGF-15
							P58	Asialoglycoprotein
P59	Sulfation		0-75%
							P60	Ubiquitination
P61	The addition of ubiquitin sample molecule
							P62	Primary structure
P63	Secondary structure
							P64	Tertiary structure
P65	Quaternary structure
							P66	Chemical stability
P67	Heat endurance

Table 3

Pharmacological characteristics list

Ty	Pharmacological characteristics	EPO	Flt3-L   igand	PDGF-B   Fc	PDGF-B	VEGF-165
							T1	Therapeutic effect
T2	Dose therapeutically effective   (TCID50)
							T3	Bioavilability
T4	Controlled from maintenance is administered into The time for the treatment of level
							T5	Absorption rate
T6	Discharge rate
							T7	Special activity
T8	Heat endurance
							T9	Lyophilized stability
T10	Serum/plasma is stable Property
							T11	Serum half-life
T12	Solubility in blood flow
							T13	Immune response feature	When with non-human cell The standard of middle expression Carry out ELISA surveys Timing is with underestimating egg White concentration.				When with non-human cell The standard of middle expression Carry out ELISA surveys It is regularly used to over-evaluate Count protein concentration.
T14	Immunogenicity
							T14A	Neutralizing antibody suppresses
T15	Side effect
							T16	Receptor/ligand affinity Power
T17	Receptor/ligand is activated Effect
							T18	Tissue or cell category Specificity
T19	Biomembrane or barrier Penetration capacity is (for example Intestines, lung, blood brain screen

Ty	Pharmacological characteristics	EPO	Flt3-L   igand	Flt3-   Fc	PDGF-B	VEGF-165
								Barrier, skin etc.)
T19A	Generate the ability of blood vessel
							T20	Tissue resorption
T21	The stability of degraded
							T22	Freeze-thaw stability
T23	Protease stability
							T24	Ubiquitin stability
T25	Administration is reduced
							T26	Mode of administration
T27	With other pharmaceutical excipients Or the compatibility of carrier
							T28	In organism or environment In residual
T29	It is steady during preservation It is qualitative
							T30	In organism or environment In toxicity etc.
T31	To different types of thin The different biologies of born of the same parents Learn effect
							T32	Propagation	Than in Escherichia coli Middle expression RhEPO is to TF-1 Cell has bigger Proliferation activity				Than in non-human cell Middle expression RhVEGF-165 pairs HUVEC cells have There is bigger propagation Activity
T33	Differentiation
							T34	Apoptosis
T35	The growth of cell size
							T36	Cell factor is adhered
T37	Cell is adhered
							T38	Cell propagation (Cell   spreading)
T39	Cell mobility
							T40	Migration and intrusion
T41	Chemotaxis

Ty	Pharmacological characteristics	EPO	Flt3-L   igand	Flt3-   Fc	PDGF-B	VEGF-165
							T42	Cell swallows
T43	Signal transduction
							T44	Albumen is raised to acceptor / part
T45	JAK/STAT approach Activation
							T46	Ras-erk approach Activation
T47	The activation of AKT approach
							T48	PKC PKC approach and The activation of PKA approach
T49	The activation of PKA approach
							T50	Src is activated
T51	Fas is activated
							T52	TNFR is activated
T53	NFkB is activated
							T54	P38MAPK is activated
T55	C-fos is activated
							T56	Secretion
T57	Acceptor caves in
							T58	Acceptor reciprocation
T59	The up-regulation of surface markers Or lower
							T60	Before FACS/side scattering The change of feature
T61	The change of subgroup ratio
							T62	Differential gene expression
T63	Meronecrosis
							T64	Cell agglutination
T65	Cellular rejection
							T66	With the knot of heparin sulfate Close
T67	With glycosylation structure With reference to
							T68	With chondroitin sulfate With reference to

Ty	Pharmacological characteristics	EPO	Flt3-L  igand	Flt3-    Fc	PDGF-B	VEGF-165
							T69	With extracellular matrix With reference to (such as glue Former, fibronectin)
T70	With the knot of artificial material Close (such as support)
							T71	And the combination of carrier
T72	With the knot of confactor Close
							T73	Individually or containing Other protein it is mixed To stem cell in compound Propagation, differentiation and/ Or the effect of self-renewing Should

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
		bFGF	Basic Fibroblast Growth Factor, FGF2
BSA	Bovine serum albumin(BSA)
		cDLC	Composite fuel part is chromatographed
CSF	Colony stimulating factor
		DCS	Donor calf serum
DeoxGlc
		1,5-anhydroglucitol
DLC			The false affinity chromatography of fuel part
	DSC	Differential scanning heating power is determined
ECD			Extracellular domain
	EGF	EGF
ELISA			Enzyme Linked Immunoadsorbent Assay

Referred to as	Description
		EPO	Hematopoietin；Erythropoietin(EPO) α；Epoetin Alfa；Recombined human Kidney erythropoietin(EPO)；Dynepo
EST	The sequence label of expression
		Fc	Crystalline fragments or constant region for immunoglobulin
FCS	Hyclone
		FGF2	Basic Fibroblast Growth Factor, bFGF
Flt3	Fms samples EGFR-TK 3；The acceptor of Fms samples EGFR-TK 3；Flt3 by Body；Flt3R；EC 2.7.1.112；Stem-like cell EGFR-TK 1；   STK-1；CD135 antigens；FLK2
		Flt3- parts	The part of Fms samples EGFR-TK 3；Flt3 parts (Flt3L)；SL cells The factor；The part of Fms- related tyrosine kinases 3；FLT3LG；FLEX.
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
		IdoA	Iduronic acid
IEC	Ion-exchange chromatography
		IEF	Isoelectric focusing
IFN	Interferon
		Ig	Immunoglobulin
IL	Interleukin
		lacNAc	N-acetyl lactosamine
lacdiNAc	N、N’-diacetyllactosediamine

Referred to as	Description
		LC	Fluid is chromatographed
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 acid)
PBS	Phosphate buffer solution
		PCS	Photon correlation spectroscopy
PDGF-AA	Platelet-derived growth factor A homodimers
		PDGF-AB	Platelet-derived growth factor AB heterodimers
PDGF-BB	Platelet-derived growth factor B homodimer
		PDGF-B	Platelet-derived grouth factor beta；PDGFB；PDGF-2；Ape sarcoma virus (v-sis) oncogene isoreagent；ssv；Oncogene sis, c-sis；With Becaplermi (restructuring)
PNGase	Tire-N4- (N- acetyl group-β-D-glucosaminyl) asparagine acid amides enzyme
		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
TNFR	Tumor Necrosis Factor Receptors
		VEGF	VEGF；VEGF-A；VPF
Xyl	Xylose

Table 5

Amino acid abbreviation s

Amino acid	Trigram subcode	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

Table 6

Stem cell list

Cell type
	Common cell types s
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

Cell type
	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
	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)

Cell type
	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.

The diagram for propagation-induced activity that Fig. 2 is comparison EPO of the invention with the people EPO that is expressed with non-human system after culture TF-1 cells 3 days.In 0.01-10.ng/ml concentration ranges, EPO of the invention proliferation activity (rhombus, triangle) is higher than the R D people EPO of Bacillus coli expression corresponding activity.Control curve (circle).

Fig. 3 is the diagram that VEGF-165 of the VEGF-165 of the present invention propagation-induced activity with being expressed with non-human system is compared after HUVEC cells are cultivated 3 days.In 0.1-5.0ng/ml concentration ranges, VEGF-165 of the invention corresponding activity of the proliferation activity (square) than the Peprotech VEGF-165 of expression in escherichia coli is high.

The EPO that Fig. 4 diagram represents the present invention is compared with the EPO expressed in non-human system the external of immunoreactivity feature.Absorbance-concentration curve of the EPO standard samples (square) of EPO (rhombus) and R＆D systems the CHO expression of the present invention is drawn (R＆DSystems human, EPO Quantikine [registration mark]) with ELISA kit.Error line represents the standard error of average value.

Fig. 5 diagram represents that the external of the VEGF-165 immunoreactivity features that the VEGF-165 of the present invention is expressed with non-human system is compared.Absorbance-concentration curve of the people VEGF-165 standard samples (rhombus) of VEGF-165 (square) and R＆D the system E. colis expression of the present invention is drawn with ELISA kit, (R＆D Systems human VEGF-165 DuoSet [registrar graticule]).Error line represents the standard error of average value.

Detailed description of the invention

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 particularly a kind of induction desired biochemical 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 can have two or more protein or chimeric molecule, selected from the group for containing or comprising EPO, EPO-Fc, Flt3- part, Flt3- parts-Fc, Flt3, Flt3-Fc, PDGF-B, PDGF-B-Fc, VEGF-165 and VEGF-165-Fc, preserve respectively.

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, " pharmacology is 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.

The present invention focuses on the protein or its chimeric molecule of separation, and it has measurable biochemical parameter (P_x) feature, wherein this feature represents, associates or formed the pharmacological characteristics (T of one or more characteristics_y) basis.The protein or chimeric molecule of separation, which are selected from, contains or comprises EPO, EPO-Fc, Flt3- part, Flt3- parts-Fc, Flt3, Flt3-Fc, PDGF-B, PDGF-B-Fc, VEGF-165 and VEGF-165-Fc group.EPO, EPO-Fc, Flt3- part as used herein, Flt3- parts-Fc, Flt3, Flt3-Fc, PDGF-B, PDGF-B-Fc, VEGF-165 and VEGF-165-Fc include whole polypeptide and its fragment being related to.Especially, term " PDGF-B " includes the dimer " PDGF-BB " for PDGF-B and its difference 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 biochemical character of measurable biochemical parameters, { [P_x]₁, [P_x]₂、··[P_x] n, wherein P_xRepresent measurable biochemical parameter and " n " be >=1 integer, wherein [P_x]₁To [P_x]_nIndividually one different measurable biochemical parameter, the numerical value of the one or more measurable biochemical characters of any of which represents, associates or formed the pharmacological characteristics of a characteristic, T_y, or series of features pharmacological characteristics ([T_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 biochemical 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 biochemical 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 ratio (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), s rc activation (T₅₀), fa s 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 biochemical character.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, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 109, 111, 113, 115, 117, 119, 123, 125, 126, 128, 129, 131 and 132, or have with above-named any bar sequence coded by least about nucleotide sequence of 60% homogeneity or the nucleotide sequence that can hybridize with any of the above-described sequence or their complementary type under low stringency conditions.

Another aspect of the present invention provides a kind of polypeptide of separation, and it selected from sequence table by including SEQ ID NO：134th, 135,136 and 137, then pass through the nucleotide sequence coded of their own mRNA of various cell processes splicing.

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 I D NO selected from sequence table：27th, 29,31,33,35,37,39,43,45,47,49,51,53.55,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,109,111,113,115,117,119,123,125,126,128,129,131 and 132 nucleotide sequence with least 60% similitude, or optimal arrangement (optimal alignment) is afterwards and/or can be with SEQ I D NO：27th, 29,31,33,35,37,39,43,45,47,49,51,53.55,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,109,111,113,115,117,119,123,125,126,128,129,131 and 132 or their complementary type one or more nucleotide sequences hybridized under low high stringency conditions.

In specific 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：28th, a 30,32,34,36,38,40,44,46,48,50,52,54,56,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,110,112,114,116,118,120,124,127,130 and 133 shown amino acid sequence or multiple, or with SEQ ID NO：28th, 30,32,34,36,38,40,44,46,48,50,52,54,56,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,110,112,114,116,118,120,124,127,130 and 133 one or more amino acid sequences with least about 60% similitude after optimal arrangement.

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：29th, the nucleotide sequence in 31,33,35,45,47,49,51,67,69,71,73,75,77,79,81,113,125,126,128 and 129, it is directly or nucleotide sequence and the constant region (Fc) of encoding human immunoglobulin or the nucleotide sequence of framework region through one or more coding protein linkers known in the art are connected, basic such as SEQ ID NO：1st, 3,5,7,9,11,13,15,17 or 19 it is one or more shown in.

On the other hand, the invention provides a kind of protein molecule of separation or its fragment, including selected from SEQ ID NO：30th, 32,34,36,46,48,50,52,68,70,72,74,76,78,80,82,114,127 and 130 amino acid sequence, it is directly or the constant region (Fc) through one or more protein linkers and human immunoglobulin(HIg) known in the art or framework region connection basic such as SEQ ID NO：2nd, 4,6,8,10,12,14,16,18 or 20 it is one or more shown in.

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：28th, 30,32,34,36,38,40,44,46,48,50,52,54,56,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,110,112,114,116,118,120,124,127,130 and 133 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 B lackwell 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 high stringency 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 nucleic acid construct 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 bacterial gpt gene (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, biochemical and/or conformational levels are relevant.There is nonidentity in amino acid levels, " similitude " includes and mutual structure, function, biochemical and/or conformational levels still relevant amino acid.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 arrangement 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 arrangement of sequence can be by the computerization of algorithm or by checking and being realized by the optimal arrangement (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 AcidsRes 25 as disclosed in Altschul：389th, 1997) being discussed in detail for sequence analysis can find (In in Ausubel etc. Unit19.3：Current Protocols in Molecular Biology、JohnWiley & 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 arrangements 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 fromHitachi Software Engineering 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 rigor 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 rigor 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 stringency conditions.Optional stringency conditions can be used in the place of needs, such as medium 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 rigor, 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 Tm of double-stranded DNA often successively decreases, the quantity of 1 DEG C of base mismatch pair increases by 1% (Bonner andLaskey, Eur J Biochem 46：83、1974.Formamide is optional under these hybridization conditions.Accordingly, rigorous level is defined below in specific embodiment：Low rigor is 6 × SSC buffer solutions, and 0.1%w/v SDS are at 25-42 DEG C；Middle rigor is 2 × SSC buffer solutions, and 0.1%w/v SDS are at 20 DEG C to 65 DEG C of temperature range；High rigor 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 for many kinds of substance examined by biochemical analysis.

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 terminator codon 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 (Bernstein Methods 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.(Rando Biochim Biophys Acta 1300(1)：5-16、1996；Gelb et al.CurrOpin Chem 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 biochemical character changed, and it changes activity (the Clarke Curr Opin Cell Biol 5 of protein：977983、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 Lewis a 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 Chem278 (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

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

Antigen title	O antigen polysaccharide o structure
		Blood group I	Gal(β1-4)GlcNAc(β1-3)[Gal(β1-4)GlcNAc(β1-6)]   Gal(β1-4)GlcNAc(β1-3)Gal(β1-R
Lewis a(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
		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

Sugar antigens determinant list

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 pI.

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 focused on 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 biochemical characteristic of its chimeric molecule.

Any " measurable biochemical 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 biochemical 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 biochemical 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 biochemical characteristic 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 biochemical character, 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 biochemical characters (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 biochemical character 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 Chroma togrA 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, As p-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 (Ramah 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.Infraredand Raman Spectra of Polyatomic Molecules, Van NostrandReinhold, 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：350th, 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 strong 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 Biol 248 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 (Kwon and Yu, Biophim Biophys Acta 1335 are detected with fluorescence：265-272,1997), to determine HAS Tm (Farruggia et al.Int J Biol Macromol 20：43-51,1997), and detect that MerP stretches interaction (Aronsson et al.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 biochemical forms 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 Chroma togr 540 of near ultraviolet region：1991) and fluorescence 187-198,.

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 nucleic acid 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 Pres s, 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 (PhilliesAnal Chem 62：1049A-1057A、1990).Protein aggregate (Li et al.Bi ochemistry 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.

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 for representing from about 33, extracting in 000 human gene well confirmed.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 biochemical forms 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 ligand, 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 Bcl2 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, the integrins of β 1) 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 biochemical forms 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 (lamellipodium) 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 biochemical forms 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, Methods Enzymol202 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 biochemical forms 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 Labora tory Manual, Harlow and Lane (eds.), ColdSpr ing Harbor Laboratory 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. JMol 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. Protein Engineering10：423、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 AcadSci USA 85：5879th, 1988) and Ward et al. (Nature 334：544th, 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 IgGl 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 USA 81：6851-6855、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. Nature332：323-329、1988；Presta、Curr OpStruct Biol2：593-596、1992；Liu et al. Proc Natl Acad Sci USA 84：3439、1987；Larrick et al. Bio/Technology 7：934、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 (pH7.5) 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 specific embodiment, with biochemical parameter (P_x) and pharmacological characteristics (T_x) feature come characterize the present invention EPO, include 8 arrive 60kD apparent molecular weight (P₁), such as 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, and it is in a specific embodiment 23-43kD.EPO pI (P₂) scope is from about 2 to about 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, and it is in a specific embodiment 3-10, including at least about 2 to about 30 isoforms (isoform), 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 isoforms, and be in a specific embodiment 6-25 isoform (P₃).The EPO of present invention carbohydrate weight percentage (P₅) it is 10-90%, and it is in a specific embodiment 21-58%.The EPO of present invention removal N- connection oligosaccharides apparent molecular weights (P₆) between 13 and 28kDa, and arrive 25kDa in a specific embodiment for 15.The EPO of present invention removal N- and O- connection oligosaccharides apparent molecular weight (P₇) between 13 and 26kDa, and arrive 23kDa in a specific embodiment for 15.The EPO of the present invention acid monose percentage composition (P₈) it is 10-50%, and it is in a specific embodiment 19-50%.The EPO of present invention monose (P₉) and sialic acid (P₁₀) content, when normalizing to N- acetylgalactosamines (GalNAc), for 1: 0-3 trehalose, 1: 0.1-8GlcNAc, 1: 0.1-4 galactolipin, 1: 0-5 mannose, 1: 0-5N- n acetylneuraminic acid n (NeuNAc), and it is in a specific embodiment 1: 0.1-2 trehalose, 1: 0.1-6GlcNAc, 1: 0.1-3 galactolipin, 1: 0.1-4 mannose, 1: 0.1-2NeuNAc；When normalizing to 3 times of mannoses, for 3: 0.1-6 trehalose, 3: 0-8GalNAc, 3: 2-17GlcNAc, 3: 1.0-6 galactolipin, 3: 0.1-9NeuNAc, and be in a specific embodiment, 3: 0.5-5 trehalose, 3: 0-6GalNAc, 3: 2-14GlcNAc, 3: 1-5 galactolipin, 3: 1-7NeuNAc.Neutral percentage (the P of the EPO of present invention N- connections oligosaccharides₁₃) it is 30 to 99% 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, it is 50 to 90% in a specific embodiment, it is 50 to 85% in a further embodiment.Acid percentage (the P of the EPO of present invention N- connections oligosaccharides₁₄) it is 1 to 70% such as 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, it is in a specific embodiment 10 to 50%, is 15 to 35% in a further embodiment.Neutral percentage (the P of the EPO of present invention O- connections oligosaccharides₁₅) be 50 to 100% such as 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, be 67 to 86% in a specific embodiment, be 71 to 81% in a further embodiment.Acid percentage (the P of the EPO of present invention O- connections oligosaccharides₁₆) be 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, be 14 to 33% in a specific embodiment, be 19 to 29% in a further embodiment.The EPO of present invention N- glycosylation sites (P₂₁) it is N-51, N-65, N-118 and N-110 (from the Base Serial Number of signal sequence).The EPO of present invention immunoreactivity feature (profile) (T₁₃) different with the people EPO that non-human cell's system is expressed, especially, when being detected using the people EPO expressed containing non-human cell's system ELI SA kits, EPO of the invention protein concentration is underestimated.The EPO of present invention multiplication capacity (T₃₂) different with the people EPO that non-human cell's system is expressed, especially, the multiplication capacity (T of EPO of the invention to TF-1 cells₃₂) exceed the people EPO that non-human cell's system is expressed.

In a specific embodiment, with biochemical parameter (P_x) and pharmacological characteristics (T_x) feature come characterize the present invention Flt3 parts, include 1 to 250 apparent molecular weight (P₁), 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, and arrive 35kDa in a specific embodiment for 18.PI (the P of the Flt3 parts of the present invention₂) it is 2 to 14, such as 2,3,4,5,6,7,8,9,10,11,12,13,14, and it is in a specific embodiment 3 to 6 and about 2 to 50, 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, and in a specific embodiment be 8-35 isoforms (P₃).Carbohydrate weight percentage (the P of the Flt3 parts of the present invention₅) 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, 99%, and it is in a specific embodiment 5 to 48%.Contents of monosaccharides (the P of the Flt3 parts of the present invention₉) and sialic acid content (P₁₀), when normalizing to N- acetylgalactosamines (GalNAc), for 1: 0-1 trehalose, 1: 0.1-5GlcNAc, 1: 0.1-4 galactolipin, 1: 0.1-5 mannose, 1: 0-5N- n acetylneuraminic acid n (NeuNAc), and it is in a specific embodiment 1: 0-0.5 trehalose, 1: 0.1-4GlcNAc, 1: 0.1-3 galactolipin, 1: 0.1-4 mannose, 1: 0-2NeuNAc；When normalizing to 3 times of mannoses, for 3: 0.1-6 trehalose, 3: 0.1-6GalNAc, 3: 0.1-12GlcNAc, 3: 0.1-7 galactolipin, 3: 0-5NeuNAc, and be in a specific embodiment, 3: 0-0.5 trehalose, 3: 0.1-5GalNAc, 3: 0.1-10GlcNAc, 3: 0.1-5.5 galactolipin, 3: 0-2NeuNAc.The sialic acid of the Flt3 parts of the present invention accounts for the percentage composition (P of contents of monosaccharides₁₀) it is 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%, and it is in a specific embodiment 0 to 25%.The sulfate of the Flt3 parts of the present invention accounts for the percentage composition (P of contents of monosaccharides₅₉) it is 0-75%, and it is in a specific embodiment 0-70%.Neutral percentage (the P of the N- connections oligosaccharides of the Flt3 parts of the present invention₁₃) it is 50 to 100%, it is in a specific embodiment 80 to 100%, and be 90 to 100% in an other embodiments.Acid percentage (the P of the N- connections oligosaccharides of the Flt3 parts of the present invention₁₄) it is 0 to 50%, it is in a specific embodiment 0 to 20%, and be 0 to 10% in an other embodiments.Neutral percentage (the P of the O- connections oligosaccharides of the Flt3 parts of the present invention₁₅) it is 30 to 75%, it is in a specific embodiment 35 to 70%, and be 40 to 65% in an other embodiments.Acid percentage (the P of the O- connections oligosaccharides of the Flt3 parts of the present invention₁₆) it is 25 to 70%, it is in a specific embodiment 30 to 65%, and be 35 to 60% in an other embodiments.

In a specific embodiment, with biochemical parameter (P_x) and pharmacological characteristics (T_x) feature come characterize the present invention Flt3-Fc, include 1 to 350 apparent molecular weight (P₁), 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, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350kDa, and arrive 150kDa in a specific embodiment for 85.The Flt3-Fc of present invention pI (P₂) it is 2 to 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and it is in a specific embodiment 4 to 8 and about 2 to 70, 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 isoforms, and it is in a specific embodiment 10-40 isoforms (P₃).The Flt3-Fc of present invention carbohydrate weight percentage (P₅) it is 0 to 99%, it is in a specific embodiment 0 to 45%.The Flt3-Fc of present invention removal N- connection oligosaccharides apparent molecular weights (P₆) between 85 and 145kDa, and arrive 140kDa in a specific embodiment for 85.The Flt3-Fc of present invention removal N- and O- connection oligosaccharides apparent molecular weight (P₇) between 85 and 130kDa, and arrive 125kDa in a specific embodiment for 85.The Flt3-Fc of present invention contents of monosaccharides (P₉) and sialic acid content (P₁₀), when normalizing to N- acetylgalactosamines (GalNAc), for 1: 0-4 trehalose, 1: 0.1-27GlcNAc, 1: 0.1-10 galactolipin, 1: 0.1-8 mannose, 1: 0-5N- n acetylneuraminic acid n (NeuNAc), and it is in a specific embodiment 1: 0-3 trehalose, 1: 0.5-20GlcNAc, 1: 0.5-7 galactolipin, 1: 0.5-6 mannose, 1: 0-1NeuNAc；When normalizing to 3 times of mannoses, for 3: 0-3 trehalose, 3: 0.1-5GalNAc, 3: 0.1-41GlcNAc, 3: 0.1-7 galactolipin, 3: 0-5NeuNAc, and be in a specific embodiment, 3: 0-2 trehalose, 3: 0.1-3GalNAc, 3: 0.5-30GlcNAc, 3: 0.5-6 galactolipin, 3: 0-1NeuNAc.The Flt 3-Fc of present invention sialic acid accounts for the percentage composition (P of contents of monosaccharides₁₀) it is 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%, and it is in a specific embodiment 1 to 25%.Neutral percentage (the P of the Flt3-Fc of present invention N- connections oligosaccharides₁₃) it is 50 to 95%, it is in a specific embodiment 70 to 90%, and be 75 to 85% in an other embodiments.Acid percentage (the P of the Flt3-Fc of present invention N- connections oligosaccharides₁₄) it is 5 to 50%, it is in a specific embodiment 10 to 30%, and be 15 to 25% in an other embodiments.Neutral percentage (the P of the Flt3-Fc of present invention O- connections oligosaccharides₁₅) it is 40 to 90%, it is in a specific embodiment 50 to 85%, and be 60 to 80% in an other embodiments.Acid percentage (the P of the Flt3-Fc of present invention O- connections oligosaccharides₁₆) it is 10 to 60%, it is in a specific embodiment 15 to 50%, and be 20 to 40% in an other embodiments.

In a specific embodiment, with biochemical parameter (P_x) and pharmacological characteristics (T_x) feature come characterize the present invention PDGF-B, include 1 to 350 apparent molecular weight (P₁), 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, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350kDa, it is 12-30kDa in a specific embodiment.The PDGF-B of present invention pI (P₂) it is 2 to 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14with about2 to 70, suchas 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, l 2, 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 isoform (P₃).The PDGF-B of present invention carbohydrate weight percentage (P₅) 0 to 99%, it is in a specific embodiment 0 to 60%.

In a specific embodiment, with biochemical parameter (P_x) and pharmacological characteristics (T_x) feature come characterize the present invention VEGF-165, include 1 to 250 apparent molecular weight (P₁), 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 19.The VEGF-165 of present invention pI (P₂) it is 2 to 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, it is 4 to 9 and about 2 to 75 in a specific embodiment, 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 isoforms, it is 5 to 40 isoform (P in a specific embodiment₃).The VEGF-165 of present invention carbohydrate weight percentage (P₅) it is 0 to 99%, it is in a specific embodiment 0 to 46%.The VEGF-165 of present invention contents of monosaccharides (P₉) and sialic acid content (P₁₀), when normalizing to N- acetylgalactosamines (GalNAc), for 1: 0.1-6 trehalose, 1: 0.1-11GlcNAc, 0.1-6 mannoses, 1: 0-5N- n acetylneuraminic acid n (NeuNAc) are arrived in 1: 0.1-10 galactolipin, 1: 1, and are in a specific embodiment 1: 0.1-5 trehalose, 1: 1-8GlcNAc, 1: 0.1-4 galactolipin, 1: 0.1-5 mannose, 1: 0-3NeuNAc；When normalizing to 3 times of mannoses, for 3: 0.1-8 trehalose, 3: 0.1-5GalNAc, 3: 1-14GlcNAc, 3: 0.1-6 galactolipin, 3: 0-5NeuNAc, and be in a specific embodiment, 3: 0.1-6 trehalose, 3: 0.1-3GalNAc, 3: 2-10GlcNAc, 3: 0.5-4 galactolipin, 3: 0-3NeuNAc.The VEGF-165 of present invention sialic acid accounts for the percentage composition (P of contents of monosaccharides₁₀) it is 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%, it is in a specific embodiment 5-25%.Neutral percentage (the P of the VEGF-165 of present invention N- connections oligosaccharides₁₃) it is 50-90%, it is in a specific embodiment 60-85%, and be 65-80% in an other embodiments.Acid percentage (the P of the VEGF-165 of present invention N- connections oligosaccharides₁₄) it is 10-50%, it is in a specific embodiment 15-40%, and be 20-35% in an other embodiments.Neutral percentage (the P of the VEGF-165 of present invention O- connections oligosaccharides₁₅) it is 50-100%, it is in a specific embodiment 90-100%, and be 95-100% in an other embodiments.Acid percentage (the P of the VEGF-165 of present invention O- connections oligosaccharides₁₆) it is 0-50%, it is in a specific embodiment 0-10%, and be 0-5% in an other embodiments.The VEGF-165 of present invention immunoreactivity feature (T₁₃) different with the people VEGF-165 that non-human cell's system is expressed, especially, when being detected using the people VEGF-165 expressed containing non-human cell's system ELISA kit, VEGF-165 of the invention protein concentration is overestimated.The VEGF-165 of present invention multiplication capacity (T₃₂) different with the people VEGF-165 that non-human cell's system is expressed, especially, the multiplication capacity (T of VEGF-165 of the invention to HUVEC cells₃₂) exceed the people VEGF-165 that non-human cell's system is expressed.

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.

+3×Gal(1-)GlcNAc(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+″+3×Gal(1-)GlcNAc(1-)″

+3×Gal(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+″+3×Gal(1-)GlcNAc(1-)+Fuc(1-)″

+3×Gal(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+″+3×Gal(b1-4)GlcNAc(b1-3)″

+3×Gal(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)[Fuc(a1-6)]GlcNAc+″+3×Gal(b1-4)GlcNAc(b1-3)″

+3×Gal(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)GlcNAc+″+3×Gal(b1-4)GlcNAc(b1-3)+Gal(b1-3)GlcNAc(b1-3)″

+3×Gal(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+″+3×Gal(b1-4)GlcNAc(b1-3)+Gal(b1-3)GlcNAc(b1-3)″

+4×Gal(1-)GlcNAc(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+″+4×Gal(1-)GlcNAc(1-)″

+4×Gal(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+″+4×Gal(1-)GlcNAc(1-)+Fuc(1-)″

+5×Gal(1-)GlcNAc(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+″+5×Gal(1-)GlcNAc(1-)″

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)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ " Where j+k=14 ＆ j, k ＞=1 "

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 "

Where j+k=14 ＆ k, j ＞=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) GlcNAc+ " Where j+k=14 ＆ k, j ＞=1 "

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)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ " Where j+k=14 ＆j, k ＞=1 "

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 "

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) { GlcNAc (b1-3) Gal (b1-4) } jGlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] G1cNAc+ " Where j+k=14 ＆ j, k ＞=1 "

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) GlcNAc+ " Where j+k=14 ＆ j, k ＞=1 "

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 "

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) { 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 "

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)] G1cNAc+ " Wherej+k=14 ＆ j, k ＞=1 "

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) [Fuc (a1-6)] GlcNAc+ " Where j+k=14 ＆ j, k ＞=1 "

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) { GlcNAc (b1-3) Gal (b1-4) } jGl cNAc (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 a1-3Man al-6Man b1-4GlcNAcb1-4GlcNAc

Glycan structures Man (a1-3) Man (a1-6) Man (b1-4) GlcNAc (b1-4) GlcNAcNeuAc a2-u Gal b1-4GlcNAcb1-2Man a1-3Man b1-4GlcNAc

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 Gl cNAc (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) [HSO3 (- 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) GlcNAc

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

+2×Man

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

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(a2-)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) Gl cNAc (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)] [Gl cNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) Gl cNAc

Glycan structures NeuAc (a2-3) 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

+NeuAc

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

+2×NeuAc

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+ "+2 × 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

+NeuAc(2-6)

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

+Fuc(a1-3)

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)Gl cNAc(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

+NeuAc(a2-6)

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

+3×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) GlcNAc

+″+3×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 (bl-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 (bl-4) GlcNAc (b1-2)] 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) GlcNAc+ "+Fuc (a1-2) "

+Fuc(a1-3)

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

+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+ "+NeuAc (a2-3)+NeuAc (a2-6) "

Glycan structures NeuAc (a2-6) Gal (bl-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

+Fuc+2×NeuAc(a2-)

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+2×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 (a2-3) 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

+3×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) Man (a1-6)] Man (b1-4) GlcNAc (b1-4)+3 × NeuAc of GlcNAc+ " (a2-)″

+HSO3(-6)+2×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+ "+HSO3 (- 6)+2 × NeuAc (a2-3)+NeuAc (a2-6) "

+2×HSO3(-6)+2×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+ "+2 × HSO3 (- 6)+2 × 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

+Gal(b1-2)GlcNAc(b1-3)+3×NeuAc

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+ "+Ga l (b1-2) GlcNAc (b1-3)+3 × NeuAc "

+NeuAc(a2-)

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-)″

+NEuAc(a2-3)+NeuAc(a2-6)

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+ "+NeuAc (a2-3)+NeuAc (a2-6) "

+HSO3(-6)+2×NeuAc(a2-)

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)+2 × 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

+3×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)+3 × NeuAc of GlcNAc+ " (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 (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

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

+3×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)+3 × NeuAc of GlcNAc+ " (a2-)″

+3×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) [Fuc (a1-6)]+3 × NeuAc of GlcNAc+ " (a2-)″

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

+4×NeuAc(a2-)

Glycan structures Fuc (a1-3) [Ga1-l (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)] Maan (b1-4) GlcNAc (b1-4)+4 × NeuAc of GlcNAc+ " (a2-)″

+2×Fuc

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+″+2×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

+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+″+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) [Ga l (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

+2×Gal(b1-4)GlcNAc(b1-3)+2×NeuAc

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+ "+2 × Gal (b1-4) GlcNAc (b1-3)+2 × NeuAc "

+Gal(b1-4)GlcNAc(1-)+4×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+″+Gal(b1-4)GlcNAc(1-)+4×NeuAc(a2-)″

+5×NeuAc(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+″+5×NeuAc(a2-)″

+Gal(b1-4)GlcNAc(b1-3)

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+ "+Gal (b1-4) GlcNAc (b1-3) "

+2×Fuc+Gal(b1-4)GlcNAc(1-)

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+″+2×Fuc+Gal(b1-4)GlcNAc(1-)″

+2×Gal(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)Man(a1-)]Man(b1-4)GlcNAc(b1-4)GlcNAc+″+2×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 b1-3 GalNAc

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) GalNAc

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-)] GalNAcdelta4,5GlcA 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) Ga1 (b1-4) Xyl

Glycan structures delta4,5GlcA (b1-3) [HSO3 (- 4)] GalNAc (b1-4) GlcA (b1-3) Ga1 (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)] Ga lNAc

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 a1-2Gal b1-3GlcNAcb1-3GalNAc

Glycan structures Fuc (a1-2) Gal (b1-3) 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)] Ga lNAcGal b1-4GlcNAcb1-3Gal bl-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 ul-u GaNAcu1-u GalNAc

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

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

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)] GalNAc

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 (a2-3) 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

+Fuc(a1-2)

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

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)] GalNAc

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

+Fuc

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(a 1-3)]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

+Fuc

Glycan structures Gal (1-)GlcNAc(1-)Gal(1-)[Fuc(1-)]GlcNAc(1-)[NeuAc(2-)Gal(1-)]Ga lNAc+″+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 structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-)Gal(b1-4)GlcNAc(b1-6)[NeuAc(a2-3)Gal(b1-3)]GalNAc

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

+2×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)] GalNAc+ "+2 × 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 biochemical forms of chimeric molecule, a kind of one or more transgenosis for encoding enzyme or a variety of enzymes, producing required biochemical forms are including but not limited to introduced into host cell.This transgenosis includes polytype sialyltransferase, such as ST3Gal1, ST3Gal2, ST3Gal3, ST 3Gal4, ST3Gal5, ST3Gal6, ST6Gal1, ST6Gal2, ST6GalNAc1, ST6Ga lNAc2, ST6GalNAc3, ST6GalNAc4, ST6GalNAc5, ST8Sia1, 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 sialylLewis x 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. PNAS 95 by adding fucosyltransferase transgenosis into the cells of HEK 293：12283-12288、1998)

In one 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-EPO, sialylated-EPO-Fc, sialylated-Flt3- parts, sialylated-Flt3- parts-Fc, sialylated-Flt3, sialylated-Flt3-Fc, sialylated-PDGF-B, sialylated-PDGF-B-Fc, sialylated-VEGF-165, sialylated-VEGF-165-Fc.

Especially, with biochemical 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 normalizing to GalNAc；It is 3: 0.1-100NeuNAc when normalizing to 3 times of mannoses.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, 98 or 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 one 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 one 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 one embodiment, the human cell line for having FUT3 (" marine alga glycated protein ", " fucosylated-protein ") using conversion produces its chimeric molecule in protein or invention.The example of marine alga glycated protein includes marine alga saccharification-EPO, marine alga saccharification-EPO-Fc, marine alga saccharification-Flt3- parts, marine alga saccharification-Flt3- parts-Fc, marine alga saccharification-Flt3, marine alga saccharification-Flt3-Fc, marine alga saccharification-PDGF-B, marine alga saccharification-PDGF-B-Fc, marine alga saccharification-VEGF-165 and/or marine alga saccharification-VEGF-165-Fc.

Especially, with biochemical parameter (P_x) feature characterize the sialylated albumen of the present invention, including monose (P₉) and sialic acid content (P₁₀), arrive 0.1-100NeuNAc when being normalized with GalNAc for 1；When with 3 times of mannose normalization 0.1-100NeuNAc is arrived for 3.

In one 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 one 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 EPO, Flt3- part, Flt3, PDGF-B and VEGF-165, 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, pcDNA3.1/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 DH10B 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, pI RESbleo3, 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 and 70oC 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 Qiagen EndofreePlasmid 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, HEK 293c18, HEK 293-T, HEK 293CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene companies), 293A (Invitrogen companies), Hela cells and its any redundant organism, HepG2, PA-1Jurkat, THP-1, HL-60, H9, HuT78, 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 biochemical forms of chimeric molecule, a kind of one or more transgenosis for encoding enzyme or a variety of enzymes, producing required biochemical forms 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), Excell 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 (fedbatch) 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 pI 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 Cu2+, Co2+, Zn2+, Mn2+, Mg2+ or the Ni2+ of chromatogram supporter stationary phase by covalently bound cheland (such as diglycinee).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 biochemical forms 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 fucosidase 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 from EPO be contained, and EPO-Fc, Flt3- parts are selected in Flt3- parts-Fc, Flt3, Flt3-Fc, PDGF, PDGF-Fc, VEGF-165, VEGF-165-Fc list.

Peptide chain additional iodine isotope (such as 123I) of the iodination to protein or its chimeric molecule can be used.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 that 99mTc is attached on the protein of the present invention or chimeric molecule, 99mTcO4- for example is reduced by using go back original reagent (such as stannous chloride), 99mTc labelled proteins or chimeric molecule are then carried out by bifunctional chelating agent (such as diethylenetriamine pentaacetic acid (DTPA)).

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.Vocabulary " biological surface " used herein, it is contemplated on body or internal any surface.The example of the local mixture adaptable " biological surface " of the present invention includes any epithelial surface, such as skin, 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.

Pharmaceutically acceptable carrier and/or diluent include any and all solvent, dispersant, and coating, antibacterium and antifungal agent wait and blend delayed absorption agent etc..The use of this medium and reagent is known in the art to pharmaceutically active substance, and is incompatible without what conventional media or reagent and conditioning agent, their applications in pharmaceutical composition have been conceived to.The reactive compound of supplement can also be incorporated into composition.

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 I I,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 SchinzelType,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 ACSV) 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,ACS I,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,Adam Complex,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,AGRTriad,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 alpha-L-fucosidase deficiency fucoside (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,AN 1,AN 2,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 Compelling Helioophthalmic Outburst syndromes,Autosomal dominant inheritance desmin distal myopathy is simultaneously tardy,Autosomal dominant EDS,Autosomal dominant emery-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,Babins ki-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,BeckwithWiedemann syndromes,Beckwith syndrome,Begnez-Cesar syndromes,Behcet syndromes,Behcet diseases,Behr 1,Behr 2,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-Ravich 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 (CPT I ＆ 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 Cataract x,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,CDG1A,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,Chiaril types (chiari deformities I),ChiariII types (chiari deformities II),Chiari I 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,chondrohys trophia,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/47XXY (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-22qll),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,CoffinSiris 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 V atp synthases 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,CS ID,CSO,CST syndromes,Hair crimping ankyloblepharon nail dysplasia,Curschmann-Battern-Steinert,CurthMacklin type fish scales hystric,Curth Macklin 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 I I 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 s ine myositiss,Dermatomyositis,Dermatosparaxis,Dermatostomatitis S tevens Johnson 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,Dienoy1- 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,DOC 4,DOC 6 (Harlequin types),The Curth-Macklin types of DOC 8,DOC11 phytane acid types,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,DOC 16Unilateral Hemidysplasia types,DOC 18,DOC 19,DOC 20,DOC 24,Sinus Le Shi bodies-myelopathy,Dolichos pondylic 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,Dorfman Chanarin 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 Melchior Clausen 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 ElfinFacie,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,erythroblas tophthisis,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 Es tren-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 AdultLiver Form,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 fucoside (storing up),Sick 2 type of fucoside (storing up),Sick 3 type of fucoside (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,GA II & 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,Gangliosidosis GM11 types,Gangliosidosis GM12 types,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,Gers tmann 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 XO,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,Hajdu Cheney,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,Hard E syndromes,HARD syndromes,Harelip,Harlequin fetus,Harlequin types DOC 6,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-Waldens trom 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,HemorrhagicaAleukia,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 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,homogentisic acidura,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,HSN I,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,HyalinePanneuropathy,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 intermittensjuvenalis,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 Urea Synthesis ArgininoSuccinic 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 PigmentiAchromians,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-Marsidi 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,Kleeblattschade l 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,Labhar t-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),LenzDysmorphogenetic Syndrome,Lenz dysplasia,Lenz nanophthalmus syndromes,Lenz syndromes,Leopard syndrome,Donohue's syndrome,Pia-arachnoid hemangioma,Leptospiral jaundice,Leri-Weill Disease,Leri-Weill dyschondrosteosis,Leri-Weil Syndrome,Lermoyez syndrome,Leroy Disease,Lesch NyhanSyndrome,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,LimbMalforma tions-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 Cyst Syndrome (- 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 Pseudohermaphroditism),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-Bickel Syndrome,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-Unverricht  DiseaseIncluded,Lupus,Lupus,Lupus erythematosus,Luschka-Magendie ForaminaAtresia,Lyell's syndrome,Lyell syndrome,Lymphadenoid goitre,The protein losing enteropathy of lymphangiectasis,Lymphangioleiomyoma,Lymphangioleiomyoma,Lymphangioma,Lymphatic malformation,Lynch Syndromes,Lynch Syndrome I,LynchSyndrome 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,Maladiede 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,sDisease,Mandibular ameloblastoma,Faciomandibular dysostosis,Mannosidosis,Surface-point-finger pattern corneal dystrophy (Map-Dot-Fingerprint Type CornealDystrophy),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,Marie Strumpell 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 Lamy Syndrome,Maroteaux Type AcromesomelicDysplasia,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 (MentalRetardation-Deafness-Skeletal Abnormalities-Coarse Face withFull 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 (MentalRetradat ion-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-SingletonSyndrome,Limb injury syndrome,Glomerular mesangium immunoglobulin-a type nephrosis,Mesenteric fat dystrophia,Mesiodens-Cataract Syndrome,MesodermalDysmorphodystrophy,Mesomelic Dwarfism-Madelung Deformity,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-NephrosisGalloway 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-Radecki Syndrome,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 TypeI),ML Disorder Type II,MLDisorder 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 (MonomodalVisual Amnesia),Mononeuritis multiplex,Mononeuritis,Single nerve endings lesion,Monosomy 3p2 (Monosomy 3p2),Monosomy 9p Partial,Monosomy 11qPartial,Monosomy 13q Partial,Monosomy 18q Syndrome,MonosomyX,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 (Mosaic Tetrasomy 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 I S ScheieSyndrome,MPS II,MPS IIA,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 SevereIntermediate Mild Maroteaux-Lamy (severe ... in light),Mucopolysaccharide (storing up) disease VII,Mucopolysaccharide (storing up) disease VII sly 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 DisorderII,MPS Disorder III,MPS DisorderVI,MPS Disorder Type 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,MyoencephalopathyRagged-Red Fiber Disease (... ragged red fibers disease),Myofibromatosis,Congenital Myofibromatosis,Myogenicity face-omoplate-fibula syndrome,Myoneurogas to ntestinalDisorder and Encephalopathy (encephalopathic),Myopathy AMC,Muscle [poison] base deficit disease,Muscle central area fibrosis,Myopathy Congenital (congenital myopathy ...) Nonprogressive,myopathy Congenital Nonprogress ivewith Central 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 Falls Syndrome (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 (NBIA I),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,NevII,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),NIIkawakuroki syndromes (N IIkawakuroki 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,Opitz BBB/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,OpitzOculogenitolaryngeal syndromes,Opitz trigonocephalias (deformity) syndrome,(eye distance is wide for opitz syndrome,Hypospadia syndrome),Opsoclonus,Opsoclonus-myoclonia,Optha lmoneuromyelitis,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] 1- tips [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),PapillotonicPsuedotabes,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 Shokeir II 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,PHI 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,PiliTorti and nerve deafness,Pili Torti- 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,q31or 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,Rautens trauch-Wiedemann syndromes,Rautens trauch-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,Potter type III 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 (PH1),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,Restrictive Cardiomyopathy,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 Gap Defects 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,Robert s 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-Pas sarge 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 IgA 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 I gA defects,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-Magee 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,SMA3,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 (SCAII),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 Naegell 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 Diaphysaire Tibio-Peroniere,Toxopachyos teose,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,II I type Gaucher disease (familial splenic anemias,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),IIA type Wa Erdunbao syndromes,IIB type Wa Erdunbao syndromes,II I type 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-Rautens trauch 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,W N S,Wohlfart diseases,Wohlfart-Kugelberg-Welander disease (heredity peri position nerve originality muscular atrophy),Wolf syndrome,Wolf-Hirschhorn chromosomal regions (W H C R),Wolf-Hirschhorn syndromes,Wolff-Parkinson-White syndrome,Wolfram syndrome,Primary familial xanthomatosis (lysosomal storage disease caused by acid lipase deficiency),Woody Guthrie ' 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,A I X P A type xeroderma pitmentosums,Type B II XPB xeroderma pitmentosums,Type E V XPE xeroderma pitmentosums,Type C IIIXPC xeroderma pitmentosums,Type D IV XPD xeroderma pitmentosums,Type F VI XPF xeroderma pitmentosums,Type G V II 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 further embodiment, the pharmaceutical composition comprising the EPO of separation or its chimeric molecule can be combined to treat anaemia individually or with other biological preparation, medicine or therapy, such as：Chronic anaemia, capilary hemolytic anemia, the anaemia (including dialysis or patient without dialysis) of adjoint chronic deficiency of the kidney, with the anaemia of end-stage renal disease, with or by anaemia caused by dialysis, ALL, amyelonic pernicious patient is due to anaemia caused by the chemotherapy given, anaemia caused by HIV or treatment, anaemia caused by cancer chemotherapy, anaemia caused by radiotherapy or adjoint hormone therapy or immunotherapy, transfused blood due to external source and reduce the anaemia patient for carrying out non-cardiac non-vascular operation, due to antiviral therapy (such as to hepatitis C and AIDs treatment), inflammatory bowel disease, lose blood (such as due to operation, intestine gastric ulcer or childbirth), rheumatic arthritis, myelocyte suppresses therapy, anaemia caused by chronic or congestive heart failure and malaria.

In a further embodiment, the pharmaceutical composition comprising the EPO of separation or its chimeric molecule can be combined to carry out non-hematopoietic treatment individually or with other biological preparation, medicine or therapy, such as：General maintenance, protection and reparation nervous system (such as damage of local cerebral ischemia, cerebral concussion, experimental autoimmune encephalomyelitis (EAE), kainic acid Salt treatment epilepsy, epilepsy, cerebral ischemia, spinal cord injury, hypoxemia, low back pain, diabetic neuropathy, multiple sclerosis, schizophrenia)；And (such as Patients with Apoplexy After also has brain damage caused by prevention perinatal injury) for the purpose of protecting nerve, retinosis, beta Thalassemia, neural porpharia, glaucoma, neonatal necrotizing enterocolitis, enhanced autologous stem cell transplantation are such as：Enhancing platelet reactivity and quantity in excessive drinking property hepatic sclerosis；

In a further embodiment, the pharmaceutical composition of Flt3- parts or its chimeric molecule comprising separation can individually or with other biological preparation, medicine or therapy are combined to treat the disease of including but not limited to cancer, such as acute myeloid leukemia (AML), liver or Pulmonary metastasis focuses, colon cancer, melanoma, clear-cell carcinoma, cervical carcinoma, prostate cancer, orchioncus, oophoroma, the peritonaeum metastasis of cancer, lung cancer, celiothelioma, liver cancer, lymphocytic lymphoma, alpastic anemia, marrow lymthoma, breast cancer, aleukemic leukemia, lymthoma, development of bone marrow exception and myeloproliferative disease, the superfluous life of thick liquid cell, neutrophilic granulocytopenia, graft-versus-host reaction disease and asthma.And the Flt3- parts or the pharmaceutical composition of its chimeric molecule comprising separation can be combined individually or with other biological preparation, medicine or therapy prevent diabetes or monocytosis Lee department make a mistake (family name) bacterium, leishmaniasis, human immunodeficiency virus (HIV), the adjuvant that iodine and hepatitis B and HIV are inoculated with is immunized caused by herpesviral.

In a further embodiment, pharmaceutical composition comprising the Flt3 of separation or its chimeric molecule such as Flt3-Fc can individually or with other biological preparation, medicine or therapy are combined to treat acute myelogenous leukemia (AML), acute promyelocytic leukemia (APL), B and T cell acute lymphoblastic leukemia (ALL), chronic granulocytic leukemia (CML), myelodysplasia (MDS), medullary sarcoma, breast cancer, the disease also in need for suppressing specific cell type hyperplasia, such as stem cell, bone marrow cell, lymphoid cell, monocyte, blood platelet dendritic cells, NK, also relate to the disease of Flt3R signal transductions, but it is not limited to wherein.And may play the role of the time-to-live of extension transplanting tissue in allograft comprising the Flt3 of separation or its chimeric molecule such as Flt3-Fc.

In a further embodiment, the pharmaceutical composition of PDGF-B comprising separation or homologous heterodimers or its chimeric molecule can individually or with other biological preparation, medicine or therapy are combined to control treatment trauma and ulcer, including chronic trauma and ulcer, with the ulcer of werner syndrome, local ischemic damage treatment after miocardial infarction, varicose ulcer, foot wound, pressure ulcer, venous stasis ulcers, chorionitis ulcer, chronic injury in io-osteohypertrophy's syndrome, ulcerative lichen planus, the related CNS local ischemic damages of apoplexy, periapical inflammation damnification, into the hemangioma (such as in infancy) of ulcer；Also periodontal regenerative (for example being engaged with bone graft) can be strengthened；Strengthen the effectiveness of dentistry implant therapy；Alleviate the illeffects of ischaemic in Colonic anastomosis healing；Accelerate the rehabilitation of the adjoint gangrenous pyaphysia of myelodysplastic syndrome；The also treatment of alzheimer disease and multiple sclerosis.

In a further embodiment, the pharmaceutical composition comprising the VEGF-165 of separation or its chimeric molecule can be combined individually or with other biological preparation, medicine or therapy includes the symptom of balloon injury, graft implantation and blood vessel graft with injury of blood vessel to treat；After the coronal and peripheral arterial vascular diseases of prevention after skin revascularization hemadostewnosis or ISR；For that can not carry out the patient of traditional revascularization operation, such as disperse coronary artery disease carries out curative revascularization；Control the related hyperplasia of superstition wound in arterial transplantation, prevention Post operation flap necrosis, prevention Post operation DupuytrenShi is sick, promote bore regenerating and fracture and Cartilage healing；Promote pancreatic islets transplantation angiogenesis and re-form to increase the long-term surviving after pancreatic islets transplantation；In compressing or other such as spinal cord injuries receptors；Promote nerve regneration, the brain damage by improving cerebrovascular regeneration and Neuroprotective Therapy in Treating Acute cerebral ischaemia or such as apoplexy, head injury, brain damage cerebrovascular malformation are developed after the damage of diabetic neuropathy；Treat the ischaemic of patient wi；Systemic chorionitis；Promote natural on-off cycles of hair growth.

In a further embodiment, the pharmaceutical composition comprising the VEGF-165 of separation or its chimeric molecule can join to treat with angina pectoris treatment, diabetic neuropathy and the treatment of acra macrovesicle disease, the treatment of the Charcot's syndrome as caused by peripheral arterial disease individually or with other biological preparation, medicine or therapy；The disease and/or symptom of chronic or fro diabetic ulcer of foot healing；Avoid acute liver failure and other hepar damnifications that toxicity is induced；Avoid Renal vascular bead endothelial cell damage in nephrosis；Treat erectile dysfunction and amyotrophic lateral sclerosis (ALS) and other nerve degenerative diseases.

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 isomers 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.

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.

The present invention is further illustrated by with non-limiting embodiment below.

Embodiment 1

The clone of the protein of the present invention

(a) preparation of pIRESbleo3-Fc constructs

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 joint with Fc regions.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.

(c) Megaprep Carrier-proteins or Carrier-protein-Fc preparation

The 750ml inoculation of the sterile LB fluid nutrient mediums containing ampicillin (100 μ g/ml) has converted Carrier-protein or the Carrier-protein-Fc E.Coli μ l of overnight culture 750.Culture is in 16 hours of 37 DEG C of shaken cultivations.Plasmid is prepared according to Qiagen Endofree PlasmidMega Kit (Qiagen Mega Prep Kit #12381).

Table 8

Protein-Fc and related cloning information

Protein	CDNA originates	Forward direction is drawn Thing	Reversely draw Thing	Restriction enzyme Enzyme site	Carrier	Size   (bp)
							EPO	RG001720,   Invitrogen	SEQ ID   NO：25	SEQ ID   NO：26	EcoRV,   BamHI	pIRESbleo3   (Cat.No. 6989-1, BD   Biosciences)	582
Flt3- Part	PUMVC 3-hFL,   Aldevron	SEQ ID   NO：41	SEQ ID   NO：42	EcoRV,   BamH1	pIRESbleo3   (Cat.No. 6989-1, BD   Biosciences)	574
							Flt3	Clone ID 5272266,   Invitrogen	SEQ ID   NO：57	SEQ ID   NO：58	NotI, BamH1	pIRESbleo3-Fc   (Cat.No. 6989-1, BD   Biosciences)	1607
PDGF-B	Clone ID 4584154,   Invitrogen	SEQ ID   NO：107	SEQ ID   NO：108	EcoRV,   EcoRI	pIRESbleo3   (Cat.No. 6989-1, BD   Biosciences)	762
							VEGF-165	PORF-hVEGF,   Integrated   Sciences	SEQ ID   NO：121	SEQ ID   NO：122	EcoRV,   BamHI	pIRESbleo3   (Cat.No. 6989-1, BD   Biosciences)	604

Selectively, the nucleotide sequence for the protein being cloned into carrier (such as pIRESbleo3 or pCEP4) can be expanded with primer, the primer adds the restriction enzyme site for allowing the DNA sequence dna of encoding proteins matter to be cloned into Fc nucleotide sequences upstream in carrier-Fc (such as pIRESbleo3-Fc or pCEP4-Fc), so that protein and Fc nucleotide sequences are merged with meeting frame directly or through joint.

Embodiment 2

(a) EPO of the invention preparation and purification

(i) EPO of the invention preparation

At the 0th day, be with the embryo HK cells of conversion 3 × 10⁷Cell is inoculated with five 500cm²Tissue culture dishes (Corning), the cell such as HEK 293, HEK 293c18, HEK 293T, 293CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene) 293A (Invitrogen).Cell is inoculated into EagleShi culture mediums/HamShi nutritional blends F12 (DMEM/F12) (JRH Biosciences) of every flat board 90ml DulbeccoShi improvement, culture medium with the addition of 10% (v/v) heat-inactivated fetal bovine serum (FCS, JRH Biosciences), 10mMHEPES (Sigma), 4mM L- glutamine (Ameresco) and 1% (v/v) Pen .- Strep (JRH Biosciences).

First day, transfected with calcium phosphate.Before transfection, the culture medium in each flat board is replaced with DMEM/F12 fresh 120ml.Calcium phosphate/DNA precipitation is prepared, CaCl is contained by pIRESbleo (Invitrogen) DNAs and 3720 μ l that add the 1200 μ g genes of EPO containing someone₂Sterilized water make final volume be 30ml (solution A).Solution A point drop-wise is added in 30ml 2 × Hepes buffer salines (HBS) (solution B) with 10ml pipettes.In adition process, solution B is gently blown and beaten.Mixture was in 25 DEG C of vortex oscillation cultures 20 minutes.12ml mixtures point drop-wise is added to each flat board.After 4 hours, culture medium containing transfection medium is removed and 100ml DMEM/F12 is added per flat board, it wherein with the addition of 10% (v/v) heat-inactivated donor calf serum (DCS, JRH Biosciences), 10mM HEPES, 4mM L- glutamines, 1% (v/v) Pen .- Strep and 3.5mM HCl make for pH7.0, and the incubated overnight at 37 DEG C.

2nd day, DMEM/F12 culture mediums are removed, 100mM PMSF (1% (v/v)) and 500mMEDTA (1% (v/v)) are added in the culture of collection.Content in flat board per flat board 50ml serum-free DMEM/F12 culture mediums with washing twice and 100ml fresh serum free DMEM/F12 culture mediums added per flat board (DMEM/F12 culture mediums with the addition of 40mM N- acetyl-D-MANNOSE amine, 10mM L- glutamines, 4.1g/L mannose, 1% (v/v) Pen .- Strep and ITS solution (5mg/L bovine insulins, the human transferrin of 5mg/L parts iron saturation and 5 μ g/ml selenium；Sigma).Flat board is in 37 DEG C of cultures.

3rd day, collect serum-free DMEM/F12 culture mediums and each flat board adds 100ml fresh serum free DMEM/F12 culture mediums.100mM PMSF (1% (v/v)) and 500mM M EDTA (1% (v/v)) are added in the culture medium of collection and mixture is in 4 DEG C of storages.

4th day, collect the serum-free DMEM/F12 culture mediums in flat board.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the culture medium of collection.5ml mixtures are used for ELISA and remaining mixture and are combined together with the serum-free gleanings of the 3rd day.Before particulate is removed using 0.45 micron low-protein adsorption filter (Durapore, Millipore), by the 200mM MES/50mMgCl for adding 1/10th volumes₂The combining gleanings of pH6 are adjusted to pH6.Mixture is in -70 DEG C of storages or uses immediately.

(ii) EPO of the invention purifying

The process of dyestuff-aglucon (DLC) chromatogram is used as the first step for purifying EPO.In order to be effectively combined and discharge in the micro form of mass purification, immobilized active dyestuff storehouse is used to screen EPO.Then, suitable dye-protein matter is combined examines in small-scale cylindricality formula.

In small scale purification, the sample for the cells and supernatant that 5ml thaws passes through 0.5ml dye ligand posts under conditions of pH6.0 or 7.3.In the step of the optimization, for the maximum recovery in cut, optimal dyestuff ball-cell factor and pH are combined and are selected, for the scale expansion in large batch of DLC.

For extensive DLC, reactive dye numbering 1High (Zymatrix) is selected as the reactive dye to EPO with best combination and elution property.The cells and supernatant of filtering has 3ml or 6ml 50mM MES/5mM MgCl respectively under gravity stream by 4.0ml or 8.0ml₂Cylinder (Alltech, Extract Clean Filtercolumns) of the pre-balance to pH6 DLC resins.4 DEG C are stored in until ELISA results confirm to purify successfully by the bulk flow of sample.Pillar buffer A (20mM MES/5mM MgCl₂PH6) rinse until cut shows clarification (non-faint yellow).EPO is eluted with three kinds of elution buffers according to order below.

Elution 1：Buffer solution C (50mM Tris-Cl/10mM EDTA pH8)

Elution 2：EN1.0(50mM Tris-Cl/10mM EDTA/1.0M NaCl pH 8)

Elution 3：EN2.0(50mM Tris-Cl/10mM EDTA/2.0M NaCl pH 8)

The cut of elution is analyzed by using the Silver stain SDS PAGE of 4-20%Tris- glycine gels (Invitrogen) and by EPO ELISA (medac).It was found that EPO is eluted in buffer solution EN1.0.The contaminating protein matter for going out 90% by SDS PAGE discriminatory analysises is removed in first purification step.DLC fraction collections containing EPO are used for SEC.

Size exclusion chromatography method is carried out to the DLC cuts of combination with the preparation scales 16/70 of Superdex 75 or the preparation scales of Sephadex 200 (Pharmacia, Uppsala, Sweden) post.The equal strength stream of 50mM MES buffer solutions (pH6.5) is used under 1.5ml/min flow velocitys.Total process time is 120min, is eluting peak at 20-100 minutes.The cut of elution is analyzed by using Silver stain SDS PAGE and the EPO ELISA (medac) of 4-20%Tris- glycine gels (Invitrogen).Eluting peak is only before comprising volume, and discovery in about 48 minutes contains EPO.

Cut containing cell factor quickly removes salt plug (Pharmacia) desalination in PBS with HiPrep 26/10.

The EPO of purifying shows to be defined as at least 95% with the apparent molecular weight between 32kDa and 38kDa and by Silver stain SDS PAGE pure.EPO final concentration is estimated as 10.5 μ g/ml by EPO ELISA (medac).

(b) preparation and purification of Flt3- parts of the invention

(i) preparation of Flt3- parts of the invention

0th day, by 5 500cm²Tissue culture dishes (Corning) use 3 × 10 that the embryo HK cells of conversion is⁷Cell is inoculated with, and the cell is, for example, HEK 293, HEK 293c18, HEK293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene) or 293A (Invitrogen).Cell is inoculated into every flat board 90ml Dulbecco ' s Modified Eagle ' s culture mediums/Ham ' s Nutrient Mixture F12 (DMEM/F12) (JRH Biosciences), it is 4mM L- glutamines (Amresco) and 1% (v/v) Pen .- Strep (benzyl penicillin 5000U/ml, the μ g/ml of streptomycin sulphate 5000) (JRH Biosciences) that culture medium, which with the addition of through concentration,.Flat board is in 37 DEG C and 5%CO₂Incubated overnight.

1st day, conversion was carried out with calcium phosphate.Before conversion, the culture medium of each flat board adds 10% (v/v) DCS, 4mM L- glutamines with 120ml, and the fresh DMEM/F12 of 1% (v/v) Pen .- Strep is replaced.Calcium phosphate/DNA precipitation is to contain CaCl by adding pIRESbleo3 (Invitrogen) DNAs and 3720 μ l of the 1200 μ g parts of Flt3- containing someone₂The sterilized water of (2.5M) reaches final volume 30ml (solution A) to prepare.Solution A is added in 2 × HEPES of 30ml buffered salines (HBS) (solution B) with 10ml pipettes point drop-wise.In adition process, solution B is gently blown and beaten.Mixture was in 25 DEG C of vortex oscillation cultures 20 minutes.12ml mixtures point drop-wise adds each flat board.After 4 hours, culture medium containing transfection mixture is removed and each flat board adds 100ml and with the addition of 10% (v/v) DCS, 4mM L- glutamines, 1% (v/v) Pen .- Strep, with final concentration 3.5mM HCl DMEM/F12, there is culture medium end pH to be 7.Flat board is in 37 DEG C and 5%CO₂Lower incubated overnight.

2nd day, scavenger-cell culture supernatant.Flat board content is washed twice with every flat board 50ml DEME/F12 culture mediums and each flat board adds 100ml and with the addition of 40mM N- acetyl-D-MANNOSE amine (New Zealand Pharmaceuticals), 10mM L- glutamines, 4.1g/L mannoses (Sigma), and 1% (v/v) Pen .- Strep fresh serum-free DMEM/F12 culture mediums.Flat board is in 37 DEG C and 5%CO₂Incubated overnight.

3rd day, collect cells and supernatant and each flat board adds the fresh serum-free DMEM/F12 culture mediums that 100ml with the addition of 40mM N- acetyl-D-MANNOSE amine, 10mM L- glutamines, 4.1g/L mannoses, and 1% (v/v) Pen .- Strep.Flat board is in 37 DEG C and 5%CO₂Incubated overnight.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the cells and supernatant of collection and mixture is in 4 DEG C of storages.

4th day, collect cells and supernatant.The gleanings that 100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the cells and supernatant of collection and with the 3rd day before-protein adsorption filter (Durapore, Millipore) low with 0.45 micron removes particulate are combined together.Mixture can be stored at -70 DEG C or used immediately.

(ii) purifying of Flt3- parts of the invention

1 liter of the cells and supernatant of filtering concentrates 20 times with tangential flow filtration (TFF) device (Pelicon XL, Ultracell, Millipore).Sample crosses 150cm with 150ml/min speed pumps²Regenerated cellulose film, while retaining nominal molecular weight 5KDa up to sample concentration to volume is 100ml.Concentrating sample is concentrated to 100ml again after the 50mM HEPES pH8 dialysis by adding same volume.The diafiltration step obtains whole concentrate 50ml twice repeatedly.

Flt3- parts be purified by by the cells and supernatant of the concentration from TFF pass through anion-exchange column (Bio-Rad Laboratories, Uno S12 pre-equilibrations to 50mM HEPES pH8 (Sigma) complete.Then in conjunction with the 50mM HEPES pH8 of Flt3- part gradients eluted on post to the 50mM HEPES pH8 containing 1M NaCl.The cut of acquisition carries out apparent molecular weight and level of purification analysis and quantitative by Flt3- parts ELISA (R ＆ D Systems) by using the 1D SDS PAGE of 4-20% gradient Tris-glycine gels (Invi trogen).

Be further purified is to carry out size exclusion chromatography completion to the I EC cuts of the combination containing Flt3- parts by using the post (Pharmacia, Uppsala, Sweden) of 75 preparation scales of Superdex 16/70.The equal strength stream of 1 × Dulbecco ' s phosphate buffer normal salines (DPBS Modified) (JRHBiosciences) is used under 1.5ml/min flow velocitys.Total process time is to have eluting peak between 120 minutes, 20 and 100 minutes.The cut of elution is analyzed by using the Silver stain SDS PAGE (Invitrogen) and Flt3- parts ELISA (R ＆ DSystems) of 4-20%Tris- glycine gels.Eluting peak at about 42 minutes finds to contain Flt3- parts.

The Flt3- parts of purifying show with the apparent molecular weight between 28-36KDa and are at least 99% pure, as evaluated by Silver stain SDS PAGE.Flt3- final ligand concentrations are between 0.79-2.5 μ g/ml, as Flt3- part ELI SA (R ＆ D Systems) are evaluated.

(c) Flt3-Fc of the invention preparation and purification

(i) Flt3-Fc of the invention preparation

0th day, by three 500cm²The 3 × 10 of the embryo HK cells system of inoculation conversion in tissue culture dishes (Corning)⁷Cell, such as HEK 293, HEK 293c18, HEK 293T, 293CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene) or 293A (Invitrogen).Cell is inoculated into every flat board 90ml Dulbecco ' s ModifiedEagle ' s culture mediums/Ham ' s Nutrient Mixture F12 (DMEM/F12) (JRHBiosciences), 10% (v/v) heat-inactivated fetal bovine serum (FCS, JRHBiosciences), 4mM L- glutamines (Amresco) and 1% (v/v) Pen .- Strep (benzyl penicillin 5000U/ml, the μ g/ml of streptomycin sulphate 5000) (JRH Biosciences) are with the addition of in culture medium.Flat board is in 37 DEG C and 5%CO₂Lower incubated overnight.

1st day, transfection was carried out with calcium phosphate.Before transfection, the culture medium in each flat board with the addition of the fresh DEME/F12 displacements of 10% (v/v) heat inactivation FCS, 4mM L- glutamines, and 1% (v/v) Pen .- Strep with 120ml.Calcium phosphate/DNA precipitation contains CaCl by adding pIRESbleo3 (Invitrogen) DNAs and 3720 μ l of the 1200 μ g genes of Flt3-Fc containing someone₂The sterilized water of (2.5M) is prepared to final volume 30ml (solution A).Solution A is added in 2 × HEPES of 30ml buffered salines (HBS) (solution B) with 10ml pipettes point drop-wise.In adding procedure, the bubble in solution B is gently blown and beaten.Mixture cultivates 20 minutes and vortex mixing at 25 DEG C.12ml mixtures point drop-wise adds each plate.After 4 hours, remove the culture medium containing transfection mixture and addition 100ml with the addition of 10% (v/v) heat inactivation FCS, 4mM L- glutamines, 1% (v/v) Pen .- Strep and final concentration 3.5mM HCl DEME/F12, make culture medium that there is end pH to be 7 in each flat board.Flat board is in 37 DEG C and 5%CO₂Incubated overnight.

2nd day, scavenger-cell culture supernatant.Content in flat board is washed twice with every flat board 50mlDMEM/F12 culture mediums and the fresh serum-frees of 100ml and the DMEM/F12 culture mediums without phenolsulfonphthalein is added per flat board, with the addition of 40mM N- acetyl-D-MANNOSE amine (NewZealand Pharmaceuticals), 10mM L- glutamines, 4.1g/L mannoses (Sigma) and 1% (v/v) Pen .- Strep.Flat board is in 37 DEG C and 5%CO₂Lower incubated overnight.

3rd day, collect cells and supernatant and each flat board adds the fresh serum-frees of 100ml and the DEEM/F12 culture mediums that with the addition of 40mM N- acetyl-D-MANNOSE amine, 10mM L- glutamines, 4.1g/L mannoses and 1% (v/v) Pen .- Strep without phenolsulfonphthalein.Flat board is in 37 DEG C and 5%CO₂Lower incubated overnight.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the cells and supernatant of collection and by mixture in 4 DEG C of storages.

4th day, collect cells and supernatant.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the cells and supernatant of collection and are combined together with the gleanings of the 3rd day.Before-protein adsorption filter (Durapore, Millipore) low with 0.45 micron removes particulate, adjust the gleanings of combination by adding 2M Tris-HCl pH8 (Sigma) to final concentration 100mM and arrive pH8.Mixture is in -70 DEG C of storages or uses immediately.

(ii) Flt3-Fc of the present invention purifying

PH adjusted 1 liter of the culture medium containing Flt3-Fc is by the Protein A Sepharose post (Pharmacia) with 1ml column volumes under gravity stream, and it is pre-processed to pH8 with 100mM Tris-HCl.Washed with the post buffer solution (100mM Tris-HCl pH 8) of 20 column volumes, Flt3-Fc is eluted in the 0.5ml cuts containing 0.1M citric acids (Sigma) pH2.2 and added 150 μ l 2M Tri s-HCl pH8 by each cut is neutralized to pH 8 immediately.Cut is by using 4

The Silver stain SDS PAGE of 20% gradient Tris-glycine gels (Invitrogen) are analyzed and quantified by using bovine serum albumin(BSA) (Pierce) in the AAS of 280nm absorptivity.Collect the purified fraction containing Flt3-Fc and dialysed more than 4 days with 1L PBS pH7.4, changed daily twice.

The Flt3-Fc of purifying has found the apparent molecular weight with about 110-135KDa by using the SDS PAGE of the Deep Purple fluorescent stainings of 4-20% gradient Tris-HCl gels (Bio-Rad) and is at least 99% pure.Flt3-Fc final concentration of 247 μ g/ml by AAS as assessed.

(d) PDGF-B of the invention preparation and purification

(i) PDGF-B of the invention preparation

0th day, by five 500cm²The 3 × 10 of the embryo HK cells system of tissue culture dishes (Corning) inoculation conversion⁷Cell, such as HEK 293, HEK 293c18, HEK 293T, 293CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene), 293A (Invitrogen) or its any derivative.Cell is inoculated into every flat board 90ml Dulbecco ' s Modified Eagle ' s culture mediums/Ham ' s Nutrient Mixture F12 (DMEM/F12) (JRH Biosciences), and culture medium with the addition of 10% (v/v) donor ox calf serum (DCS, JRH Biosciences), 4mM L- glutamines (Amresco) and 1% (v/v) Pen .- Strep (benzyl penicillin 5000U/ml, the μ g/ml of streptomycin sulphate 5000) (JRH Biosciences).Flat board is in 37 DEG C and 5%CO₂Lower incubated overnight.

1st day, transfection was carried out with calcium phosphate.Before transfection, culture medium in each flat board with 120ml it is fresh with the addition of 10% (v/v) DCS, 4mM L- glutamines, and 1% (v/v) Pen .- Strep DMEM/F12 displacements.DNA and 3000 μ l 2.5M CaCl that calcium phosphate/DNA precipitation passes through the addition 1200 μ g genes of PDGF-B containing someone₂It is prepared by sterile 1xTE to final volume 30ml (solution A).Solution A is added in 30ml 2 × HEPES buffer salines (HBS) (solution B) with 10ml pipettes point drop-wise.In adition process, the bubble in solution B is gently blown and beaten.Mixture is cultivated 20 minutes at 25 DEG C.12ml mixtures point drop-wise is added in each flat board.After 4 hours, remove the culture medium containing transfection mixture and each flat board adds the DMEM/F12 that 100ml with the addition of 10% (v/v) FCS, 4mM L-glutamine, 1% (v/v) Pen .- Strep and final concentration 4.0mM HCl, the whole pH of culture medium is 7.Flat board is in 37 DEG C and 5%CO₂Lower incubated overnight.

2nd day, scavenger-cell culture supernatant.Content in flat board with washed twice per flat board 50mlDMEM/F12 culture mediums and add the fresh serum-frees of 100ml per flat board with the addition of 40mM N- acetyl-D-MANNOSE amine (DMEM/F12 culture mediums of New Zealand Pharmaceuticals 7mM L- glutamines (Amresco), 0.5g/L mannoses (Sigma) and 1% (v/v) Pen .- Strep.Flat board is in 37 DEG C and 5%CO₂Lower incubated overnight.

3rd day, collect cells and supernatant and the DMEM/F12 culture mediums that with the addition of 40mM N- acetyl-D-MANNOSE amine, 7mM L- glutamines, 0.5g/L mannoses and 1% (v/v) Pen .- Strep of 100ml fresh serum frees are added in each flat board.Flat board is in 37 DEG C and 5%CO₂Lower incubated overnight.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the cells and supernatant of collection and mixture is in 4 DEG C of storages.

4th day, collect cells and supernatant.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the cells and supernatant of collection and combined with the gleanings of the 3rd day, before-protein combination filter (Durapore, Millipore) low with 0.45 micron removes particulate.Mixture is stored or used immediately at 4 DEG C.If storing for a long time, supernatant is placed in -70 DEG C.

(ii) PDGF-B of the invention purifying

Ion-exchange chromatography (IEC) process as PDGF-B purifying the 1st step.

SP Ago-Gels FF (Amersham Biosciences) is elected to be with the ion exchange resin to PDGF-B best combinations and elution property.The cells and supernatant of filtering arrives pH 7 SP Ago-Gel 4.0ml cylinders (Alltech, Extract Clean Filter columns) under gravity stream by the use 20mM HEPES pH7 pretreatments equipped with 3ml.Pillar, which is cleaned with 20mM HEPESpH7 to cut, shows clarification (non-faint yellow).PDGF-B presses following sequentially eluting with five kinds of elution buffers.

Buffer solution 1：20mM HEPES pH 7/0.2M NaCl

Buffer solution 2：20mM HEPES pH 7/0.4M NaCl

Buffer solution 3：20mM HEPES pH 7/0.6M NaCl

Buffer solution 4：20mM HEPES pH 7/0.8M NaCl

Buffer solution 5：20mM HEPES pH 7/1M NaCl

Eluting fraction is analyzed by using the Silver stain SDS-PAGE and anti-human PDGF-B ELISA (R ＆ D Systems) of 4-20%Tris- glycine gels (Invitrogen).It was found that PDGF-B is attached to SP Ago-Gels and eluted in buffer solution 2 and 3.Collect the cut containing PDGF-B and dilute 1 part of sample with 1 part of 2M ammonium sulfate/20mM HEPES pH 7, for hydrophobic interaction chromatography.

Hydrophobic interaction chromatography (HIC) is carried out to the PDGF-B cuts of collection, using with HIC phenyl HP posts (Amersham Biosciences) pretreated 1M ammonium sulfate/20mM HEPES pH 7.It is used to elute PGDF-B from 1M ammonium sulfate/20mM HEPES pH7 to 20mM HEPES pH7 30 minutes linear gradients carried out with 1ml/min flow velocitys.Eluting fraction is analyzed by using the Silver stain SDS-PAGE and anti-human PDGF-B ELISA (R ＆D Systems) of 4-20%Tris- glycine gels (Invitrogen).Broad peak at about 0.25M ammonium sulfate finds to contain PDGF-B.Collect these cuts and be concentrated to~1ml volumes with 30-kDa MWCO Centricon inspissators (Millipore).

Size exclusion chromatography is carried out to the HIC cuts of concentration, Superdex 75 preparation scale 16/70 (Pha rmacia, Uppsala, Sweden) post is used.The equal strength stream of phosphate buffered saline (PBS) (PBS) or 1% ammonium hydrogen sulfate is used in 1ml/min flow velocitys.Eluting fraction is analyzed by using the Silver stain SDS-PAGE and anti-PDGE-BELISA (R ＆ D Systems) of 4-20%Tris-glycine gels (Invitrogen).

The PDGF-B of purifying there is 10-30KDa apparent molecular weights and be at least 95% purity.

(e) VEGF-165 of the present invention preparation and purification

(i) VEGF-165 of the present invention preparation

0th day, by five 500cm²The embryo HK cells of tissue culture dishes (Corning) inoculation conversion is 3 × 10⁷Cell, such as HEK 293, HEK 293c18, HEK 293T, 293CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene) or 293A (Invitrogen).Cell is inoculated into every flat board 90ml Dulbecco ' s ModifiedEagle ' s culture mediums/Ham ' s Nutrient Mixture F12 (DMEM/F12) (JRHBiosciences), and culture medium with the addition of 10% (v/v) donor calf serum (DCS, JRHBiosciences), 4mM L- glutamines (Amresco) and 1% (v/v) Pen .- Strep (benzyl penicillin 5000U/ml, streptomycin sulphate 5mg/mi) (JRHBiosciences).Flat board is in 37 DEG C and 5%CO₂Lower incubated overnight.

1st day, transfected with calcium phosphate.Before transfection, the culture medium in each flat board is replaced with the fresh DMEM/F12 that with the addition of 10% (v/v) DCS, 4mM L- glutamines and 1% (v/v) Pen .- Strep of 120ml.Calcium phosphate/DNA precipitation contains CaCl by adding pIRESbleo3 (Invitrogen) DNAs and 3720 μ l of the 1200 μ g genes of VEGF-165 containing someone₂The sterilized water of (2.5M) is prepared to final volume 30ml (solution A).Solution A is added in 2 × HEPES of 30ml buffered salines (HBS) (solution B) with 10ml pipettes point drop-wise.In adition process, solution B is gently blown and beaten.Mixture vortex oscillation culture 20 minutes at 25 DEG C.12ml mixtures point drop-wise is added to each flat board.After 4 hours, remove the culture medium containing transfection mixture and the DMEM/F12 that 100ml with the addition of 10% (v/v) DCS, 4mM L- glutamines, 1% (v/v) Pen .- Strep and final concentration 3.5mM HCl is added per flat board, there is culture medium end pH to be 7.Flat board is in 37 DEG C and 5%CO₂Lower incubated overnight.

2nd day, scavenger-cell culture supernatant.Content in plate is washed twice with often plate 50mlDMEM/F12 culture mediums and each plate adds the DMEM/F12 culture mediums that with the addition of 40mM N- acetyl-D-MANNOSE amine (New Zealand Pharmaceuticals), 10mML- glutamines (Amresco), 4.1g/L mannoses (Sigma) and 1% (v/v) Pen .- Strep of the fresh serum-frees of 100ml.Plate is in 37 DEG C and 5%CO₂Lower incubated overnight.

3rd day, collect cells and supernatant and the DMEM/F12 culture mediums that with the addition of 40mM N- acetyl-D-MANNOSE amine, 10mM L- glutamines, 4.1g/L mannoses and 1% (v/v) Pen .- Strep of the fresh serum-frees of 100ml are added per flat board.Flat board is in 37 DEG C and 5%CO₂Lower incubated overnight.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the cells and supernatant of collection and mixture is in 4 DEG C of storages.

4th day, collect cells and supernatant.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) are added in the cells and supernatant of collection and are combined together with the gleanings of the 3rd day.With reference to gleanings pass through add 1/10th volumes 200mM MES/50mM MgCl₂PH 6 is arrived in pH6 regulations, before-protein adsorption filter (Durapore, Millipore) low with 0.45 micron removes particulate.Mixture is in -70 DEG C of storages or uses immediately.

(ii) VEGF-165 of the invention purifying

Dyestuff-aglucon chromatogram (DLC) process is used as the first step for purifying VDGF-165.In order to be effectively combined and discharge in the micro form of Batch purification, immobilized active dyestuff storehouse is used to screen VEGF-165.Then, suitable dye-protein matter is combined examines in small-scale cylindricality formula.

In small scale purification, the 5ml samples of the cells and supernatant of defrosting pH be 6 or 7.3 time by 0.5ml dyestuffs-with pilum.In the Optimization Steps, for the maximum recovery of cut, optimal dyestuff pearl-cell factor and pH combination, the scale expansion for high-volume DLC are selected.

For extensive DLC, reactive dye numbering 49High (Zymatrix) is elected to be with the best combination and the reactive dye of elution property to VEGF-165.Filtered cells and supernatant is under gravity stream by being respectively provided with 3ml or 6ml 50mM MES/5mM MgCl₂Pre-process 4.0ml the or 8.0ml cylinders (Alltech, Extract Clean Filtercolumns) of pH 6 DLC fixed dyes.Chromatographic column cleans (20mM MES/5mM MgCl with buffer A₂PH 6) show clarification (non-faint yellow) until cut.VEGF-165 is eluted with three kinds of elution buffers according to following order.

Elution 1：Buffer solution C (50mM Tris-Cl/10mM EDTA pH 8)

Elution 2：EN1.0(50mM Tris-Cl/10mM EDTA/1.0M NaCl pH 8)

Elution 3：EN2.0(50mM Tris-Cl/10mM EDTA/2.0M NaCl pH 8)

The cut of elution is analyzed by using the Silver stain SDS PAGE of 4-20%Tris- glycine gels (Invitrogen) and by VEGF-165ELISA (R ＆ D Systems).VEGF-165 has found to elute in buffer solution EN1.0 and buffer solution EN2.0.Collecting the DLC cuts containing VEGF-165 is used for size exclusion chromatography.

Size exclusion chromatography is carried out to the DLC cuts of combination, uses the preparation scale post (Pharmacia, Uppsala, Sweden) of 75 preparation scales of Superdex 16/70.The equal strength stream of 1 x Dulbecco ' s phosphate buffered salines (DPBS Modified) (JRH Biosciences) is used under 1.5ml/min flow velocity.Total process time is 120 minutes, has eluting peak between 20 and 100 minutes.The cut of elution is analyzed by using the Silver stain SDS PAGE of 4-20%Tris- glycine gels (Invitrogen) and by VEGF-165ELISA (R ＆ D Systems).VEGF-165 has found to elute in the peak of 44 to 47 minutes of process in post.

The VEGF-165 of purifying is had found pure with the apparent molecular weight between 20-32kDa and at least 99% by Silver stain SDS-PAGE analyses.VEGF-165 is assessed by VEGF-165ELISA (R ＆ D Systems) finds that there is maximum concentration to be 24.5 μ g/ml.

Embodiment 3

(a) EPO of the invention feature

(i) two-way polyacrylamide gel electrophoresis

The sample collected by embodiment 2 (a) changes buffer solution into (18MOhm) water of repurity by dialysis or desalting column (Pharmacia HR 10/10Fast Desalting Column), and is dried with SpeedVac inspissators.Then, sample is re-dissolved in 240 μ l MSD buffer solutions (5M urea, 2M thiocarbamides, 65mM DTT, 2% (w/v) CHAPS, 2% (w/v) DMPT 3-10,0.2% (v/v) carrier ampholyte, 40mM Tris, 0.002% (w/v) bromophenol blue, water) and centrifuged 8 minutes under 15000g.

Isoelectric focusing (IEF) is carried out with prefabricated 11cm or prefabricated 17cm pH of latex gel 3-10 retentive force IEF 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 is to 11cm adhesive tape in 100V 1 hour, 200V 1 hour, 600V 2 hours, 1000V 2 hours, and 2000V2 hours, 3500V 12 hours and 100V were carried out for more than 12 hours, or are carried out 17cm adhesive tape 85kV a few hours with (use identical V ramp-up process).

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 pH8.8,6M 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 × 8cm × 1mm is thick) 10-20%v/v Tris glycine gradient gels (BioRad) second.17cm adhesive tape is separated into the 10-20%v/v Tris glycine gradient gels 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 the agarose of the 0.5%v/v containing the bromophenol blue as trace dyestuff.

SDS-PAGE is carried out with Criterin or Protean II electrophoresis systems (BioRad) (200V 1h (will be disengaged from gel end until buffer solution is proceeded to) are used for 11cm gels and 15mA constant currents per gel 21h for 17cm gels).Buffer solution used is 192mM glycine, and 0.1% (w/v) SDS, 24.8mM Tris alkali is under pH 8.3.

After the completion of second fix 30 minutes to gel-in 10%v/v methanol (MeOH) and 7% acetic acid (Hac) overnight.Then, gel is dyed at least 3 hours with Sypro Ruby gel stains (BioRad) and decolourized at least 30 minutes with 10%v/v MeOH and 7%v/v HAc.Optionally, after fixed, gel Deep Purple fluorescent dyeings.Gel is in 300mM Na₂CO₃、35mMNaHCO₃Culture 2 × 30 minutes, then, is cultivated at least 1 hour in the dark in the Deep Purple dyestuffs of 1: 200 dilution.Then, gel is decolourized by cultivating 2 × 15 minutes in 10%v/v MeOH, 7%v/v HAc.During two, gel is imaged with FX laser densitometers (BioRad) and suitable filter.

Software I mageJ (http://rsb.info.nih.gov/ij/) be used to analyze the relative intensities of protein spotses on each gel.Optical densitometric method is carried out to the spot of the selection area of each gel and background subtraction is carried out with the appropriate area of each gel of protein spot.Volume integral is carried out to each destination protein spot and thus calculates spot quality center.Calculate the percentage specific strength of each protein spotses and the associated value of the spotted intensity of institute is reached 100% by normalization, relative to other spots in gel, the intensity of each protein spot is measured.

The molecular weight of each spot is by the measurement of each distance between spot and each gel bottom and with being also used for relatively determining for the distance that the Precision or Kaleidoscope molecular weight markers of gel are shown.With 4^thPolynomial exponential function is applied to accurately mark to make up the difference for plaque of protein point location respectively.With this method, the molecular weight of each spot can be accurately determined.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 relation between the pI values of adhesive tape and the physical distance of gel is linear, the pI values corresponding to the different pKa values of isoform spot are easily determined.

The unique EPO isoforms of each protein spotses correspondence.The relative intensity of these isoforms of the display apparent molecular weight of table 9 and 10, pI values and two kinds of gels.The value listed corresponds to the intensity weighted center in the selection area containing spotted each gel, and is the pI and molecular weight of protein abundant reflection therefore.

Table 9

The molecular weight and pI values of EPO isoforms

Spot number	Isoelectric point (pI)	Molecular weight MW (kDa)	Relative intensity %) (normalized value)
				2	4.19	30.82	13.68
3	4.42	30.38	17.04
				4	4.63	30.26	12.64
5	4.84	30.11	9.39
				6	5.06	30.09	10.02
7	5.31	30.06	10.04
				8	5.60	29.79	9.80

Table 10

The molecular weight and pI values of EPO isoforms

Spot number	Isoelectric point (pI)	Molecular weight MW (kDa)	Relative intensity %) (normalized value)
				2	4.20	35.87	0.57
3	4.37	35.93	1.88
				4	4.53	35.90	2.62
5	4.68	35.47	4.36
				6	4.84	35.17	5.06
7	5.03	34.81	5.92
				8	4.20	32.17	0.21
9	4.37	31.92	0.90
				10	4.52	31.91	1.74
11	4.67	31.41	2.82
				12	4.84	31.16	3.07
13	5.03	30.92	2.85
				14	5.24	33.11	9.85
15	5.46	32.69	8.79
				16	5.68	32.10	10.37
17	5.98	31.34	13.06
				18	6.45	30.50	9.17
19	7.08	30.05	7.68
				20	7.58	29.55	1.13
21	8.07	29.04	5.91
				22	8.62	29.54	1.48
23	9.15	29.02	0.57

(ii) unidirectional polyacrylamide gel electrophoresis

The sample collected by embodiment 2 is dried, (10% glycerine, 0.1%SDS, 10mM DTT, 63mM tris-HCl) is then redissolved in 60 μ l1D 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 carried out will be disengaged from gel until buffer solution is proceeded to Criterion electrophoresis systems (BioRad) in 200V.The buffer solution used is 192mM glycine, 0.1% (w/v) SDS, 24.8mM Tris alkali under pH 8.3.

The gel of completion is fixed overnight in 10% methanol (MeOH) and 7.5% acetic acid (HAc), then uses 200mM Na₂CO₃(washing for 2 × 15 minutes) alkalization.Then, gel is dyed at least 1 hour according to manufacturer's technical specification with Deep Purple (Fluorotechnics production code member RPN6306V) and decolourized at least 30 minutes with 10%MeOH and 7.5%HAc.Gel is imaged with Typhoon TrioVariable Mode Imager (Amersham Biosciences) and suitable filter.

EPO apparent molecular weight is between 23 and 38kDa.

The apparent molecular weight of the EPO (as observed by SDS-PAGE) after N- connections oligosaccharides (by PNGase processing) is discharged between 16 and 20kDa.The apparent molecular weight (as observed by SDS-PAGE) for discharging the EPO after N- connections and O- connections oligosaccharides (by glucosides ferment treatment) is between 15.5 and 20kDa.

(III) the N- end sequencings of protein

Protein band, which cuts from two-way gel or unidirectional gel and is put into 0.5ml test tubes and adds 100ml, leaches buffer solution (100mM sodium acetates, 0.1%SDS, 50mM DTT pH 5.5).Gel slice was in 37 DEG C of shaken cultivations 16 hours.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 Biosys tems, 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.expa sy.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.Fragment ion (missing of amino acid of the correspondence from precursor peptide) is recorded and handled with MassLynx Version 3.4 (Micromass).Amino acid sequence is inferred to by using the mass discrepancy of y- or b- ions ' gradient ' series of MassSeq (Micromass) program and determined by manual translation.Then, peptide sequence is used to search for NCBI and TrEMBL databases, uses BLASTP programs " shortnearly exact matches ".The minimum value of two pairing peptides is necessary for providing given firmly believing for homogeneity.The homogeneity of hydrogel spots is proved to be EPO.

Further, it was observed that 1Da move into trypsase peptide aggregation in, the asparagine residue (N) of 3NX (S/T/C) motif found in the theoretical amino acid sequence for showing people EPO is modified to asparatate (D), with known to PNGaseF, the N connection oligosaccharides combined with removing, the ability for inducing residues of the N as D to modify is consistent.Therefore, the site that EPO of the invention N glycosylations are proved is N-51, N-65 and N-110 (having signal sequence starting point open numbering).

(b) feature of Flt3- parts of the present invention

(i) two-way polyacrylamide gel electrophoresis

The sample collected by embodiment 2 (b) is through handling and being analyzed according to above-described embodiment 3 (a) (i).Obtained each protein spot on 2D gels corresponds to unique isoform of Flt3- parts.Table 11 shows the relative intensity of apparent molecular weight, pI values and these isoforms.Intensity weighted center in listed selection region of the value correspondence containing spotted gel, is the abundant reflection of the molecular weight of pI and protein therefore.

Table 11

The molecular weight and pI values of the isoform of Flt3- parts

Spot number	Isoelectric point (pI)	Molecular weight (kDa)	Relative intensity (%) (normalized value)
				2	3.93	28.37	1.98
3	4.09	28.16	3.80
				4	4.26	28.05	5.36
5	4.45	27.92	6.08
				6	4.66	28.00	7.78
7	4.87	28.08	4.86
				8	5.11	28.13	3.64
9	5.32	28.06	1.85
				10	5.47	29.35	0.71
11	5.66	28.36	1.01
				12	3.93	25.27	1.74
13	4.11	25.02	3.44
				14	4.29	24.96	4.10
15	4.48	24.94	4.82
				16	4.68	24.98	6.72
17	4.88	25.18	5.33
				18	5.12	25.33	4.72
19	5.35	25.32	2.98
				20	5.67	25.39	1.97
21	3.93	22.32	0.73
				22	4.11	22.06	1.88
23	4.31	22.02	2.43
				24	4.50	22.03	2.74
25	4.68	22.06	5.14

Spot number	Isoelectric point (pI)	Molecular weight (kDa)	Relative intensity (%) (normalized value)
				26	4.89	22.14	3.93
27	5.11	22.40	3.47
				28	5.36	22.75	2.01
29	5.67	22.68	1.24
				30	5.28	21.01	0.90
31	5.47	20.72	1.20
				32	5.73	20.47	1.46

(ii) unidirectional polyacrylamide gel electrophoresis

The sample collected by embodiment 2 (b) is handled according to the above embodiments 3 (a) (ii).

(III) the N- ends sequencing of protein

Prepare the protein band obtained by 2D or 1D gels and handled according to above-described embodiment 3 (a) (III).Obtained sequence is used for the identification for determining Flt3- ligandin matter.

(iv) peptide mapping fingerprinting method

The peptide mapping fingerprinting method of the Flt3- parts of the present invention is carried out according to above-described embodiment 3 (a) (iv).It is Flt3- parts that the homogeneity of hydrogel spots, which is confirmed,.

(c) Flt3-Fc of the invention feature

(i) two-way polyacrylamide gel electrophoresis

The sample collected by embodiment 2 (c) is handled and analyzed according to above-described embodiment 3 (a) (i).Each protein spot on 2D gels corresponds to unique Flt3-Fc isoforms.Table 12 and 13 illustrates the relative intensity of apparent molecular weight, pI and these isoforms.Listed value corresponds to the intensity weighted center containing spotted each gel selection region, is the abundant reflection of protein pI and molecular weight therefore.

Table 12

The molecular weight and pI values of Flt3-Fc isoforms

Spot number	Isoelectric point (pI)	Molecular weight (kDa)	Relative intensity (%) (normalized value)
				16	5.375	141.606	1.847
17	5.471	138.626	1.589
				18	5.549	137.976	1.522
19	5.680	126.297	1.376
				20	5.768	124.581	1.269
21	5.887	125.038	1.877
				22	6.028	122.222	3.242
23	6.194	121.575	1.792
				24	6.300	120.820	1.305
25	6.467	119.890	0.709
				26	6.692	117.102	0.603
27	5.377	115.220	1.086
				28	5.468	111.931	0.827
29	5.557	113.781	0.958
				30	5.685	109.927	1.971
31	5.778	109.803	1.835
				32	5.887	107.946	3.450
33	6.029	105.727	4.186
				34	6.200	104.621	3.191
35	6.323	103.692	1.540
				36	6.464	103.051	1.231
37	6.698	101.605	1.131
				38	6.809	101.040	0.611

Table 13

The molecular weight and pI values of Flt3-Fc isoforms

Spot number	Isoelectric point (pI)	Molecular weight (kDa)	Relative intensity (%) (normalized value)
				2	5.14	128.34	0.566
3	5.18	128.66	0.868
				4	5.24	128.41	1.553
5	5.30	127.49	2.475
				6	5.36	126.16	3.153
7	5.41	125.59	3.578
				8	5.46	125.08	4.942
9	5.52	124.49	5.773
				10	5.58	123.99	7.038
11	5.64	123.52	7.333
				12	5.71	123.10	6.899
13	5.77	122.54	6.830
				14	5.84	121.83	6.983
15	5.91	121.41	6.797
				16	5.97	120.67	6.037
17	6.05	119.91	5.611
				18	6.12	119.15	5.086
19	6.20	118.67	4.162
				20	6.27	118.07	3.872
21	6.36	117.09	2.881
				22	6.44	115.79	2.568
23	6.53	114.14	1.862
				24	6.62	112.87	1.994
25	6.72	112.63	1.140

(ii) unidirectional polyacrylamide gel electrophoresis

The sample collected by embodiment 2 (c) is handled according to above-described embodiment 3 (a) (ii).

The apparent molecular weight (as observed by SDS-PAGE) of the Flt3-Fc after N connections oligosaccharides (by PNGase processing) is discharged between 85 and 135kDa.The apparent molecular weight of the Flt3-Fc (as observed by SDS-PAGE) after N connections and O connections oligosaccharides (by glucosides ferment treatment) is discharged between 85 and 120kDa.

(III) the N- end sequencings of protein

Protein band from 2D or 1D gels is prepared and handled according to above-described embodiment 3 (a) (III).Obtained sequence is used for the homogeneity for determining Flt3-Fc protein.

(iv) peptide mapping fingerprinting

The Flt3-Fc of present invention peptide mapping fingerprinting is prepared according to above-described embodiment 3 (a) (iv).The homogeneity of hydrogel spots is defined as Flt3-Fc.

(d) PDGF-B of the invention feature

(i) two-way polyacrylamide gel electrophoresis

The sample collected by embodiment 2 (d) is handled and analyzed according to above-described embodiment 3 (a) (i).

(ii) unidirectional polyacrylamide gel electrophoresis

The sample collected by embodiment 2 (c) is handled according to above-described embodiment 3 (a) (ii).

(III) the N- end sequencings of protein

Protein band from 2D or 1D gels is prepared and handled according to above-described embodiment 3 (a) (III).Obtained sequence is used for the homogeneity for determining Flt3-Fc protein.

(iv) peptide mapping fingerprinting

PDGF-B of the present invention peptide mapping fingerprinting is carried out according to above-described embodiment 3 (a) (iv).The homogeneity of hydrogel spots turns out to be PDGF-B.

(e) VEGF-165 of the invention feature

(i) two-way polyacrylamide gel electrophoresis

The sample collected by embodiment 2 (e) is handled and analyzed according to above-described embodiment 3 (a) (i).Each protein spot on obtained 2D gels corresponds to unique VEGF-165 isoform.Table 14 shows the relative intensity of the apparent molecular weight of two kinds of samples derived from the present invention, pI values and these isoforms.The intensity that listed value corresponds in the selection region of the gel containing spottiness strengthens center, is the abundant reflection of protein pI and molecular weight therefore.

Table 14

The molecular weight and pI values of VEGF-165 isoforms

Spot number	Isoelectric point (pI)	Molecular weight (kDa)	Relative intensity (%) (normalized value）
				2	3.93	28.37	1.98
3	4.09	28.16	3.80
				4	4.26	28.05	5.36
5	4.45	27.92	6.08
				6	4.66	28.00	7.78
7	4.87	28.08	4.86
				8	5.11	28.13	3.64
9	5.32	28.06	1.85
				10	5.47	29.35	0.71
11	5.66	28.36	1.01
				12	3.93	25.27	1.74
13	4.11	25.02	3.44
				14	4.29	24.96	4.10
15	4.48	24.94	4.82
				16	4.68	24.98	6.72
17	4.88	25.18	5.33
				18	5.12	25.33	4.72
19	5.35	25.32	2.98
				20	5.67	25.39	1.97
21	3.93	22.32	0.73
				22	4.11	22.06	1.88
23	4.31	22.02	2.43
				24	4.50	22.03	2.74
25	4.68	22.06	5.14
				26	4.89	22.14	3.93
27	5.11	22.40	3.47
				28	5.36	22.75	2.01

(ii) unidirectional polyacrylamide gel electrophoresis

The sample collected by embodiment 2 (e) is handled according to above-described embodiment 3 (a) (ii).

(III) the N- end sequencings of protein

Protein band from 2D or 1D gels is prepared and handled according to above-described embodiment 3 (a) (III).Obtained sequence is used for the homogeneity for determining VEGF-165 protein.

(iv) peptide mapping fingerprinting

The VEGF-165 of present invention peptide mapping fingerprinting is carried out according to above-described embodiment 3 (a) (iv).The homogeneity of hydrogel spots turns out to be VEGF-165.

Embodiment 4

(a) EPO of the invention amino acid, Monsaccharide, oligosaccharides, phosphate, sulfate and isoform are constituted

(i) it is used for amino acid, Monsaccharide, oligosaccharides, phosphate, the preparation for the sample that sulfate and isoform are analyzed

For monose and the identification of glycosylated and phosphorylation and sulphation posttranslational modification, glycosyl is removed from polypeptide backbone by the method for hydrolysis or enzyme first.Sample buffer component is also removed, and replaces to eliminate the inhibitory action of hydrolysis and enzyme digestion reaction before analysis starts with water.The EPO solution purified in PBS is dialysed 4 days on a large scale by 4 liters of deionization ultrafiltration water (18MOhm), liquid is changed daily twice, with specified molecular cut off (NMWC) 5KDa regenerated cellulose dialysis membrane (Spectrapore).After dialysis, solution is dried with Savant Speed Vac (New York, USA).Then, dried sample, which is resuspended with 2ml deionization ultrafiltration water (18MOhm) and is divided into each several part, is used for various analyses.

(ii) analyzed by the amino acid composition of vapor phase hydrolysis method

Amino acid in sample is analyzed with the pre-column derivatization by 6- aminoquinolines base-n-hydroxysuccinimide base-carbamate (AQC).Separate stable Fluorescent amino acid derivative and purified by anti-phase (C18) HPLC.Operation is carried out according to Waters AccQTag amino acid analysis methods.

Take the sample of 100 μ l EPO products and dried in Speed Vac.Then, dry sample is hydrolyzed 24 hours at 110 DEG C.After hydrolysis, sample is dried again before following derivative.Drying sample is re-dissolved in adding 15 μ L AQC derivative reagents after 10 μ L amino acid internal standards (butyrine, AABA), 35 μ L borate buffer solutions of addition.Reactant mixture is heated 12 minutes in constant-temperature incubation device at 50 DEG C.Derivative amino sample is transferred to the automatic sampler of HPLC system, and HPLC system includes the separation assemblies of Waters Alliance 2695 of series connection, the fluorescent probes of Waters 474 and Waters 2487Dual λ absorptance detectors.Control and analysis software are WatersEmpower Pro Module (Waters Corporation, Milford.MA, USA).Sample is used chromatographic parameter known in the art (such as suitable eluent and gradient current) by Waters AccQTag posts (15cm × 3.9mm ID).

(III) neutral and amino monosaccharide composition analysis

Take the sample of 100 two kinds of EPO products of μ l and handled with two kinds of different modes to discharge monose.Every kind of three parts of processing method.

1. 100 DEG C are heated to 2M trifluoroacetic acid (TFA) to hydrolyze 4 hours to discharge neutral sugar (galactolipin, glucose, fucose and mannose).

2. 100 DEG C are heated to 4M HCl to hydrolyze 4 hours to discharge amino sugar (N- acetyl-amine-galactose, N- acetyl-aminoglucose).

All hydrolysates are lyophilized with Speed Vac systems, are re-dissolved in 200 μ l and contain in the water of 0.8nmol internal standard compounds.Internal standard compound for neutral and amino sugar is 2-DG.Then, sample centrifuges 30 minutes to remove protein fragments under 10,000g.Supernatant is transferred to new test tube and by high pH anion exchange chromatographies, using the systems of LC 50 and GP50 pumps and ED50 pulse current detectors (Dionex Ltd) together.Neutral and amino glycan analysis is carried out with DionexCarboPac PA-20 posts.Eluted more than 20 minutes with 10mM equal strength hydroxide concentrate.The process elutes generation system with Dionex EG50 and completed.

(iv) acid monosaccharide composition analysis

Take the sample of 100 μ l EPO products and handle to discharge sialic acid monosaccharide with the following method.Processing method handles three parts.

Sample hydrolyzes 40 minutes to discharge N- acetyl and N- glycoloylneuraminic acids with 0.1M TFA at 80 DEG C.Hydrolysate is freezed with Speed Vac, 200 μ l is re-dissolved in and contains in the water of 0.8nmol internal standard compounds.Internal standard compound for analyzing sialic acid is lactobionic acid.Then, sample centrifuges 30 minutes to remove protein fragments under 10,000g.Supernatant is transferred to new test tube and by high pH anion exchange chromatographies, using the systems of Dionex LC 50 and GP50 pumps and ED50 pulse current detectors together.Saliva acid analysis is carried out with Dionex CarboPac PA1, is used chromatographic parameter known in the art (such as suitable eluent and gradient current).

(v) analysis of oligosaccharides composition

In order to analyze oligosaccharides composition, two kinds of 300 μ l, three parts of samples are taken, and handle as follows.

The release of N- connection oligosaccharides is completed with BPTI-N4- (N- acetyl-β-GLUCOSAMINE base) asparagine acid amides enzyme (PNGase).1/5^thThe denaturing soln (2%SDS (Sigma)/1M beta -mercaptoethanols (Sigma)) of volume 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.

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.

Further processing, to remove buffer solution composition, uses CarboPac graphitised carbon SPE posts for N- connections and O- connections sample.Column equilibration and elution requirement are as follows.Post is with 1 column volume 80%v/v acetonitriles (Sigma) pre-equilibration, then with the water balance of two column volumes.

Sample fills post and with the H of two column volumes under gravity stream₂O is rinsed.In order to elute neutral oligosaccharides, 2ml 50%v/v acetonitriles are used in post.In order to elute acidic oligosaccharide, 2ml 50%v/v acetonitriles/0.1%v/v formic acid is used in post.The oligosaccharides of any reservation is eluted by adding 2ml 80%v/v acetonitriles/0.1%v/v formic acid.

Dried by the cut 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 (Harrisonand Packer Methods Mol Biol 125：The method of 211-216,2000) description is carried out.

Taking the sample of 100 μ l EPO 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 IonPac AS11 anion-exchange columns.

Hydroxide ion and SO are neutralized with Dionex self-regeneration anion micro-membrane suppressors (SRMS-1)₄And PO₄Ion is detected with conductance monitor.

(vii) the further separation of EPO isoforms

The further separation of EPO isoforms 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.EPO isoforms find to be eluted in the peak of different shaped.

(vIII) result

Amino acid is constituted

EPO products have obtained the amino acid composition shown in table 15 through hydrolysis, derivative and described anti-phase high performance liquid chromatography analysis.As a result represented with the percentage of the presence of various amino acid in the total amount and sequence (including SD) of weight.

Table 15

Monose

Each monose is obtained by EPO amino acid backbone hydrolysis and analyzed by described high pH anion-exchange chromatographies (HP AEC) to obtain following composition analysis.Result from sample is respectively for GalNAc and three times mannose normalization (table 16-17).Table 19 is the summary of the result from three parts of samples.

Table 16

Table 17

Table 18

Table 19

Amino acid composition data combination monose data obtain different types of content (table 20).

Table 20

(b) amino acid of Flt3- parts of the invention, monose, oligosaccharides, phosphate, the analysis of sulfate and isoform composition

(i) it is used for amino acid, monose, oligosaccharides, phosphate, the preparation for the sample that sulfate and isoform are analyzed

The solution of the Flt3- parts of purifying in PBS is handled according to above-described embodiment 4 (a) (i).

(ii) it is made up of gas phase hydrolysis method analysis of amino acid

The sample of Flt3- part products is handled according to above-described embodiment 4 (a) (ii).

(III) analysis that neutral and amino monose is constituted

The sample of Flt3- part products is handled according to above-described embodiment 4 (a) (III).

(iv) analysis of acid monose composition

The sample of Flt3- part products is handled according to above-described embodiment 4 (a) (iv).

(v) analysis of oligosaccharides composition

The sample of Flt3- part products is handled according to above-described embodiment 4 (a) (v).

(vi) analysis of sulfate and phosphate composition

The sample of Flt3- part products is handled according to above-described embodiment 4 (a) (vi).

(vii) the further separation of Flt3- parts isoform

The further separation of Flt3- part isoforms is carried out according to above-described embodiment 4 (a) (vii).Flt3- parts isoform is eluted in the peak of different shaped.

(vIII) result

Amino acid is constituted

Flt3- parts derive through hydrolysis and following amino acid composition (table 21) are obtained by described rp-hplc analysis.As a result represented with the presence percentage (including standard deviation (SD)) of various amino acid in weight total amount and sequence.Glycine is known pollutant in amino acid analysis, and it can change amino acid composition with non-natural.It is included in, as a result still shows variant with theoretical value, especially L, I, P and R, it is lower than desired value, and K and D, it is higher than desired value.

Table 21

Monose and sulfate

Each monose, phosphate and sulfate are obtained by the amino acid backbone hydrolysis of Flt3- parts and obtain following composition analysis by high pH anion chromatographics (HP AEC) analysis as mentioned.The result obtained by sample is respectively to GalNAc and the mannose of three times normalization (table 22-24).Table 25 derives from the summary of the result of three parts of samples.Glucose is the normal composition of common pollutant and not N or O connection oligosaccharides.

Table 22

Table 23

Table 24

Table 25

Amino acid composition data combination monose and phosphate and sulfate data are to obtain the content (table 26) of various species.

Table 26

(c) Flt3-Fc of the invention amino acid, monose, oligosaccharides, phosphate, the analysis of sulfate and isoform composition

(i) it is used for amino acid, monose, oligosaccharides, phosphate, the preparation for the sample that sulfate and isoform are analyzed.

The Flt3-Fc solution purified in PBS is handled according to above-described embodiment 4 (a) (i).

(ii) amino acid composition analysis of vapor phase hydrolysis method is passed through

The sample of Flt3-Fc products is handled according to above-described embodiment 4 (a) (ii).

(III) analysis that neutral and amino monose is constituted

The sample of Flt3-Fc products is handled according to above-described embodiment 4 (a) (III).

(iv) analysis of acid monose composition

The sample of Flt3-Fc products is handled according to above-described embodiment 4 (a) (iv).

(v) analysis of oligosaccharides composition

The sample of Flt3-Fc products is handled according to above-described embodiment 4 (a) (v).

(vi) the further separation of Flt3-Fc isoforms

The further separation of Flt3-Fc isoforms is carried out according to above-described embodiment 4 (a) (vii).Flt3-Fc isoforms show to elute in the peak of different shaped.

(vii) result

Amino acid is constituted

Flt3-Fc derives through hydrolysis and following amino acid composition (table 27) is obtained by described rp-hplc analysis.Glycine is known pollutant in amino acid analysis, and it can change amino acid composition with non-natural.It is included in, the result of some amino acid differs still further from theoretical value, includes the pollutant of less content present in sample.As a result represented with the presence percentage (including standard deviation (SD)) of various amino acid in weight total amount and sequence.

Table 27

Amino acid composition

Monose

Each monose, is obtained by Flt3-Fc amino acid backbone hydrolysis and obtains following composition analysis by high pH anion chromatographics (HP AEC) analysis as mentioned.Glucose is common pollutant and is not normally to show in N or O connection oligosaccharides.The GlcNAc levels of measurement are also high and are probably due to pollutant.The result obtained by sample is respectively to GalNAc and the mannose of three times normalization (table 28-30).Table 31 is derived from the summary of the result of three parts of samples.

Table 28

Table 29

Table 30

Table 31

Amino acid composition data combination monose and phosphate and sulfate data obtain the content (table 32) of various species.

Table 32

(d) PDGF-B of the invention amino acid, monose, oligosaccharides, phosphate, the analysis of sulfate and isoform composition

(i) it is used for amino acid, monose, oligosaccharides, phosphate, the preparation for the sample that sulfate and isoform are analyzed.

The PDGF-B solution purified in PBS is handled according to above-described embodiment 4 (a) (i).

(ii) amino acid composition analysis of vapor phase hydrolysis method is passed through

The sample of PDGF-B products is handled according to above-described embodiment 4 (a) (ii).

(III) analysis that neutral and amino monose is constituted

The sample of PDGF-B products is handled according to above-described embodiment 4 (a) (III).

(iv) analysis of acid monose composition

The sample of PDGF-B products is handled according to above-described embodiment 4 (a) (iv).

(v) analysis of oligosaccharides composition

The sample of PDGF-B products is handled according to above-described embodiment 4 (a) (v).

(vi)

(vii) the further separation of Flt3-Fc isoforms

The further separation of Flt3-Fc isoforms is carried out according to above-described embodiment 4 (a) (vii).Flt3-Fc isoforms show to elute in the peak of different shaped.

(e) VEGF-165 of the invention amino acid, monose, oligosaccharides, phosphate, the analysis of sulfate and isoform composition

(i) it is used for amino acid, monose, oligosaccharides, phosphate, the preparation for the sample that sulfate and isoform are analyzed.

The VEGF-165 solution purified in PBS is handled according to above-described embodiment 4 (a) (i).

(ii) amino acid composition analysis of vapor phase hydrolysis method is passed through

The sample of VEGF-165 products is handled according to above-described embodiment 4 (a) (ii).

(III) analysis that neutral and amino monose is constituted

The sample of VEGF-165 products is handled according to above-described embodiment 4 (a) (III).

(iv) analysis of acid monose composition

The sample of VEGF-165 products is handled according to above-described embodiment 4 (a) (iv).

(v) analysis of oligosaccharides composition

The sample of VEGF-165 products is handled according to above-described embodiment 4 (a) (v).

(vi) analysis of sulfate and phosphate composition

The sample of VEGF-165 products is handled according to above-described embodiment 4 (a) (vi).

(vii) the further separation of VEGF-165 isoforms

The further separation of VEGF-165 isoforms is carried out according to above-described embodiment 4 (a) (vii).VEGF-165 isoforms show to elute in the peak of different shaped.

(vIII) result

Monose

Each monose is obtained by VEGF-165 amino acid backbone hydrolysis and analyzed by described high pH anion-exchange chromatographies (HP AEC) to obtain following composition analysis.Result from sample is respectively to GalNAc and the mannose of three times normalization (table 33-35).Table 36 is the summary of the result from three parts of samples.Notice that glucose is universal pollutant and not normal N or O connections oligosaccharides is constituted.

Table 33

Table 34

Table 35

Table 36

Amino acid composition data combination monose and phosphate and sulfate data, to obtain the content (table 37) of various species.

Table 37

Embodiment 5

Saccharic amount dactylogram

Target protein is separated with the 2D gel electrophoresis technologies of such as embodiment 3 and is adsorbed onto on Kynoar (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 MSⁿSpectrum.These fragments can obtain the prediction of structure, using methods known in the art, for example, passing through the use of GlycosidIQ program bags.

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 containing alpha-2,6- sialyltransferase

The cDNA for encoding alpha-2,6- sialyltransferase (alpha2,6ST) is expanded by PCR by poly (A)-primed cDNA.PCR primer is connected in suitable carrier, such as pIRESpuro4 or pCEP4, to produce alpha2,6ST plasmid.The cDNA of clone is sequenced and its homogeneity is confirmed by being compared with disclosed alpha-2,6ST cDNA sequence.DNA sequencing is carried out with known method.

Mammalian host cell, includes cell strain system (cell line-molecules of interest) alpha2,6ST plasmid transfection of the identical pedigree of the high-level molecules of interest of expression, 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 the inspection active for intracellular alpha2,6ST.In order to separate expression alpha2,6ST individual cells strain, cell gleanings pass through such as Kronman (Gene121：The limitation dilution method clone of 295-304,1992) description.Single cell strain system is selected at random, and cell expands and detects alpha2,6ST activity of strain.

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 alpha2,6ST cell line-molecules of interest cell and carry out external and/or Half-life in vivo biologicall test (see embodiment 10).Molecules of interest from high expression alpha2,6ST cell shows increased external and/or Half-life in vivo compared with derived from no molecules of interest of identical parental cell line or the molecules of interest derived from other cell lines by any transgeneic procedure.

(b) the genetically modified host cell system containing fucosyltransferase

The cDNA, such as FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, FUT10, FUT11 of fucosyltransferase (FT) are encoded, is expanded by PCR by poly (A)-primed cDNA.PCR primer is connected in suitable carrier, such as pIRESpuro4 or pCEP4, to produce alpha2,6ST plasmid.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, it is 1992) 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. (Glycobiology15 (1)：87th, 2005) it is described in detail.Selectively, 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 Slex structure.

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 the mRNA profile different with the cell factor or the profile of receptor-inducible that are produced by separate sources, such as E.coli, yeast or Chinese hamster ovary celI.

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 37 degree are cultivated 4 hours, 12 hours etc.).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

(clinic) is tested in vivo

(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) with recombinant human erythropoietin (rhEPO) and recombinant human interferon alpha 2-beta (rhIFN-B) composition treatment multiple sclerosis

The individual subjects of in vivo studies described here are people and especially, are the people for showing the acute Demyelinating Condition consistent with multiple sclerosis.Clinical treatment is strictly controlled to ensure that individual will not be placed in unnecessary risk and they are fully informed their effects in test.

It is preferred that, in order to compensate the psychological impact for receiving treatment, treatment is carried out under double-blind fashion.Volunteer is assigned randomly to recombinant human interferon alpha 2 B (rhIFN-B) or recombinant human erythropoietin (rhEPO)+rhIFN-B treatment groups.Further, in order to prevent doctor from there is prejudice in the treatment, it is IFN-B or rhEPO+rhIFN-B that they, which are not apprised of the medicine being administered on them,.Such random fashion is used, there is each volunteer identical chance to be given new treatment or control.

Volunteer receives one section of the injection suitable time of rhIFN-B or rhEPO+rhIFN-B treatments, starting (base line measurement before any treatment) in combination with the morbid state or the biological parameter of health status shown, terminate (after final treatment), and be measured during experiment in the interval of rule.This measurement includes the level of the body fluid compared with level before treatment, tissue or the IFN-B in organ and EPO.Other measurements include, but it is not limited to, the index of morbid state or the health status for the treatment of, such as multiple sclerosis function combination score (MSFC) body weight, blood pressure, the serum titer or toxicity and ADME of anti-IFN beta neutralizing antibodies (NAbs) (absorb, distribution, metabolism and excretion) measurement.

Record each patient information include the age (year), sex, the species of the family history (Yes/No) of high (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 rhINF-B and rhEPO+rhIFN-B treatments.General, in off-test, the volunteer treated with rhEPO+rhIFN-B shows that their morbid state or the index of disease are intended to the positive, with only compared with the volunteer that rhIFN-B is treated.

(c)-stent in the treatment coronary artery stenosis (CAS) is coated with VEGF

The individual subjects of in vivo studies described here are warm-blooded vertebrates, and it includes people.Especially, supplement has patient with sympotoms with coronary artery stenosis.

Volunteer passes through inner support coronary angioplasty (CAS), and one kind operation includes conduit or pipe is inserted into inguinal artery, and then the artery of conduit through body is reached to the plaque location in heart in coronary artery.Balloon artery is used, then Stent Implantation is to cover patch and keep artery open.Clinical test is strictly controlled to ensure that individual is not placed into unnecessary risk, and they are fully informed their effects in test.It is preferred that, in order to compensate the psychological impact for receiving special treatment, experiment is carried out under double-blind fashion.Volunteer is assigned to " non-coating stent " or VEGF- coating stents " treatment group.Support used in latter treatment group uses the preparation of the VEGF-165 containing the present invention to be coated with techniques known in the art.In order to prevent doctor from there is prejudice in the treatment, it is " non-coating stent " or VEGF- coating stents that they, which are not apprised of the treatment carried out on them, " operation.This random fashion is used, there is each volunteer identical chance to receive existing or new treatment.

The biological parameter relevant with coronary artery stenosis is starting (base line measurement before any treatment), terminates (after final treatment), and be measured during experiment in the interval of rule.Such measurement includes, but it is not limited to, Magnetic resonance imaging (MRI) for identifying coronary flow feature, body weight, blood pressure, the serum titer of the pharmacology indicator of disease, such as special heart disease indicator or toxicity and ADME (absorb, distribution, metabolism and excretion) measurement.Record each patient information include the age (year), sex, the species of the family history (Yes/No) of high (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 research masculinity and femininity number approximate equality.Volunteer with some features is evenly distributed for the CAS treatments with not coated support and the CAS treatments with the coated supports of VEGF-.General, in off-test, the volunteer through the CAS treatments with VEGF- coating stents shows that impaired flow characteristic coronarius is intended to the positive, compared with through the volunteer with the CAS treatments of not coated support.

Embodiment 12

(a) comparison of EPO of the invention biological activity and the EPO expressed with non-human system biological activity

TF-1 cells are used for the biological activity for comparing EPO of the invention and the people EPO expressed with non-human system.

TF-1 cells are placed in the hole of 96 hole tissue culturing plates, with the concentration of 10000 cells in 100 μ l culture mediums.Cell 0-1000ng/ml EPO processing of the invention, and cultivated 3 days at 37 DEG C.Then with MTS analytic approach measurement cell quantity.

In order to which MTS is analyzed, the mono- Solution Cell Proliferation analyses (Promega) of CellTiter96 Aqueous are used.In this analysis, the tetrazolium salt compound MTS present in electronics coupled reagent (phenazine methosulfate) (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) formazan product is reduced to by cell biological.The concentration of formazan reads absorbance measurement of the generation solution in 490nm by using spectrophotometer (E Maxprecision microplate reader, Molecular Devices).

Above-mentioned analysis is used in E.coli systems (Cat286-EP；R＆ D Systems) the middle people EPO repetitions expressed.

ED50s is calculated respectively after absorbance curve is fitted and obtains EPO concentration with 4- parametric fit equations.

The EPO of the present invention for 0.002-0.005ng/ml (Fig. 2), compared (0.035-0.036ng/ml with R＆D systems EPO ED50；Fig. 2).Therefore, EPO of the invention shows the proliferation activity (Fig. 2) of high 7-18 times of the R＆D system people EPO than being expressed in E.coli.

(b) biological activity of Flt3- part of the biological activity of Flt3- parts of the invention with being expressed with non-human system is compared

Flt3- parts have been reported in proliferative induction in the pre B cell system of Flt3 transfections.In 96 orifice plates, the Flt3- parts of pre B cell/hole various concentrations of 10000Flt3 transfections are handled 72 hours at 37 DEG C.Then cell quantity is analyzed with MTS or determined by flow cytometry.

In order to which MTS is analyzed, the mono- Solution Cell Proliferation analyses (Promega) of CellTiter96 Aqueous are used.In this analysis, it is present in tetrazolium salt compound MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium in electronics coupled reagent (phenazine methosulfate)) formazan product is reduced to by cell biological.Formazan concentration reads the absorbance measurement of generation solution by using spectrophotometer (E Maxprecision microplate reader, Molecular Devices) under 490nm.

For flow cytometry, living cells FACScan flow cytometries (BectonDickinson Immunocytometry Systems, San Jose, CA) disclosed method (Dedov et al.Apoptosis 8：399-406,2003) counting.Data analysis is carried out with CellQuest Software.

ED50 is calculated after absorbance curve is fitted and obtains Flt3- ligand concentration values with 4- parametric equations.

The Flt3- parts of above-mentioned analysis non-human cell's system, such as E.coli, yeast or expressing cho cell repeat and find that the ED50s of difference is significantly different.

(c) Flt3-Fc of the Flt3-Fc of the invention biological activity with being expressed with non-human system biological activity is compared

Flt3-Fc, which has been reported, suppresses the propagation that the rhFlt-3 parts for the pre B cell system that Flt-3 is transfected are induced.Flt3-Fc combination Flt3- parts, and therefore, it is possible to the combination of Reverse transcriptase Flt3- parts and cell Flt3 acceptor sites, show Flt3- part bioinactivations.

The pre B cell of Flt-3 transfections is placed in the hole of 96 hole tissue culturing plates, with the concentration of the cell of 1ml culture mediums 10000.The preculture 2ng/ml Flt3- part -0-1000ng/ml Flt3-Fc mixtures processing of cell various concentrations, then, is cultivated 72 hours at 37 DEG C.Then, cell quantity is analyzed with MTS or flow cytometry is determined.

In order to which MTS is analyzed, the mono- Solution Cell Proliferation analyses (Promega) of CellTiter96 Aqueous are used.In this analysis, it is present in tetrazolium salt compound MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium in electronics coupled reagent (phenazine methosulfate)) formazan product is reduced to by cell biological.Formazan concentration reads the absorbance measurement of generation solution by using spectrophotometer (E Maxprecision microplate reader, Molecular Devices) under 490nm.

For flow cytometry, living cells FACScan flow cytometers (BectonDickinson Immunocytometry Systems, San Jose, CA) disclosed method (Dedov et al.Apoptosis 8：399-406,2003) counting.Data analysis is carried out with CellQuest Software.

ND50s is calculated after absorbance curve is fitted and obtains Flt3-Fc concentration values with 4- parametric equations.

The Flt3-Fc of above-mentioned analysis non-human cell's system, such as E.coli, yeast or expressing cho cell is repeated and is found that the ED50s of difference is significantly different.

(d) PDGF-B biological activity and the PDGF-B expressed with non-human system biological activity of the invention compare

PDGF-B, which has been reported, suppresses NR6R-3T3 fibroblasts and Human umbilical vein endothelial cells (HUVEC) propagation.In 96 orifice plates, the PDGF-B of 10000 NR6R-3T3 cells/well various concentrations is handled 72 hours at 37 DEG C.Selectively, HUVEC cells are placed in the hole of 96 hole tissue culturing plates, with the concentration of the cell of 100 μ l culture mediums 3000.The PDGF-B of cell various concentrations is handled 72 hours at 37 DEG C.Then, cell quantity is determined with MTS analyses.

In order to which MTS is analyzed, the mono- Solution Cell Proliferation analyses (Promega) of the Aqueous of CellTiter 96 are used.In this analysis, it is present in tetrazolium salt compound MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium in electronics coupled reagent (phenazine methosulfate)) formazan product is reduced to by cell biological.Formazan concentration reads the absorbance measurement of generation solution by using spectrophotometer (E Maxprecision microplate reader, Molecular Devices) under 490nm.

ED50 is calculated after absorbance curve is fitted and obtains PDGF-B concentration values with 4- parametric equations.

The PDGF-B of above-mentioned analysis non-human cell's system, such as E.coli, yeast or expressing cho cell is repeated and is found that the ED50s of difference is significantly different.

(e) VEGF-165 biological activity and the VEGF-165 expressed with non-human system biological activity of the invention compare

Human umbilical vein endothelial cells (HUVEC) are used for the biological activity for the people VEGF-165 for comparing VEGF-165 of the invention and being expressed with non-human system.

HUVEC cells are placed in the hole of 96 hole tissue culturing plates, with the concentration of every cell of 100 μ l culture mediums 3000.Cell is handled with 0-10ng/ml VEGF-165 of the invention and cultivated 3 days at 37 DEG C.Then cell quantity is determined with MTS analyses.

In order to which MTS is analyzed, the mono- Solution Cell Proliferation analyses (Promega) of the Aqueous of CellTiter 96 are used.In this analysis, it is present in tetrazolium salt compound MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium in electronics coupled reagent (phenazine methosulfate)) formazan product is reduced to by cell biological.Formazan concentration reads the absorbance measurement of generation solution by using spectrophotometer (E Maxprecision microplate reader, Molecular Devices) under 490nm.

The people VEGF-165 that above-mentioned analysis uses the E.coli from PeproTech (Cat.#100-20) to express is repeated.

ED50s is calculated after absorbance curve is fitted and obtains VEGF-165 concentration values with 4- parametric equations.

The VEGF-165 of present invention ED50 is 0.18-0.26ng/ml (Fig. 3), the ED50 (1.8-2.7ng/ml with PeproTechVEGF-165；Fig. 3) compare.Therefore, VEGF-165 of the invention shows the proliferation activity (Fig. 3) of high 10 times of the R＆D systems than being expressed in E.coli.

Embodiment 13

(a) the internal of immunoreactivity feature is compared between the people EPO that EPO of the invention is expressed with non-human system

The EPO of present invention protein evaluation Bradford protein analyses (Bradford, M.Anal Biochem 72：248-254,1976) measure.

The EPO of the present invention, it is normalized with Bradford analysis results, it is diluted and in R＆D system people EPO Quantikine

Examined in ELISA kit (Cat.#DEP00), according to the technical specification of manufacturer.The people EPO that the ELISA kit that foregoing is directed to is expressed with Chinese hamster ovary (CHO) is as standard.

R＆D system people EPO Quantikine

The concentration evaluation that the result of ELISA kit provides the EPO of the present invention is about 335pg/ml (Fig. 4) under 0.663 OD450nm, when the standard curve from the CHO- people EPO expressed is assessed.However, concentration of the making up the difference actual EPO of the present invention is about 1338pg/ml, in similar OD450nm is assessed (Fig. 4).

As a result represent to pass through R＆D system people EPO Quantikine

The EPO concentration of the invention that ELISA kit is obtained about underestimates 4 times, a kind of commercial reagents box, the antibody of the people EPO standards expressed with CHO- and the people EPO of anti-CHO- expression, it is used for the level for assessing the EPO that natural human is expressed in laboratory sample and people patient sample.

This result shows the different immunoreactivity features of the EPO of the present invention and people's EPO molecules of non-human cell's expression.

(b) the external of immune response feature is compared between Flt3- parts of the invention and the Flt3- parts expressed with non-human system

The protein evaluation standard protein analytical technology of the Flt3- parts of the present invention, such as Bradford protein analyses (Bradford Anal Biochem 72：248-254,1976) measure.

The Flt3- parts of the present invention, it is normalized with standard protein analysis result, it is diluted and inspection, R＆D system people's Flt3- parts DuoSet in business can acquire ELISA kit

ELISA kit (Cat#DY308), according to the technical specification of manufacturer.The ELISA kit that foregoing is directed to is used in the people's Flt3- parts expressed in mouse NSO cells as standard.

As a result show, R＆D system people's Flt3- parts DuoSet

ELISA kit inaccurately have rated the concentration of the Flt3- parts of the present invention under OD450nm, when comparing with the corresponding Flt3- ligand concentrations of the present invention for analyzing measure by standard protein.

On structure level, such result represents the Flt3- parts of the present invention and the different immunoreactivity features of people's Flt3- ligand moleculars that non-human cell expresses.

(c) the external of immunoreactivity feature is compared between Flt3 of the invention and the people Flt3 or chimera Flt3 molecules expressed with non-human system

The Flt3 of present invention protein evaluation standard method, such as Bradford protein analyses (Bradford 1976 is as above) are determined.

The Flt3 of the present invention, it is normalized with standard protein analysis result, it is diluted and in the suitable of the Flt3 or chimera Flt3 standards expressed supplemented with non-human cell, commercially available Flt3 quantitative immunologicals are determined in operation and examined, such as anti-Flt3 ELISA kits, are used according to the technical specification of manufacturer.Selectively, the quantitative immunoassay procedures for obtaining component progress originated from commercially available are used for the level for identifying the Flt3 of the present invention.For example, anti- Flt3-FcELISA is developed, employment Flt3 Mab (R＆D Systems Cat#MAB812) are as capture antibody, and as detection antibody and in mouse NSO cells (R＆D Systems Cat#368-ST-050/CF), the recombined human Flt3-Fc of expression is used as protein standards to biotinylated people Flt3 Pab (R＆D Systems Cat#BAF812).The Flt3 of present invention protein concentration, it is normalized with standard protein analysis result, analyzed with the reagent that foregoing is directed to ELISA method known in the art.

It is different from what is determined by standard protein analysis method by commercially available ELISA kit or by using the Flt3 protein concentrations of the invention of the quantitative immunological measure of the component progress in source, when the capture and/or detection antibody that are used in commercially available ELISA kit or immunoassay procedures show the people Flt3 or chimera Flt3 protein that anti-non-human cell expresses.

On structure level, such result represents that the Flt3 of present invention immune response feature is different from the people Flt3 or chimera Flt3 molecules that non-human cell expresses.

(d) the external of immunoreactivity feature is compared between PDGF-B of the invention and the PDGF-BB expressed with non-human system

The PDGF-B of present invention protein evaluation is determined with standard protein analytical technology, for example, Bradford protein analyses (Bradford 1976 is as above).

The PDGF-B of the present invention, analyzes normalized with standard protein, is diluted and is examined in commercially available ELISA kit, for example, R＆D system people PDGF-BB Quantikine

ELISA kit (Cat#DBB00), according to the technical specification of manufacturer.The ELISA kit that foregoing is directed to uses the people PDGF-BB expressed in E.coli to be standard.

As a result show, R＆D system people PDGF-BB QuantikineELISA kit inaccurately evaluates the PDGF-B of present invention concentration under OD450nm, when comparing with the corresponding PDGF-B concentration of the present invention analyzed and identified as mentioned by standard protein.

On structure level, such result can represent that the PDGF-B of present invention immunoreactivity feature is different from people's PDGF-BB molecules that non-human cell expresses.

(e) the external of immunoreactivity feature is compared between VEGF-165 of the invention and the people VEGF-165 expressed with non-human system

The VEGF-165 of present invention protein evaluation is used to be determined selected from following technology：Bradford protein analyses (Bradford 1976 is as above), Lowry protein analyses, A280 spectrophotometric analysis, anti-VEGF elisa assay.

The VEGF-165 of the present invention, it is normalized with the standard protein evaluation analysis that foregoing is directed to, it is diluted and R＆D system people VEGF DuoSet

ELISA kit (Cat.#DY293) is examined according to the technical specification of manufacturer.The ELISA kit that foregoing is directed to uses the people VEGF-165 expressed in E.coli to be standard.

R＆D system people VEGF DuoSet

ELISA kit result gives the VEGF-165 of present invention concentration evaluation, about 2000pg/ml under about 0.15 OD450nm (Fig. 5), when assessing from people's VEGF-165 standard curves that E.coli is expressed.However, under similar OD450nm assessments, VEGF-165 of the invention actual concentration of making up the difference is about 500pg/ml (Fig. 5).

These results show that the VEGF-165 of the present invention about assesses 4 times by too high, pass through R＆D system people VEGF DuoSet

ELISA kit, a kind of commercial reagents box of people's VEGF-165 protein standards of use E.coli expression and the antibody for the people VEGF-165 for showing anti-E.coli expression.

The result shows that the VEGF-165 of present invention immunoreactivity expresses the difference of people's VEGF-165 molecules with non-human cell.

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.Selectively, the solution insulin containing protein sample digests in ammonium bicarbonate buffers (10-25mM, pH7.5-9).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/z400-2000,1.0s) 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/z50-2000).

LC/MS/MS data are searched for Mascot (Matrix Science, London, UK) and Protein Lynx 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.Selectively, 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, the spleen cell cultures supernatant cultivated together with the protein of the protein of the present invention or 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 Quantikine of R＆D systems

ELISA 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 × 10⁵CD4⁺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, Peptide Cutter (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.

Selectively, 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 albumen in the present invention is prepared from the genome of restructuring or the cDNA carriers of alternative splicing

From including SEQ ID NO：The gene order that epo protein in the coding present invention is selected in 134,135,136 or 137 enumerate is expanded by PCR, and is cloned into suitable expression vector, such as：pIRESbleo3、pCMV-SPORT6、pUMCV3、pORF、pORF9、pcDNA3.1/GS、pCEP4、pIRESpuro3、pIRESpuro4、pcDNA3.1/Hygro(+)、pcDNA3.1/Hygro(-)、pEF6/V5-His.Selectively, cDNA alternative splicing transcripts are obtained from people's cell or tissue, and are cloned into above-mentioned appropriate expression vector.Described in embodiment 1 (c), these recombinant precursors are prepared, for transfected human cells.Described in embodiment 2, the albumen of the invention from recombinant dna construct is purified.

Embodiment 17

Compare it is of the invention in VEGF-165 and non-human system expression VEGF-165 in vivo bioactivity

Known VEGF is a kind of effective vascular permeability derivant.Miles vascular permeabilities analysis (Wise et al.PNAS96 are carried out to the guinea pig of anesthesia using the method delivered：3071-3076、1999).First, to experimental animal the μ l 0.5%Evans blue dye of injection of heart 500 PBS, dye liquor is entered blood circulation.Then, the VEGF-165 in the present invention for specifying consumption is subcutaneously injected in the region of the back shaving hair removal hair of experimental animal (0-100ng albumen is dissolved in the suitable buffer solutions of 100-150 μ l).After 20-30 minutes, animal is put to death, the skin of injection areas is removed.The skin removed at room temperature with formamide culture 3-4 days, finally detects absorbance of the resulting solution at 620nm with spectrophotometer, and the amount to the dye liquor of leaching is measured.

Above-mentioned experiment is repeated with the VEGF-165 of non-human system expression, the VEGF-165 of such as Bacillus coli cells expression.

As a result show, the VEGF-165 in the present invention can induce stronger vascular permeability (being analyzed as mentioned by Miles vascular permeabilities) relative to the VEGF-165 of non-human system expression.

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 component and compound and any step or feature described in two or more are optionally combined.

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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.)

Boehm, Ingrid (only to the U.S.)

Domagala, Teresa A (only to the U.S.)

Lee, Carol M Y (only to the U.S.)

Simpson, Raina J (only to the U.S.)

Pilkington, Glenn R (only to the U.S.)

Liddell, Catherine A (only to the U.S.)

<120>Molecule and its chimeric molecule

<130>12683720/EJH/HPM

<160>137

<170>PatentIn version3.1

<210>1

<211>714

<212>DNA

<213>People (human)

<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 tctcccggac 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 ProIle 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 Pro 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 Hi s 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

<211>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 Scr 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 Ash 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>28

<212>DNA

<213>Artificial sequence

<400>25

agctcggata tcaccatggg ggtgcacg                                         28

<210>26

<211>28

<212>DNA

<213>Artificial sequence

<400>26

gtgggatcca agctcgcctc atctgtcc                                         28

<210>27

<211>81

<212>DNA

<213>People

<400>27

atgggggtgc acgaatgtcc tgcctggctg tggcttctcc tgtccctgct gtcgctccct      60

ctgggcctcc cagtcctggg c                                                81

<210>28

<211>27

<212>PRT

<213>People

<400>28

Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu

1               5                   10                  15

Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly

            20                  25

<210>29

<211>498

<212>DNA

<213>People

<400>29

gccccaccac gcctcatctg tgacagccga gtcctggaga ggtacctctt ggaggccaag    60

gaggccgaga atatcacgac gggctgtgct gaacactgca gcttgaatga gaatatcact    120

gtcccagaca ccaaagttaa tttctatgcc tggaagagga tggaggtcgg gcagcaggcc    180

gtagaagtct ggcagggcct ggccctgctg tcggaagctg tcctgcgggg ccaggccctg    240

ttggtcaact cttcccagcc gtgggagccc ctgcagctgc atgtggataa agccgtcagt    300

ggccttcgca gcctcaccac tctgcttcgg gctctgcgag cccagaagga agccatctcc    360

cctccagatg cggcctcagc tgctccactc cgaacaatca ctgctgacac tttccgcaaa    420

ctcttccgag tctactccaa tttcctccgg ggaaagctga agctgtacac aggggaggcc    480

tgcaggacag gggacaga                                                  498

<210>30

<211>166

<212>PRT

<213>People

<400>30

Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu

1               5                   10                  15

Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His

            20                  25                  30

Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe

        35                  40                  45

Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp

    50                  55                  60

Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu

65                  70                  75                  80

Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp

                85                  90                  95

Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu

            100                 105                 110

Arg Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala

        115                 120                 125

Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val

    130                 135                 140

Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala

145                 150                 155                 160

Cys Arg Thr Gly Asp Arg

                165

<210>31

<211>498

<212>DNA

<213>Artificial sequence

<400>31

gccccaccac gcctcatctg tgacagccga gtcctggaga ggtacctctt ggaggccaag  60

gaggccgaga atatcacgac gggctgtgct gaacactgca gcttgaatga gaatatcact  120

gtcccagaca ccaaagttaa tttctatgcc tggaagagga tggaggtcgg gcagcaggcc  180

gtagaagtct ggcagggcct ggccctgctg tcggaagctg tcctgcgggg ccaggccctg  240

ttggtcaact cttcccagcc gtgggagccc ctgcagctgc atgtggataa agccgtcagt  300

ggccttcgca gcctcaccac tctgcttcgg gctctgggag cccaggagga agccatctcc  360

cctccagatg cggcctcagc tgctccactc cgaacaatca ctgctgacac tttccgcaaa  420

ctcttccgag tctactccaa tttcctccgg ggaaagctga agctgtacac aggggaggcc  480

tgcaggacag gggacaga                                                498

<210>32

<211>166

<212>PRT

<213>Artificial sequence

<400>32

Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu

1               5                   10                  15

Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His

            20                  25                  30

Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe

        35                  40                  45

Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp

    50                  55                  60

Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu

65                  70                  75                  80

Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp

                85                  90                  95

Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu

            100                 105                 110

Gly Ala Gln Glu Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala

        115                 120                 125

Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val

    130                 135                 140

Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala

145                 150                 155                 160

Cys Arg Thr Gly Asp Arg

                165

<210>33

<211>579

<212>DNA

<213>Artificial sequence

<400>33

atgggggtgc acgaatgtcc tgcctggctg tggcttctcc tgtccctgct gtcgctccct  60

ctgggcctcc cagtcctggg cgccccacca cgcctcatct gtgacagccg agtcctggag  120

aggtacctct tggaggccaa ggaggccgag aatatcacga cgggctgtgc tgaacactgc  180

agcttgaatg agaatatcac tgtcccagac accaaagtta atttctatgc ctggaagagg  240

atggaggtcg ggcagcaggc cgtagaagtc tggcagggcc tggccctgct gtcggaagct  300

gtcctgcggg gccaggccct gttggtcaac tcttcccagc cgtgggagcc cctgcagctg  360

catgtggata aagccgtcag tggccttcgc agcctcacca ctctgcttcg ggctctgcga  420

gcccagaagg aagccatctc ccctccagat gcggcctcag ctgctccact ccgaacaatc  480

actgctgaca ctttccgcaa actcttccga gtctactcca atttcctccg gggaaagctg  540

aagctgtaca caggggaggc ctgcaggaca ggggacaga                         579

<210>34

<211>193

<212>PRT

<213>Artificial sequence

<400>34

Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu

1               5                   10                  15

Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu

            20                  25                  30

Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu

        35                  40                  45

Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu

    50                  55                  60

Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg

65                  70                  75                  80

Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu

                85                  90                  95

Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser

            100                 105                 110

Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly

        115                 120                 125

Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Arg Ala Gln Lys Glu

    130                 135                 140

Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile

145                 150                 155                 160

Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu

                165                 170                 175

Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp

            180                 185                 190

Arg

<210>35

<211>579

<212>DNA

<213>Artificial sequence

<400>35

atgggggtgc acgaatgtcc tgcctggctg tggcttctcc tgtccctgct gtcgctccct    60

ctgggcctcc cagtcctggg cgccccacca cgcctcatct gtgacggccg agtcctggag    120

aggtacctct tggaggccaa ggaggccgag aatatcacga cgggctgtgc tgaacactgc    180

agcttgaatg agaatatcac tgtcccagac accaaagtta atttctatgc ctggaagagg    240

atggaggtcg ggcagcaggc cgtagaagtc tggcagggcc tggccctgct gtcggaagct    300

gtcctgcggg gccaggccct gttggtcaac tcttcccagc cgtgggagcc cctgcagctg    360

catgtggata aagccgtcag tggccttcgc agcctcacca ctctgcttcg ggctctggga    420

gcccaggagg aagccatctc ccctccagat gcggcctcag ctgctccact ccgaacaatc    480

actgctgaca ctttccgcaa actcttccga gtctactcca atttcctccg gggaaagctg    540

aagctgtaca caggggaggc ctgcaggaca ggggacaga                           579

<210>36

<211>193

<212>PRT

<213>Artificial sequence

<400>36

Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu

1               5                   10                  15

Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu

            20                  25                  30

Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu

        35                  40                  45

Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu

    50                  55                  60

Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg

65                  70                  75                  80

Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu

                85                  90                  95

Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser

            100                 105                 110

Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly

        115                 120                 125

Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Glu Glu

    130                 135                 140

Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile

145                 150                 155                 160

Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu

                165                 170                 175

Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp

            180                 185                 190

Arg

<2L0>37

<211>1308

<212>DNA

<213>Artificial sequence

<400>37

atgggggtgc acgaatgtcc tgcctggctg tggcttctcc tgtccctgct gtcgctccct    60

ctgggcctcc cagtcctggg cgccccacca cgcctcatct gtgacagccg agtcctggag    120

aggtacctct tggaggccaa ggaggccgag aatatcacga cgggctgtgc tgaacactgc    180

agcttgaatg agaatatcac tgtcccagac accaaagtta atttctatgc ctggaagagg    240

atggaggtcg ggcagcaggc cgtagaagtc tggcagggcc tggccctgct gtcggaagct    300

gtcctgcggg gccaggccct gttggtcaac tcttcccagc cgtgggagcc cctgcagctg    360

catgtggata aagccgtcag tggccttcgc agcctcacca ctctgcttcg ggctctgcga    420

gcccagaagg aagccatctc ccctccagat gcggcctcag ctgctccact ccgaacaatc    480

actgctgaca ctttccgcaa actcttccga gtctactcca atttcctccg gggaaagctg    540

aagctgtaca caggggaggc ctgcaggaca ggggacagag gatccagcaa caccaaggtg    600

gacaagaaag ttgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca    660

cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc    720

atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct    780

gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg    840

cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag    900

gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc    960

atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg    1020

cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc    1080

ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac    1140

aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc    1200

gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct    1260

ctgcacaacc actacacgca gaagagcctc tccctgtctc  cgggtaaa                1308

<210>38

<211>436

<212>PRT

<213>Artificial sequence

<400>38

Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu

1               5                   10                  15

Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu

            20                  25                  30

Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu

        35                  40                  45

Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu

    50                  55                  60

Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg

65                  70                  75                  80

Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu

                85                  90                  95

Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser

            100                 105                 110

Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly

        115                 120                 125

Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Arg Ala Gln Lys Glu

    130                 135                 140

Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile

145                 150                 155                 160

Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu

                165                 170                 175

Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp

            180                 185                 190

Arg Gly Ser Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

        195                 200                 205

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

    210                 215                 220

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

225                 230                 235                 240

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

                245                 250                 255

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

            260                 265                 270

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

        275                 280                 285

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

    290                 295                 300

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

305                 310                 315                 320

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

                325                 330                 335

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

            340                 345                 350

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

        355                 360                 365

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

    370                 375                 380

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

385                 390                 395                 400

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

                405                 410                 415

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

            420                 425                 430

Ser Pro Gly Lys

        435

<210>39

<211>1308

<212>DNA

<213>Artificial sequence

<400>39

atgggggtgc acgaatgtcc tgcctggctg tggcttctcc tgtccctgct gtcgctccct    60

ctgggcctcc cagtcctggg cgccccacca cgcctcatct gtgacagccg agtcctggag    120

aggtacctct tggaggccaa ggaggccgag aatatcacga cgggctgtgc tgaacactgc    180

agcttgaatg agaatatcac tgtcccagac accaaagtta atttctatgc ctggaagagg    240

atggaggtcg ggcagcaggc cgtagaagtc tggcagggcc tggccctgct gtcggaagct    300

gtcctgcggg gccaggccct gttggtcaac tcttcccagc cgtgggagcc cctgcagctg    360

catgtggata aagccgtcag tggccttcgc agcctcacca ctctgcttcg ggctctggga    420

gcccaggagg aagccatctc ccctccagat gcggcctcag ctgctccact ccgaacaatc    480

actgctgaca ctttccgcaa actcttccga gtctactcca atttcctccg gggaaagctg    540

aagctgtaca caggggaggc ctgcaggaca ggggacagag gatccagcaa caccaaggtg    600

gacaagaaag ttgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca    660

cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc    720

atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct    780

gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg    840

cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag    900

gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc    960

atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg    1020

cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc    1080

ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac    1140

aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc    1200

gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct    1260

ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaa                 1308

<210>40

<211>436

<212>PRT

<213>Artificial sequence

<400>40

Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu

1               5                   10                  15

Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu

            20                  25                  30

Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu

        35                  40                  45

Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu

    50                  55                  60

Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg

65                  70                  75                  80

Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu

                85                  90                  95

Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser

            100                 105                 110

Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly

        115                 120                 125

Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Glu Glu

    130                 135                 140

Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile

145                 150                 155                 160

Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu

                165                 170                 175

Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp

            180                 185                 190

Arg Gly Ser Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

        195                 200                 205

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

    210                 215                 220

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

225                 230                 235                 240

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

                245                 250                 255

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

            260                 265                 270

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

        275                 280                 285

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

    290                 295                 300

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

305                 310                 315                 320

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

                325                 330                 335

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

            340                 345                 350

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

        355                 360                 365

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

    370                 375                 380

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

385                 390                 395                 400

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

                405                 410                 415

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

            420                 425                 430

Ser Pro Gly Lys

        435

<210>41

<211>29

<212>DNA

<213>Artificial sequence

<400>41

gtcgtcgata tcgccaccat gacagtgct                                        29

<210>42

<211>29

<212>DNA

<213>Artificial sequence

<400>42

ggaaaaagat ctggatccgc ggccgctta                                      29

<210>43

<211>78

<212>DNA

<213>People

<400>43

atgacagtgc tggcgccagc ctggagccca acaacctatc tcctcctgct gctgctgctg    60

agctcgggac tcagtggg                                                  78

<210>44

<211>26

<212>PRT

<213>People

<400>44

Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu

1               5                   10                  15

Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly

            20                  25

<210>45

<211>471

<212>DNA

<213>People

<400>45

acccaggact gctccttcca acacagcccc atctcctccg acttcgctgt caaaatccgt    60

gagctgtctg actacctgct tcaagattac ccagtcaccg tggcctccaa cctgcaggac    120

gaggagctct gcgggggcct ctggcggctg gtcctggcac agcgctggat ggagcggctc    180

aagactgtcg ctgggtccaa gatgcaaggc ttgctggagc gcgtgaacac ggagatacac    240

tttgtcacca aatgtgcctt tcagcccccc cccagctgtc ttcgcttcgt ccagaccaac    300

atctcccgcc tcctgcagga gacctccgag cagctggtgg cgctgaagcc ctggatcact    360

cgccagaact tctcccggtg cctggagctg cagtgtcagc ccgactcctc aaccctgcca    420

cccccatgga gtccccggcc cctggaggcc acagccccga cagccccgca g             471

<210>46

<211>157

<212>PRT

<213>People

<400>46

Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala

1               5                   10                  15

Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val

            20                  25                  30

Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp

        35                  40                  45

Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala

    50                  55                  60

Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His

65                  70                  75                  80

Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe

                85                  90                  95

Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu

            100                 105                 110

Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu

        115                 120                 125

Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser

    130                 135                 140

Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln

145                 150                 155

<210>47

<211>474

<212>DNA

<213>Artificial sequence

<400>47

acccaggact gctccttcca acacagcccc atctcctccg acttcgctgt caaaatccgt  60

gagctgtctg actacctgct tcaagattac ccagtcaccg tggcctccaa cctgcaggac  120

gaggagctct gcgggggcct ctggcggctg gtcctggcac agcgctggat ggagcggctc  180

aagactgtcg ctgggtccaa gatgcaaggc ttgctggagc gcgtgaacac ggagatacac  240

tttgtcacca aatgtgcctt tcagcccccc cccagctgtc ttcgcttcgt ccagaccaac  300

atctcccgcc tcctgcagga gacctccgag cagctggtgg cgctgaagcc ctggatcact  360

cgccagaact tctcccggtg cctggagctg cagtgtcagc ccgactcctc aaccctgcca  420

cccccatgga gtccccggcc cctggaggcc acagccccga cagccccggc tagc        474

<210>48

<211>158

<212>PRT

<213>Artificial sequence

<400>48

Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala

1               5                   10                  15

Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val

            20                  25                  30

Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp

        35                  40                  45

Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala

    50                  55                  60

Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His

65                  70                  75                  80

Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe

                85                  90                  95

Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu

            100                 105                 110

Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu

        115                 120                 125

Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser

    130                 135                 140

Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Ala Ser

145                 150                 155

<210>49

<211>549

<212>DNA

<213>Artificial sequence

<400>49

atgacagtgc tggcgccagc ctggagccca acaacctatc tcctcctgct gctgctgctg  60

agctcgggac tcagtgggac ccaggactgc tccttccaac acagccccat ctcctccgac  120

ttcgctgtca aaatccgtga gctgtctgac tacctgcttc aagattaccc agtcaccgtg  180

gcctccaacc tgcaggacga ggagctctgc gggggcctct ggcggctggt cctggcacag  240

cgctggatgg agcggctcaa gactgtcgct gggtccaaga tgcaaggctt gctggagcgc  300

gtgaacacgg agatacactt tgtcaccaaa tgtgcctttc agcccccccc cagctgtctt  360

cgcttcgtcc agaccaacat ctcccgcctc ctgcaggaga cctccgagca gctggtggcg  420

ctgaagccct ggatcactcg ccagaacttc tcccggtgcc tggagctgca gtgtcagccc  480

gactcctcaa ccctgccacc cccatggagt ccccggcccc tggaggccac agccccgaca  540

gccccgcag                                                          549

<210>50

<211>183

<212>PRT

<213>Artificial sequence

<400>50

Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu

1               5                   10                  15

Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr Gln Asp Cys Ser Phe

            20                  25                  30

Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu

        35                  40                  45

Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu

    50                  55                  60

Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln

65                  70                  75                  80

Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly

                85                  90                  95

Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala

            100                 105                 110

Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser

        115                 120                 125

Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp

    130                 135                 140

Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro

145                 150                 155                 160

Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala

                165                 170                 175

Thr Ala Pro Thr Ala Pro Gln

            180

<210>51

<211>552

<212>DNA

<213>Artificial sequence

<400>51

atgacagtgc tggcgccagc ctggagccca acaacctatc tcctcctgct gctgctgctg  60

agctcgggac tcagtgggac ccaggactgc tccttccaac acagccccat ctcctccgac  120

ttcgctgtca aaatccgtga gctgtctgac tacctgcttc aagattaccc agtcaccgtg  180

gcctccaacc tgcaggacga ggagctctgc gggggcctct ggcggctggt cctggcacag  240

cgctggatgg agcggctcaa gactgtcgct gggtccaaga tgcaaggctt gctggagcgc  300

gtgaacacgg agatacactt tgtcaccaaa tgtgcctttc agcccccccc cagctgtctt  360

cgcttcgtcc agaccaacat ctcccgcctc ctgcaggaga cctccgagca gctggtggcg  420

ctgaagccct ggatcactcg ccagaacttc tcccggtgcc tggagctgca gtgtcagccc  480

gactcctcaa ccctgccacc cccatggagt ccccggcccc tggaggccac agccccgaca  540

gccccggcta gc                                                      552

<210>52

<211>184

<212>PRT

<213>Artificial sequence

<400>52

Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu

1               5                   10                  15

Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr Gln Asp Cys Ser Phe

            20                  25                  30

Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu

        35                  40                  45

Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu

    50                  55                  60

Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln

65                  70                  75                  80

Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly

                85                  90                  95

Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala

            100                 105                 110

Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser

        115                 120                 125

Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp

    130                 135                 140

Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro

145                 150                 155                 160

Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala

                165                 170                 175

Thr Ala Pro Thr Ala Pro Ala Ser

            180

<210>53

<211>1278

<212>DNA

<213>Artificial sequence

<400>53

atgacagtgc tggcgccagc ctggagccca acaacctatc tcctcctgct gctgctgctg  60

agctcgggac tcagtgggac ccaggactgc tccttccaac acagccccat ctcctccgac    120

ttcgctgtca aaatccgtga gctgtctgac tacctgcttc aagattaccc agtcaccgtg    180

gcctccaacc tgcaggacga ggagctctgc gggggcctct ggcggctggt cctggcacag    240

cgctggatgg agcggctcaa gactgtcgct gggtccaaga tgcaaggctt gctggagcgc    300

gtgaacacgg agatacactt tgtcaccaaa tgtgcctttc agcccccccc cagctgtctt    360

cgcttcgtcc agaccaacat ctcccgcctc ctgcaggaga cctccgagca gctggtggcg    420

ctgaagccct ggatcactcg ccagaacttc tcccggtgcc tggagctgca gtgtcagccc    480

gactcctcaa ccctgccacc cccatggagt ccccggcccc tggaggccac agccccgaca    540

gccccgcagg gatccagcaa caccaaggtg gacaagaaag ttgagcccaa atcttgtgac    600

aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc    660

ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc    720

gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc    780

gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt    840

gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc    900

aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg    960

cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac    1020

caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg    1080

gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac    1140

ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac    1200

gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc    1260

tccctgtctc cgggtaaa                                                  1278

<210>54

<211>426

<212>PRT

<213>Artificial sequence

<400>54

Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu

1               5                   10                  15

Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr Gln Asp Cys Ser Phe

            20                  25                  30

Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu

        35                  40                  45

Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu

    50                  55                  60

Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln

65                  70                  75                  80

Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly

                85                  90                  95

Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala

            100                 105                 110

Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser

        115                 120                 125

Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp

    130                 135                 140

Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro

145                 150                 155                 160

Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala

                165                 170                 175

Thr Ala Pro Thr Ala Pro Gln Gly Ser Ser Asn Thr Lys Val Asp Lys

            180                 185                 190

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

        195                 200                 205

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

    210                 215                 220

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

225                 230                 235                 240

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

                245                 250                 255

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

            260                 265                 270

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

        275                 280                 285

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

    290                 295                 300

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

305                 310                 315                 320

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

                325                 330                 335

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

            340                 345                 350

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

        355                 360                 365

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phc

    370                 375                 380

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

385                 390                 395                 400

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

                405                 410                 415

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

            420                 425

<210>55

<211>1281

<212>DNA

<213>Artificial sequence

<400>55

atgacagtgc tggcgccagc ctggagccca acaacctatc tcctcctgct gctgctgctg  60

agctcgggac tcagtgggac ccaggactgc tccttccaac acagccccat ctcctccgac    120

ttcgctgtca aaatccgtga gctgtctgac tacctgcttc aagattaccc agtcaccgtg    180

gcctccaacc tgcaggacga ggagctctgc gggggcctct ggcggctggt cctggcacag    240

cgctggatgg agcggctcaa gactgtcgct gggtccaaga tgcaaggctt gctggagcgc    300

gtgaacacgg agatacactt tgtcaccaaa tgtgcctttc agcccccccc cagctgtctt    360

cgcttcgtcc agaccaacat ctcccgcctc ctgcaggaga cctccgagca gctggtggcg    420

ctgaagccct ggatcactcg ccagaacttc tcccggtgcc tggagctgca gtgtcagccc    480

gactcctcaa ccctgccacc cccatggagt ccccggcccc tggaggccac agccccgaca    540

gccccggcta gcggatccag caacaccaag gtggacaaga aagttgagcc caaatcttgt    600

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc    660

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca    720

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac    780

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac    840

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag    900

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa    960

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag    1020

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag    1080

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc    1140

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg    1200

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc    1260

ctctccctgt ctccgggtaa a                                              1281

<210>56

<211>427

<212>PRT

<213>Artificial sequence

<400>56

Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu

1               5                   10                  15

Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr Gln Asp Cys Ser Phe

            20                  25                  30

Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu

        35                  40                  45

Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu

    50                  55                  60

Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln

65                  70                  75                  80

Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly

                85                  90                  95

Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala

            100                 105                 110

Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser

        115                 120                 125

Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp

    130                 135                 140

Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro

145                 150                 155                 160

Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala

                165                 170                 175

Thr Ala Pro Thr Ala Pro Ala Ser Gly Ser Ser Asn Thr Lys Val Asp

            180                 185                 190

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

        195                 200                 205

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

    210                 215                 220

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

225                 230                 235                 240

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

                245                 250                 255

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

            260                 265                 270

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

        275                 280                 285

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

    290                 295                 300

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

305                 310                 315                 320

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

                325                 330                 335

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

            340                 345                 350

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

        355                 360                 365

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

    370                 375                 380

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

385                 390                 395                 400

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

                405                 410                 415

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

            420                 425

<210>57

<211>34

<212>DNA

<213>Artificial sequence

<400>57

aaaagcggcc gcccggaggc catgccggcg ttgg                                  34

<210>58

<211>30

<212>DNA

<213>Artificial sequence

<400>58

aaaaggatcc tgtcttggat gaaagggaag                                     30

<210>59

<211>69

<212>DNA

<213>People

<400>59

atgccggcgt tggcgcgcga cgcgggcacc gtgccgctgc tcgttgtttt ttctgcaatg    60

atatttggg                                                            69

<210>60

<211>23

<212>PRT

<213>People

<400>60

Met Pro Ala Leu Ala Arg Asp Ala Gly Thr Val Pro Leu Leu Val Val

1                   5               10                  15

Phe Ser Ala Met Ile Phe Gly

            20

<210>61

<211>78

<212>DNA

<213>People

<400>61

atgccggcgt tggcgcgcga cgcgggcacc gtgccgctgc tcgttgtttt ttctgcaatg    60

atatttggga ctattaca                                                  78

<210>62

<211>26

<212>PRT

<213>People

<400>62

Met Pro Ala Leu Ala Arg Asp Ala Gly Thr Val Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr

            20                  25

<210>63

<211>69

<212>DNA

<213>Artificial sequence

<400>63

atgccggcgt tggcgcgcga cggcggccag ctgccgctgc tcgttgtttt ttctgcaatg    60

atatttggg                                                            69

<210>64

<211>23

<212>PRT

<213>Artificial sequence

<400>64

Met Pro Ala Leu Ala Arg Asp Gly Gly Gln Leu Pro Leu Leu Val Val

1               5               10                      15

Phe Ser Ala Met Ile Phe Gly

            20

<210>65

<211>78

<212>DNA

<213>Artificial sequence

<400>65

atgccggcgt tggcgcgcga cggcggccag ctgccgctgc tcgttgtttt ttctgcaatg    60

atatttggga ctattaca                                                  78

<210>66

<211>26

<212>PRT

<213>Artificial sequence

<400>66

Met Pro Ala Leu Ala Arg Asp Gly Gly Gln Leu Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr

            20                  25

<210>67

<211>1551

<212>DNA

<213>People

<400>67

actattacaa atcaagatct gcctgtgatc aagtgtgttt taatcaatca taagaacaat  60

gattcatcag tggggaagtc atcatcatat cccatggtat cagaatcccc ggaagacctc  120

gggtgtgcgt tgagacccca gagctcaggg acagtgtacg aagctgccgc tgtggaagtg  180

gatgtatctg cttccatcac actgcaagtg ctggtcgatg ccccagggaa catttcctgt    240

ctctgggtct ttaagcacag ctccctgaat tgccagccac attttgattt acaaaacaga    300

ggagttgttt ccatggtcat tttgaaaatg acagaaaccc aagctggaga atacctactt    360

tttattcaga gtgaagctac caattacaca atattgttta cagtgagtat aagaaatacc    420

ctgctttaca cattaagaag accttacttt agaaaaatgg aaaaccagga cgccctggtc    480

tgcatatctg agagcgttcc agagccgatc gtggaatggg tgctttgcga ttcacagggg    540

gaaagctgta aagaagaaag tccagctgtt gttaaaaagg aggaaaaagt gcttcatgaa    600

ttatttggga cggacataag gtgctgtgcc agaaatgaac tgggcaggga atgcaccagg    660

ctgttcacaa tagatctaaa tcaaactcct cagaccacat tgccacaatt atttcttaaa    720

gtaggggaac ccttatggat aaggtgcaaa gctgttcatg tgaaccatgg attcgggctc    780

acctgggaat tagaaaacaa agcactcgag gagggcaact actttgagat gagtacctat    840

tcaacaaaca gaactatgat acggattctg tttgcttttg tatcatcagt ggcaagaaac    900

gacaccggat actacacttg ttcctcttca aagcatccca gtcaatcagc tttggttacc    960

atcgtaggaa agggatttat aaatgctacc aattcaagtg aagattatga aattgaccaa    1020

tatgaagagt tttgtttttc tgtcaggttt aaagcctacc cacaaatcag atgtacgtgg    1080

accttctctc gaaaatcatt tccttgtgag caaaagggtc ttgataacgg atacagcata    1140

tccaagtttt gcaatcataa gcaccagcca ggagaatata tattccatgc agaaaatgat    1200

gatgcccaat ttaccaaaat gttcacgctg aatataagaa ggaaacctca agtgctcgca    1260

gaagcatcgg caagtcaggc gtcctgtttc tcggatggat acccattacc atcttggacc    1320

tggaagaagt gttcagacaa gtctcccaac tgcacagaag agatcacaga aggagtctgg    1380

aatagaaagg ctaacagaaa agtgtttgga cagtgggtgt cgagcagtac tctaaacatg    1440

agtgaagcca taaaagggtt cctggtcaag tgctgtgcat acaattccct tggcacatct    1500

tgtgagacga tccttttaaa ctctccaggc cccttccctt tcatccaaga c    1551

<210>68

<211>517

<212>PRT

<213>People

<400>68

Thr Ile Thr Asn Gln Asp Leu Pro Val Ile Lys Cys Val Leu Ile Asn

1               5                   10                  15

His Lys Asn Asn Asp Ser Ser Val Gly Lys Ser Ser Ser Tyr Pro Met

            20                  25                  30

Val Ser Glu Ser Pro Glu Asp Leu Gly Cys Ala Leu Arg Pro Gln Ser

        35                  40                  45

Ser Gly Thr Val Tyr Glu Ala Ala Ala Val Glu Val Asp Val Ser Ala

    50                  55                  60

Ser Ile Thr Leu Gln Val Leu Val Asp Ala Pro Gly Asn Ile Ser Cys

65                  70                  75                  80

Leu Trp Val Phe Lys His Ser Ser Leu Asn Cys Gln Pro His Phe Asp

                85                  90                  95

Leu Gln Asn Arg Gly Val Val Ser Met Val Ile Leu Lys Met Thr Glu

            100                 105                 110

Thr Gln Ala Gly Glu Tyr Leu Leu Phe Ile Gln Ser Glu Ala Thr Asn

        115                 120                 125

Tyr Thr Ile Leu Phe Thr Val Ser Ile Arg Asn Thr Leu Leu Tyr Thr

    130                 135                 140

Leu Arg Arg Pro Tyr Phe Arg Lys Met Glu Asn Gln Asp Ala Leu Val

145                 150                 155                 160

Cys Ile Ser Glu Ser Val Pro Glu Pro Ile Val Glu Trp Val Leu Cys

                165                 170                 175

Asp Ser Gln Gly Glu Ser Cys Lys Glu Glu Ser Pro Ala Val Val Lys

            180                 185                 190

Lys Glu Glu Lys Val Leu His Glu Leu Phe Gly Thr Asp Ile Arg Cys

        195                 200                 205

Cys Ala Arg Asn Glu Leu Gly Arg Glu Cys Thr Arg Leu Phe Thr Ile

    210                 215                 220

Asp Leu Asn Gln Thr Pro Gln Thr Thr Leu Pro Gln Leu Phe Leu Lys

225                 230                 235                 240

Val Gly Glu Pro Leu Trp Ile Arg Cys Lys Ala Val His Val Asn His

                245                 250                 255

Gly Phe Gly Leu Thr Trp Glu Leu Glu Asn Lys Ala Leu Glu Glu Gly

            260                 265                 270

Asn Tyr Phe Glu Met Ser Thr Tyr Ser Thr Asn Arg Thr Met Ile Arg

        275                 280                 285

Ile Leu Phe Ala Phe Val Ser Ser Val Ala Arg Asn Asp Thr Gly Tyr

    290                 295                 300

Tyr Thr Cys Ser Ser Ser Lys His Pro Ser Gln Ser Ala Leu Val Thr

305                 310                 315                 320

Ile Val Gly Lys Gly Phe Ile Asn Ala Thr Asn Ser Ser Glu Asp Tyr

                325                 330                 335

Glu Ile Asp Gln Tyr Glu Glu Phe Cys Phe Ser Val Arg Phe Lys Ala

            340                 345                 350

Tyr Pro Gln Ile Arg Cys Thr Trp Thr Phe Ser Arg Lys Ser Phe Pro

        355                 360                 365

Cys Glu Gln Lys Gly Leu Asp Asn Gly Tyr Ser Ile Ser Lys Phe Cys

    370                 375                 380

Asn His Lys His Gln Pro Gly Glu Tyr Ile Phe His Ala Glu Asn Asp

385                 390                 395                 400

Asp Ala Gln Phe Thr Lys Met Phe Thr Leu Asn Ile Arg Arg Lys Pro

                405                 410                 415

Gln Val Leu Ala Glu Ala Ser Ala Ser Gln Ala Ser Cys Phe Ser Asp

            420                 425                 430

Gly Tyr Pro Leu Pro Ser Trp Thr Trp Lys Lys Cys Ser Asp Lys Ser

        435                 440                 445

Pro Asn Cys Thr Glu Glu Ile Thr Glu Gly Val Trp Asn Arg Lys Ala

    450                 455                 460

Asn Arg Lys Val Phe Gly Gln Trp Val Ser Ser Ser Thr Leu Asn Met

465                 470                 475                 480

Ser Glu Ala Ile Lys Gly Phe Leu Val Lys Cys Cys Ala Tyr Asn Ser

                485                 490                 495

Leu Gly Thr Ser Cys Glu Thr Ile Leu Leu Asn Ser Pro Gly Pro Phe

            500                 505                 510

Pro Phe Ile Gln Asp

        515

<210>69

<211>1542

<212>DNA

<213>People

<400>69

aatcaagatc tgcctgtgat caagtgtgtt ttaatcaatc ataagaacaa tgattcatca    60

gtggggaagt catcatcata tcccatggta tcagaatccc cggaagacct cgggtgtgcg    120

ttgagacccc agagctcagg gacagtgtac gaagctgccg ctgtggaagt ggatgtatct    180

gcttccatca cactgcaagt gctggtcgat gccccaggga acatttcctg tctctgggtc    240

tttaagcaca gctccctgaa ttgccagcca cattttgatt tacaaaacag aggagttgtt    300

tccatggtca ttttgaaaat gacagaaacc caagctggag aatacctact ttttattcag    360

agtgaagcta ccaattacac aatattgttt acagtgagta taagaaatac cctgctttac    420

acattaagaa gaccttactt tagaaaaatg gaaaaccagg acgccctggt ctgcatatct    480

gagagcgttc cagagccgat cgtggaatgg gtgctttgcg attcacaggg ggaaagctgt    540

aaagaagaaa gtccagctgt tgttaaaaag gaggaaaaag tgcttcatga attatttggg    600

acggacataa ggtgctgtgc cagaaatgaa ctgggcaggg aatgcaccag gctgttcaca    660

atagatctaa atcaaactcc tcagaccaca ttgccacaat tatttcttaa agtaggggaa    720

cccttatgga taaggtgcaa agctgttcat gtgaaccatg gattcgggct cacctgggaa    780

ttagaaaaca aagcactcga ggagggcaac tactttgaga tgagtaccta ttcaacaaac    840

agaactatga tacggattct gtttgctttt gtatcatcag tggcaagaaa cgacaccgga    900

tactacactt gttectcttc aaagcatccc agtcaatcag ctttggttac catcgtagga    960

aagggattta taaatgctac caattcaagt gaagattatg aaattgacca atatgaagag    1020

ttttgttttt ctgtcaggtt taaagcctac ccacaaatca gatgtacgtg gaccttctct    1080

cgaaaatcat ttccttgtga gcaaaagggt cttgataacg gatacagcat atccaagttt    1140

tgcaatcata agcaccagcc aggagaatat atattccatg cagaaaatga tgatgcccaa    1200

tttaccaaaa tgttcacgct gaatataaga aggaaacctc aagtgctcgc agaagcatcg    1260

gcaagtcagg cgtcctgttt ctcggatgga tacccattac catcttggac ctggaagaag    1320

tgttcagaca agtctcccaa ctgcacagaa gagatcacag aaggagtctg gaatagaaag    1380

gctaacagaa aagtgtttgg acagtgggtg tcgagcagta ctctaaacat gagtgaagcc    1440

ataaaagggt tcctggtcaa gtgctgtgca tacaattccc ttggcacatc ttgtgagacg    1500

atccttttaa actctccagg ccccttccct ttcatccaag ac                       1542

<210>70

<211>514

<212>PRT

<213>People

<400>70

Asn Gln Asp Leu Pro Val Ile Lys Cys Val Leu Ile Asn His Lys Asn

1               5                   10                  15

Asn Asp Ser Ser Val Gly Lys Ser Ser Ser Tyr Pro Met Val Ser Glu

            20                  25                  30

Ser Pro Glu Asp Leu Gly Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr

        35                  40                  45

Val Tyr Glu Ala Ala Ala Val Glu Val Asp Val Ser Ala Ser Ile Thr

    50                  55                  60

Leu Gln Val Leu Val Asp Ala Pro Gly Asn Ile Ser Cys Leu Trp Val

65                  70                  75                  80

Phe Lys His Ser Ser Leu Asn Cys Gln Pro His Phe Asp Leu Gln Asn

                85                  90                  95

Arg Gly Val Val Ser Met Val Ile Leu Lys Met Thr Glu Thr Gln Ala

            100                 105                 110

Gly Glu Tyr Leu Leu Phe Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile

        115                 120                 125

Leu Phe Thr Val Ser Ile Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg

    130                 135                 140

Pro Tyr Phe Arg Lys Met Glu Asn Gln Asp Ala Leu Val Cys Ile Ser

145                 150                 155                 160

Glu Ser Val Pro Glu Pro Ile Val Glu Trp Val Leu Cys Asp Ser Gln

                165                 170                 175

Gly Glu Ser Cys Lys Glu Glu Ser Pro Ala Val Val Lys Lys Glu Glu

            180                 185                 190

Lys Val Leu His Glu Leu Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg

        195                 200                 205

Asn Glu Leu Gly Arg Glu Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn

    210                 215                 220

Gln Thr Pro Gln Thr Thr Leu Pro Gln Leu Phe Leu Lys Val Gly Glu

225                 230                 235                 240

Pro Leu Trp Ile Arg Cys Lys Ala Val His Val Asn His Gly Phe Gly

                245                 250                 255

Leu Thr Trp Glu Leu Glu Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe

            260                 265                 270

Glu Met Ser Thr Tyr Ser Thr Asn Arg Thr Met Ile Arg Ile Leu Phe

        275                 280                 285

Ala Phe Val Ser Ser Val Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys

    290                 295                 300

Ser Ser Ser Lys His Pro Ser Gln Ser Ala Leu Val Thr Ile Val Gly

305                 310                 315                 320

Lys Gly Phe Ile Asn Ala Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp

                325                 330                 335

Gln Tyr Glu Glu Phe Cys Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln

            340                 345                 350

Ile Arg Cys Thr Trp Thr Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln

        355                 360                 365

Lys Gly Leu Asp Asn Gly Tyr Ser Ile Ser Lys Phe Cys Asn His Lys

    370                 375                 380

His Gln Pro Gly Glu Tyr Ile Phe His Ala Glu Asn Asp Asp Ala Gln

385                 390                 395                 400

Phe Thr Lys Met Phe Thr Leu Asn Ile Arg Arg Lys Pro Gln Val Leu

                405                 410                 415

Ala Glu Ala Ser Ala Ser Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro

            420                 425                 430

Leu Pro Ser Trp Thr Trp Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys

        435                 440                 445

Thr Glu Glu Ile Thr Glu Gly Val Trp Asn Arg Lys Ala Asn Arg Lys

    450                 455                 460

Val Phe Gly Gln Trp Val Ser Ser Ser Thr Leu Asn Met Ser Glu Ala

465                 470                 475                 480

Ile Lys Gly Phe Leu Val Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr

                485                 490                 495

Ser Cys Glu Thr Ile Leu Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile

            500                 505                 510

Gln Asp

<210>71

<211>1551

<212>DNA

<213>Artificial sequence

<400>71

actattacaa atcaagatct gcctgtgatc aagtgtgttt taatcaatca taagaacaat  60

gattcatcag tggggaagtc atcatcatat cccatggtat cagaatcccc ggaagacctc  120

gggtgtgcgt tgagacccca gagctcaggg acagtgtacg aagctgccgc tgtggaagtg  180

gatgtatctg cttccatcac actgcaagtg ctggtcgatg ccccagggaa catttcctgt  240

ctctgggtct ttaagcacag ctccctgaat tgccagccac attttgattt acaaaacaga  300

ggagttgttt ccatggtcat tttgaaaatg acagaaaccc aagctggaga atacctactt  360

tttattcaga gtgaagctac caattacaca atattgttta cagtgagtat aagaaatacc  420

ctgctttaca cattaagaag accttacttt agaaaaatgg aaaaccagga cgccctggtc  480

tgcatatctg agagcgttcc agagccgatc gtggaatggg tgctttgcga ttcacagggg  540

gaaagctgta aagaagaaag tccagctgtt gttaaaaagg aggaaaaagt gcttcatgaa  600

ttatttggga cggacataag gtgctgtgcc agaaatgaac tgggcaggga atgcaccagg  660

ctgttcacaa tagatctaaa tcaaactcct cagaccacat tgccacaatt atttcttaaa  720

gtaggggaac ccttatggat aaggtgcaaa gctgttcatg tgaaccatgg attcgggctc  780

acctgggaat tagaaaacaa agcactcgag gagggcaact actttgagat gagtacctat  840

tcaacaaaca gaactatgat acggattctg tttgcttttg tatcatcagt ggcaagaaac  900

gacaccggat actacacttg ttcctcttca aagcatccca gtcaatcagc tttggttacc  960

atcgtagaaa agggatttat aaatgctacc aattcaagtg aagattatga aattgaccaa  1020

tatgaagagt tttgtttttc tgtcaggttt aaagcctacc cacaaatcag atgtacgtgg  1080

accttctctc gaaaatcatt tccttgtgag caaaagggtc ttgataacgg atacagcata  1140

tccaagtttt gcaatcataa gcaccagcca ggagaatata tattccatgc agaaaatgat  1200

gatgcccaat ttaccaaaat gttcacgctg aatataagaa ggaaacctca agtgctcgca  1260

gaagcatcgg caagtcaggc gtcctgtttc tcggatggat acccattacc atcttggacc  1320

tggaagaagt gttcagacaa gtctcccaac tgcacagaag agatcacaga aggagtctgg  1380

aatagaaagg ctaacagaaa agtgtttgga cagtgggtgt cgagcagtac tctaaacatg  1440

agtgaagcca taaaagggtt cctggtcaag tgctgtgcat acaattccct tggcacatct  1500

tgtgagacga tccttttaaa ctctccaggc cccttccctt tcatccaaga c           1551

<210>72

<211>517

<212>PRT

<213>Artificial sequence

<400>72

Thr Ile Thr Asn Gln Asp Leu Pro Val Ile Lys Cys Val Leu Ile Asn

1               5                   10                  15

His Lys Asn Asn Asp Set Ser Val Gly Lys Set Ser Ser Tyr Pro Met

            20                  25                  30

Val Ser Glu Ser Pro Glu Asp Leu Gly Cys Ala Leu Arg Pro Gln Ser

        35                  40                  45

Ser Gly Thr Val Tyr Glu Ala Ala Ala Val Glu Val Asp Val Ser Ala

    50                  55                  60

Ser Ile Thr Leu Gln Val Leu Val Asp Ala Pro Gly Asn Ile Ser Cys

65                  70                  75                  80

Leu Trp Val Phe Lys His Ser Ser Leu Asn Cys Gln Pro His Phe Asp

                85                  90                  95

Leu Gln Asn Arg Gly Val Val Ser Met Val Ile Leu Lys Met Thr Glu

            100                 105                 110

Thr Gln Ala Gly Glu Tyr Leu Leu Phe Ile Gln Ser Glu Ala Thr Asn

        115                 120                 125

Tyr Thr Ile Leu Phe Thr Val Ser Ile Arg Asn Thr Leu Leu Tyr Thr

    130                 135                 140

Leu Arg Arg Pro Tyr Phe Arg Lys Met Glu Asn Gln Asp Ala Leu Val

145                 150                 155                 160

Cys Ile Ser Glu Ser Val Pro Glu Pro Ile Val Glu Trp Val Leu Cys

                165                 170                 175

Asp Ser Gln Gly Glu Ser Cys Lys Glu Glu Ser Pro Ala Val Val Lys

            180                 185                 190

Lys Glu Glu Lys Val Leu His Glu Leu Phe Gly Thr Asp Ile Arg Cys

        195                 200                 205

Cys Ala Arg Asn Glu Leu Gly Arg Glu Cys Thr Arg Leu Phe Thr Ile

    210                 215                 220

Asp Leu Asn Gln Thr Pro Gln Thr Thr Leu Pro Gln Leu Phe Leu Lys

225                 230                 235                 240

Val Gly Glu Pro Leu Trp Ile Arg Cys Lys Ala Val His Val Asn His

                245                 250                 255

Gly Phe Gly Leu Thr Trp Glu Leu Glu Asn Lys Ala Leu Glu Glu Gly

            260                 265                 270

Asn Tyr Phe Glu Met Ser Thr Tyr Ser Thr Asn Arg Thr Met Ile Arg

        275                 280                 285

Ile Leu Phe Ala Phe Val Ser Ser Val Ala Arg Asn Asp Thr Gly Tyr

    290                 295                 300

Tyr Thr Cys Ser Ser Ser Lys His Pro Ser Gln Ser Ala Leu Val Thr

305                 310                 315                 320

Ile Val Glu Lys Gly Phe Ile Asn Ala Thr Asn Ser Ser Glu Asp Tyr

                325                 330                 335

Glu Ile Asp Gln Tyr Glu Glu Phe Cys Phe Ser Val Arg Phe Lys Ala

            340                 345                 350

Tyr Pro Gln Ile Arg Cys Thr Trp Thr Phe Ser Arg Lys Ser Phe Pro

        355                 360                 365

Cys Glu Gln Lys Gly Leu Asp Asn Gly Tyr Ser Ile Ser Lys Phe Cys

    370                 375                 380

Asn His Lys His Gln Pro Gly Glu Tyr Ile Phe His Ala Glu Asn Asp

385                 390                 395                 400

Asp Ala Gln Phe Thr Lys Met Phe Thr Leu Asn Ile Arg Arg Lys Pro

                405                 410                 415

Gln Val Leu Ala Glu Ala Ser Ala Set Gln Ala Set Cys Phe Ser Asp

            420                 425                 430

Gly Tyr Pro Leu Pro Ser Trp Thr Trp Lys Lys Cys Ser Asp Lys Ser

        435                 440                 445

Pro Asn Cys Thr Glu Glu Ile Thr Glu Gly Val Trp Asn Arg Lys Ala

    450                 455                 460

Asn Arg Lys Val Phe Gly Gln Trp Val Set Set Ser Thr Leu Asn Met

465                 470                 475                 480

Set Glu Ala Ile Lys Gly Phe Leu Val Lys Cys Cys Ala Tyr Asn Ser

                485                 490                 495

Leu Gly Thr Set Cys Glu Thr Ile Leu Leu Asn Ser Pro Gly Pro Phe

            500                 505                 510

Pro Phe Ile Gln Asp

        515

<210>73

<211>1542

<212>DNA

<213>Artificial sequence

<400>73

aatcaagatc tgcctgtgat caagtgtgtt ttaatcaatc ataagaacaa tgattcatca    60

gtggggaagt catcatcata tcccatggta tcagaatccc cggaagacct cgggtgtgcg    120

ttgagacccc agagctcagg gacagtgtac gaagctgccg ctgtggaagt ggatgtatct    180

gcttccatca cactgcaagt gctggtcgat gccccaggga acatttcctg tctctgggtc    240

tttaagcaca gctccctgaa ttgccagcca cattttgatt tacaaaacag aggagttgtt    300

tccatggtca ttttgaaaat gacagaaacc caagctggag aatacctact ttttattcag    360

agtgaagcta ccaattacac aatattgttt acagtgagta taagaaatac cctgctttac    420

acattaagaa gaccttactt tagaaaaatg gaaaaccagg acgccctggt ctgcatatct    480

gagagcgttc cagagccgat cgtggaatgg gtgctttgcg attcacaggg ggaaagctgt    540

aaagaagaaa gtccagctgt tgttaaaaag gaggaaaaag tgcttcatga attatttggg    600

acggacataa ggtgctgtgc cagaaatgaa ctgggcaggg aatgcaccag gctgttcaca    660

atagatctaa atcaaactcc tcagaccaca ttgccacaat tatttcttaa agtaggggaa    720

cccttatgga taaggtgcaa agctgttcat gtgaaccatg gattcgggct cacctgggaa    780

ttagaaaaca aagcactcga ggagggcaac tactttgaga tgagtaccta ttcaacaaac    840

agaactatga tacggattct gtttgctttt gtatcatcag tggcaagaaa cgacaccgga    900

tactacactt gttcctcttc aaagcatccc agtcaatcag ctttggttac catcgtagaa    960

aagggattta taaatgctac caattcaagt gaagattatg aaattgacca atatgaagag    1020

ttttgttttt ctgtcaggtt taaagcctac ccacaaatca gatgtacgtg gaccttctct    1080

cgaaaatcat ttccttgtga gcaaaagggt cttgataacg gatacagcat atccaagttt    1140

tgcaatcata agcaccagcc aggagaatat atattccatg cagaaaatga tgatgcccaa    1200

tttaccaaaa tgttcacgct gaatataaga aggaaacctc aagtgctcgc agaagcatcg    1260

gcaagtcagg cgtcctgttt ctcggatgga tacccattac catcttggac ctggaagaag  1320

tgttcagaca agtctcccaa ctgcacagaa gagatcacag aaggagtctg gaatagaaag  1380

gctaacagaa aagtgtttgg acagtgggtg tcgagcagta ctctaaacat gagtgaagcc  1440

ataaaagggt tcctggtcaa gtgctgtgca tacaattccc ttggcacatc ttgtgagacg  1500

atccttttaa actctccagg ccccttccct ttcatccaag ac                     1542

<210>74

<211>514

<212>PRT

<213>Artificial sequence

<400>74

Asn Gln Asp Leu Pro Val Ile Lys Cys Val Leu Ile Asn His Lys Asn

1               5                   10                  15

Asn Asp Ser Ser Val Gly Lys Ser Ser Ser Tyr Pro Met Val Ser Glu

            20                  25                  30

Ser Pro Glu Asp Leu Gly Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr

        35                  40                  45

Val Tyr Glu Ala Ala Ala Val Glu Val Asp Val Ser Ala Ser Ile Thr

    50                  55                  60

Leu Gln Val Leu Val Asp Ala Pro Gly Asn Ile Ser Cys Leu Trp Val

65                  70                  75                  80

Phe Lys His Ser Ser Leu Asn Cys Gln Pro His Phe Asp Leu Gln Asn

                85                  90                  95

Arg Gly Val Val Ser Met Val Ile Leu Lys Met Thr Glu Thr Gln Ala

            100                 105                 110

Gly Glu Tyr Leu Leu Phe Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile

        115                 120                 125

Leu Phe Thr Val Ser Ile Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg

    130                 135                 140

Pro Tyr Phe Arg Lys Met Glu Asn Gln Asp Ala Leu Val Cys Ile Ser

145                 150                 155                 160

Glu Ser Val Pro Glu Pro Ile Val Glu Trp Val Leu Cys Asp Ser Gln

                165                 170                 175

Gly Glu Ser Cys Lys Glu Glu Ser Pro Ala Val Val Lys Lys Glu Glu

            180                 185                 190

Lys Val Leu His Glu Leu Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg

        195                 200                 205

Asn Glu Leu Gly Arg Glu Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn

    210                 215                 220

Gln Thr Pro Gln Thr Thr Leu Pro Gln Leu Phe Leu Lys Val Gly Glu

225                 230                 235                 240

Pro Leu Trp Ile Arg Cys Lys Ala Val His Val Asn His Gly Phe Gly

                245                 250                 255

Leu Thr Trp Glu Leu Glu Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe

            260                 265                 270

Glu Met Ser Thr Tyr Ser Thr Asn Arg Thr Met Ile Arg Ile Leu Phe

        275                 280                 285

Ala Phe Val Ser Ser Val Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys

    290                 295                 300

Ser Ser Ser Lys His Pro Ser Gln Ser Ala Leu Val Thr Ile Val Glu

305                 310                 315                 320

Lys Gly Phe Ile Asn Ala Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp

                325                 330                 335

Gln Tyr Glu Glu Phe Cys Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln

            340                 345                 350

Ile Arg Cys Thr Trp Thr Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln

        355                 360                 365

Lys Gly Leu Asp Asn Gly Tyr Ser Ile Ser Lys Phe Cys Asn His Lys

    370                 375                 380

His Gln Pro Gly Glu Tyr Ile Phe His Ala Glu Asn Asp Asp Ala Gln

385                 390                 395                 400

Phe Thr Lys Met Phe Thr Leu Asn Ile Arg Arg Lys Pro Gln Val Leu

                405                 410                 415

Ala Glu Ala Ser Ala Ser Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro

            420                 425                 430

Leu Pro Ser Trp Thr Trp Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys

        435                 440                 445

Thr Glu Glu Ile Thr Glu Gly Val Trp Asn Arg Lys Ala Asn Arg Lys

    450                 455                 460

Val Phe Gly Gln Trp Val Ser Ser Ser Thr Leu Asn Met Ser Glu Ala

465                 470                 475                 480

Ile Lys Gly Phe Leu Val Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr

                485                 490                 495

Ser Cys Glu Thr Ile Leu Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile

            500                 505                 510

Gln Asp

<210>75

<211>1620

<212>DNA

<213>Artificial sequence

<400>75

atgccggcgt tggcgcgcga cgcgggcacc gtgccgctgc tcgttgtttt ttctgcaatg  60

atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat  120

aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg  180

gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct  240

gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac  300

atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta    360

caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa    420

tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata    480

agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac    540

gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat    600

tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg    660

cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa    720

tgcaccaggc tgttcacaat agatctaaat caaactcctc agaceacatt gccacaatta    780

tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga    840

ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg    900

agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg    960

gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct    1020

ttggttacca tcgtaggaaa gggatttata aatgctacca attcaagtga agattatgaa    1080

attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga    1140

tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga    1200

tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca    1260

gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa    1320

gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca    1380

tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa    1440

ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact    1500

ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt    1560

ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac    1620

<210>76

<211>540

<212>PRT

<213>Artificial sequence

<400>76

Met Pro Ala Leu Ala Arg Asp Ala Gly Thr Val Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val

            20                  25                  30

Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly

        35                  40                  45

Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly

    50                  55                  60

Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala

65                  70                  75                  80

Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp

                85                  90                  95

Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu

            100                 105                 110

Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met

        115                 120                 125

Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe

    130                 135                 140

Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile

145                 150                 155                 160

Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met

                165                 170                 175

Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro

            180                 185                 190

Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu

        195                 200                 205

Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu

    210                 215                 220

Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu

225                 230                 235                 240

Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr

                245                 250                 255

Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys

            260                 265                 270

Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu

        275                 280                 285

Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser

    290                 295                 300

Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val

305                 310                 315                 320

Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro

                325                 330                 335

Ser Gln Ser Ala Leu Val Thr Ile Val Gly Lys Gly Phe Ile Asn Ala

            340                 345                 350

Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys

        355                 360                 365

Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr

    370                 375                 380

Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly

385                 390                 395                 400

Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr

                405                 410                 415

Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr

            420                 425                 430

Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser

        435                 440                 445

Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp

    450                 455                 460

Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu

465                 470                 475                 480

Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val

                485                 490                 495

Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val

            500                 505                 510

Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu

        515                 520                 525

Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp

    530                 535                 540

<210>77

<211>1620

<212>DNA

<213>Artificial sequence

<400>77

atgccggcgt tggcgcgcga cggcggccag ctgccgctgc tcgttgtttt ttctgcaatg  60

atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat  120

aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg  180

gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct  240

gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac  300

atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta  360

caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa  420

tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata  480

agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac    540

gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat    600

tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg    660

cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa    720

tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta    780

tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga    840

ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg    900

agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg    960

gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct    1020

ttggttacca tcgtaggaaa gggatttata aatgctacca attcaagtga agattatgaa    1080

attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga    1140

tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga    1200

tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca    1260

gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa    1320

gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca    1380

tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa    1440

ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact    1500

ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt    1560

ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac    1620

<210>78

<211>540

<212>PRT

<213>Artificial sequence

<400>78

Met Pro Ala Leu Ala Arg Asp Gly Gly Gln Leu Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val

            20                  25                  30

Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly

        35                  40                  45

Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly

    50                  55                  60

Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala

65                  70                  75                  80

Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp

                85                  90                  95

Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu

            100                 105                 110

Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met

        115                 120                 125

Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe

    130                 135                 140

Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile

145                 150                 155                 160

Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met

                165                 170                 175

Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro

            180                 185                 190

Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu

        195                 200                 205

Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu

    210                 215                 220

Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu

225                 230                 235                 240

Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr

                245                 250                 255

Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys

            260                 265                 270

Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu

        275                 280                 285

Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser

    290                 295                 300

Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val

305                 310                 315                 320

Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro

                325                 330                 335

Ser Gln Ser Ala Leu Val Thr Ile Val Gly Lys Gly Phe Ile Asn Ala

            340                 345                 350

Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys

        355                 360                 365

Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr

    370                 375                 380

Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly

385                 390                 395                 400

Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr

                405                 410                 415

Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr

            420                 425                 430

Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser

        435                 440                 445

Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp

    450                 455                 460

Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu

465                 470                 475                 480

Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val

                485                 490                 495

Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val

            500                 505                 510

Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu

        515                 520                 525

Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp

    530                 535                 540

<210>79

<211>1620

<212>DNA

<213>Artificial sequence

<400>79

atgccggcgt tggcgcgcga cgcgggcacc gtgccgctgc tcgttgtttt ttctgcaatg  60

atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat  120

aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg  180

gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct  240

gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac  300

atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta  360

caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa  420

tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata  480

agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac  540

gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat  600

tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg  660

cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa    720

tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta    780

tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga    840

ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg    900

agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg    960

gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct    1020

ttggttacca tcgtagaaaa gggatttata aatgctacca attcaagtga agattatgaa    1080

attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga    1140

tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga    1200

tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca    1260

gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa    1320

gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca    1380

tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa    1440

ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact    1500

ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt    1560

ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac    1620

<210>80

<211>540

<212>PRT

<213>Artificial sequence

<400>80

Met Pro Ala Leu Ala Arg Asp Ala Gly Thr Val Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val

            20                  25                  30

Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly

        35                  40                  45

Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly

    50                  55                  60

Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala

65                  70                  75                  80

Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp

                85                  90                  95

Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu

            100                 105                 110

Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met

        115                 120                 125

Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe

    130                 135                 140

Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile

145                 150                 155                 160

Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met

                165                 170                 175

Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro

            180                 185                 190

Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu

        195                 200                 205

Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu

    210                 215                 220

Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu

225                 230                 235                 240

Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr

                245                 250                 255

Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys

            260                 265                 270

Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu

        275                 280                 285

Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser

    290                 295                 300

Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val

305                 310                 315                 320

Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro

                325                 330                 335

Ser Gln Ser Ala Leu Val Thr Ile Val Glu Lys Gly Phe Ile Asn Ala

            340                 345                 350

Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys

        355                 360                 365

Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr

    370                 375                 380

Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly

385                 390                 395                 400

Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr

                405                 410                 415

Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr

            420                 425                 430

Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser

        435                 440                 445

Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp

    450                 455                 460

Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu

465                 470                 475                 480

Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val

                485                 490                 495

Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val

            500                 505                 510

Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu

        515                 520                 525

Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp

    530                 535                 540

<210>81

<211>1620

<212>DNA

<213>Artificial sequence

<400>81

atgccggcgt tggcgcgcga cggcggccag ctgccgctgc tcgttgtttt ttctgcaatg  60

atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat  120

aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg  180

gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct  240

gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac  300

atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta  360

caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa  420

tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata  480

agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac  540

gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat  600

tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg  660

cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa  720

tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta  780

tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga  840

ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg  900

agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg  960

gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct  1020

ttggttacca tcgtagaaaa gggatttata aatgctacca attcaagtga agattatgaa  1080

attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga  1140

tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga  1200

tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca  1260

gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa  1320

gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca  1380

tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa  1440

ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact  1500

ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt  1560

ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac  1620

<210>82

<211>540

<212>PRT

<213>Artificial sequence

<400>82

Met Pro Ala Leu Ala Arg Asp Gly Gly Gln Leu Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val

            20                  25                  30

Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly

        35                  40                  45

Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly

    50                  55                  60

Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala

65                  70                  75                  80

Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp

                85                  90                  95

Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu

            100                 105                 110

Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met

        115                 120                 125

Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe

    130                 135                 140

Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile

145                 150                 155                 160

Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met

                165                 170                 175

Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro

            180                 185                 190

Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu

        195                 200                 205

Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu

    210                 215                 220

Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu

225                 230                 235                 240

Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr

                245                 250                 255

Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys

            260                 265                 270

Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu

        275                 280                 285

Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser

    290                 295                 300

Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val

305                 310                 315                 320

Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro

                325                 330                 335

Ser Gln Ser Ala Leu Val Thr Ile Val Glu Lys Gly Phe Ile Asn Ala

            340                 345                 350

Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys

        355                 360                 365

Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr

    370                 375                 380

Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly

385                 390                 395                 400

Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr

                405                 410                 415

Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr

            420                 425                 430

Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser

        435                 440                 445

Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp

    450                 455                 460

Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu

465                 470                 475                 480

Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val

                485                 490                 495

Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val

            500                 505                 510

Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu

        515                 520                 525

Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp

    530                 535                 540

<210>83

<211>2343

<212>DNA

<213>Artificial sequence

<400>83

atgccggcgt tggcgcgcga cgcgggcacc gtgccgctgc tcgttgtttt ttctgcaatg  60

atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat  120

aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg  180

gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct  240

gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac  300

atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta  360

caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa  420

tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata  480

agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac  540

gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat  600

tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg  660

cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa  720

tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta  780

tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga  840

ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg  900

agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg  960

gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct  1020

ttggttacca tcgtaggaaa gggatttata aatgctacca attcaagtga agattatgaa    1080

attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga    1140

tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga    1200

tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca    1260

gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa    1320

gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca    1380

tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa    1440

ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact    1500

ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt    1560

ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac    1620

aggatcccca aggtggacaa gaaagttgag cccaaatctt gtgacaaaac tcacacatgc    1680

ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttcctctt ccccccaaaa    1740

cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg    1800

agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat    1860

gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc    1920

accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa    1980

gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca    2040

caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc    2100

tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag    2160

ccggagaaca actaeaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc    2220

tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc    2280

gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt    2340

aaa                                                       2343

<210>84

<211>781

<212>PRT

<213>Artificial sequence

<400>84

Met Pro Ala Leu Ala Arg Asp Ala Gly Thr Val Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val

            20                  25                  30

Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly

        35                  40                  45

Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly

    50                  55                  60

Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala

65                  70                  75                  80

Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp

                85                  90                  95

Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu

            100                 105                 110

Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met

        115                 120                 125

Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe

    130                 135                 140

Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile

145                 150                 155                 160

Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met

                165                 170                 175

Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro

            180                 185                 190

Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu

        195                 200                 205

Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu

    210                 215                 220

Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu

225                 230                 235                 240

Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr

                245                 250                 255

Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys

            260                 265                 270

Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu

        275                 280                 285

Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser

    290                 295                 300

Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val

305                 310                 315                 320

Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro

                325                 330                 335

Ser Gln Ser Ala Leu Val Thr Ile Val Gly Lys Gly Phe Ile Asn Ala

            340                 345                 350

Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys

        355                 360                 365

Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr

    370                 375                 380

Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly

385                 390                 395                 400

Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr

                405                 410                 415

Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr

            420                 425                 430

Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser

        435                 440                 445

Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp

    450                 455                 460

Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu

465                 470                 475                 480

Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val

                485                 490                 495

Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val

            500                 505                 510

Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu

        515                 520                 525

Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Arg Ile Pro Lys

    530                 535                 540

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

545                 550                 555                 560

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

                565                 570                 575

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

            580                 585                 590

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

        595                 600                 605

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

    610                 615                 620

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

625                 630                 635                 640

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

                645                 650                 655

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

            660                 665                 670

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

        675                 680                 685

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

    690                 695                 700

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

705                 710                 715                 720

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

                725                 730                 735

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

            740                 745                 750

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

        755                 760                 765

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

    770                 775                 780

<210>85

<211>2343

<212>DNA

<213>Artificial sequence

<400>85

atgccggcgt tggcgcgcga cggcggccag ctgccgctgc tcgttgtttt ttctgcaatg    60

atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat    120

aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg    180

gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct    240

gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac    300

atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta    360

caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa    420

tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata    480

agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac    540

gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat    600

tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg    660

cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa    720

tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta    780

tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga    840

ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg    900

agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg    960

gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct    1020

ttggttacca tcgtaggaaa gggatttata aatgctacca attcaagtga agattatgaa    1080

attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga    1140

tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga    1200

tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca    1260

gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa    1320

gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca    1380

tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa    1440

ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact    1500

ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt    1560

ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac    1620

aggatcccca aggtggacaa gaaagttgag cccaaatctt gtgacaaaac tcacacatgc    1680

ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttcctctt ccccccaaaa    1740

cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg    1800

agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat    1860

gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc    1920

accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa    1980

gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca    2040

caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc    2100

tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag    2160

ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc    2220

tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc    2280

gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt    2340

aaa                                                                  2343

<210>86

<211>781

<212>PRT

<213>Artificial sequence

<400>86

Met Pro Ala Leu Ala Arg Asp Gly Gly Gln Leu Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val

            20                  25                  30

Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly

        35                  40                  45

Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly

    50                  55                  60

Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala

65                  70                  75                  80

Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp

                85                  90                  95

Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu

            100                 105                 110

Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met

        115                 120                 125

Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe

    130                 135                 140

Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile

145                 150                 155                 160

Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met

                165                 170                 175

Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro

            180                 185                 190

Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu

        195                 200                 205

Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu

    210                 215                 220

Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu

225                 230                 235                 240

Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr

                245                 250                 255

Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys

            260                 265                 270

Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu

        275                 280                 285

Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser

    290                 295                 300

Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val

305                 310                 315                 320

Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro

                325                 330                 335

Ser Gln Ser Ala Leu Val Thr Ile Val Gly Lys Gly Phe Ile Asn Ala

            340                 345                 350

Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys

        355                 360                 365

Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr

    370                 375                 380

Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly

385                 390                 395                 400

Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr

                405                 410                 415

Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr

            420                 425                 430

Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser

        435                 440                 445

Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp

    450                 455                 460

Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu

465                 470                 475                 480

Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val

                485                 490                 495

Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val

            500                 505                 510

Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu

        515                 520                 525

Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Arg Ile Pro Lys

    530                 535                 540

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

545                 550                 555                 560

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

                565                 570                 575

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

            580                 585                 590

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

        595                 600                 605

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

    610                 615                 620

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

625                 630                 635                 640

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

                645                 650                 655

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

            660                 665                 670

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

        675                 680                 685

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

    690                 695                 700

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

705                 710                 715                 720

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

                725                 730                 735

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

            740                 745                 750

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

        755                 760                 765

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

    770                 775                 780

<210>87

<211>2343

<212>DNA

<213>Artificial sequence

<400>87

atgccggcgt tggcgcgcga cgcgggcacc gtgccgctgc tcgttgtttt ttctgcaatg  60

atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat  120

aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg  180

gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct    240

gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac    300

atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta    360

caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa    420

tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata    480

agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac    540

gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat    600

tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg    660

cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa    720

tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta    780

tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga    840

ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg    900

agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg    960

gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct    1020

ttggttacca tcgtagaaaa gggatttata aatgctacca attcaagtga agattatgaa    1080

attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga    1140

tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga    1200

tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca    1260

gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa    1320

gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca    1380

tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa    1440

ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact    1500

ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt    1560

ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac    1620

aggatcccca aggtggacaa gaaagttgag cccaaatctt gtgacaaaac tcacacatgc    1680

ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttcctctt ccccccaaaa    1740

cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg    1800

agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat    1860

gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc    1920

accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa    1980

gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca    2040

caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc    2100

tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag    2160

ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc    2220

tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc    2280

gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt    2340

aaa                                                                  2343

<210>88

<211>781

<212>PRT

<213>Artificial sequence

<400>88

Met Pro Ala Leu Ala Arg Asp Ala Gly Thr Val Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val

            20                  25                  30

Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly

        35                  40                  45

Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly

    50                  55                  60

Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala

65                  70                  75                  80

Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp

                85                  90                  95

Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu

            100                 105                 110

Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met

        115                 120                 125

Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe

    130                 135                 140

Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile

145                 150                 155                 160

Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met

                165                 170                 175

Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro

            180                 185                 190

Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu

        195                 200                 205

Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu

    210                 215                 220

Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu

225                 230                 235                 240

Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr

                245                 250                 255

Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys

            260                 265                 270

Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu

        275                 280                 285

Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser

    290                 295                 300

Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val

305                 310                 315                 320

Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro

                325                 330                 335

Ser Gln Ser Ala Leu Val Thr Ile Val Glu Lys Gly Phe Ile Asn Ala

            340                 345                 350

Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys

        355                 360                 365

Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr

    370                 375                 380

Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly

385                 390                 395                 400

Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr

                405                 410                 415

Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr

            420                 425                 430

Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser

        435                 440                 445

Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp

    450                 455                 460

Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu

465                 470                 475                 480

Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val

                485                 490                 495

Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val

            500                 505                 510

Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu

        515                 520                 525

Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Arg Ile Pro Lys

    530                 535                 540

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

545                 550                 555                 560

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

                565                 570                 575

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

            580                 585                 590

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

        595                 600                 605

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

    610                 615                 620

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

625                 630                 635                 640

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

                645                 650                 655

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

            660                 665                 670

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

        675                 680                 685

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

    690                 695                 700

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

705                 710                 715                 720

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

                725                 730                 735

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

            740                 745                 750

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

        755                 760                 765

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

    770                 775                 780

<210>89

<211>2343

<212>DNA

<213>Artificial sequence

<400>89

atgccggcgt tggcgcgcga cggcggccag ctgccgctgc tcgttgtttt ttctgcaatg  60

atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat  120

aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg  180

gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct  240

gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac  300

atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta  360

caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa  420

tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata    480

agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac    540

gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat    600

tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg    660

cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa    720

tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta    780

tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga    840

ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg    900

agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg    960

gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct    1020

ttggttacca tcgtagaaaa gggatttata aatgctacca attcaagtga agattatgaa    1080

attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga    1140

tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga    1200

tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca    1260

gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa    1320

gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca    1380

tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa    1440

ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact    1500

ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt    1560

ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac    1620

aggatcccca aggtggacaa gaaagttgag cccaaatctt gtgacaaaac tcacacatgc    1680

ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttcctctt ccccccaaaa    1740

cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg    1800

agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat    1860

gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc    1920

accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa    1980

gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca    2040

caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc    2100

tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag    2160

ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc    2220

tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc    2280

gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt    2340

aaa                                                                  2343

<210>90

<211>781

<212>PRT

<213>Artificial sequence

<400>90

Met Pro Ala Leu Ala Arg Asp Gly Gly Gln Leu Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val

            20                  25                  30

Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly

        35                  40                  45

Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly

    50                  55                  60

Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala

65                  70                  75                  80

Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp

                85                  90                  95

Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu

            100                 105                 110

Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met

        115                 120                 125

Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe

    130                 135                 140

Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile

145                 150                 155                 160

Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met

                165                 170                 175

Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro

            180                 185                 190

Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu

        195                 200                 205

Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu

    210                 215                 220

Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu

225                 230                 235                 240

Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr

                245                 250                 255

Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys

            260                 265                 270

Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu

        275                 280                 285

Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser

    290                 295                 300

Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val

305                 310                 315                 320

Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro

                325                 330                 335

Ser Gln Ser Ala Leu Val Thr Ile Val Glu Lys Gly Phe Ile Asn Ala

            340                 345                 350

Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys

        355                 360                 365

Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr

    370                 375                 380

Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly

385                 390                 395                 400

Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr

                405                 410                 415

Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr

            420                 425                 430

Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser

        435                 440                 445

Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp

    450                 455                 460

Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu

465                 470                 475                 480

Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val

                485                 490                 495

Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val

            500                 505                 510

Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu

        515                 520                 525

Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Arg Ile Pro Lys

    530                 535                 540

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

545                 550                 555                 560

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

                565                 570                 575

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

            580                 585                 590

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

        595                 600                 605

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

    610                 615                 620

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

625                 630                 635                 640

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

                645                 650                 655

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

            660                 665                 670

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

        675                 680                 685

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

    690                 695                 700

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

705                 710                 715                 720

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

                725                 730                 735

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

            740                 745                 750

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

        755                 760                 765

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

    770                 775                 780

<210>91

<211>2412

<212>DNA

<213>Artificial sequence

<400>91

atgccggcgt tggcgcgcga cgcgggcacc gtgccgctgc tcgttgtttt ttctgcaatg  60

atatttggga tgccggcgtt ggcgcgcgac gcgggcaccg tgccgctgct cgttgttttt  120

tctgcaatga tatttgggac tattacaaat caagatctgc ctgtgatcaa gtgtgtttta  180

atcaatcata agaacaatga ttcatcagtg gggaagtcat catcatatcc catggtatca  240

gaatccccgg aagacctcgg gtgtgcgttg agaccccaga gctcagggac agtgtacgaa  300

gctgccgctg tggaagtgga tgtatctgct tccatcacac tgcaagtgct ggtcgatgcc  360

ccagggaaca tttcctgtct ctgggtcttt aagcacagct ccctgaattg ccagccacat  420

tttgatttac aaaacagagg agttgtttcc atggtcattt tgaaaatgac agaaacccaa  480

gctggagaat acctactttt tattcagagt gaagctacca attacacaat attgtttaca  540

gtgagtataa gaaataccct gctttacaca ttaagaagac cttactttag aaaaatggaa  600

aaccaggacg ccctggtctg catatctgag agcgttccag agccgatcgt ggaatgggtg  660

ctttgcgatt cacaggggga aagctgtaaa gaagaaagtc cagctgttgt taaaaaggag    720

gaaaaagtgc ttcatgaatt atttgggacg gacataaggt gctgtgccag aaatgaactg    780

ggcagggaat gcaccaggct gttcacaata gatctaaatc aaactcctca gaccacattg    840

ccacaattat ttcttaaagt aggggaaccc ttatggataa ggtgcaaagc tgttcatgtg    900

aaccatggat tcgggctcac ctgggaatta gaaaacaaag cactcgagga gggcaactac    960

tttgagatga gtacctattc aacaaacaga actatgatac ggattctgtt tgcttttgta    1020

tcatcagtgg caagaaacga caccggatac tacacttgtt cctcttcaaa gcatcccagt    1080

caatcagctt tggttaccat cgtaggaaag ggatttataa atgctaccaa ttcaagtgaa    1140

gattatgaaa ttgaccaata tgaagagttt tgtttttctg tcaggtttaa agcctaccca    1200

caaatcagat gtacgtggac cttctctcga aaatcatttc cttgtgagca aaagggtctt    1260

gataacggat acagcatatc caagttttgc aatcataagc accagccagg agaatatata    1320

ttccatgcag aaaatgatga tgcccaattt accaaaatgt tcacgctgaa tataagaagg    1380

aaacctcaag tgctcgcaga agcatcggca agtcaggcgt cctgtttctc ggatggatac    1440

ccattaccat cttggacctg gaagaagtgt tcagacaagt ctcccaactg cacagaagag    1500

atcacagaag gagtctggaa tagaaaggct aacagaaaag tgtttggaca gtgggtgtcg    1560

agcagtactc taaacatgag tgaagccata aaagggttcc tggtcaagtg ctgtgcatac    1620

aattcccttg gcacatcttg tgagacgatc cttttaaact ctccaggccc cttccctttc    1680

atccaagaca ggatccccaa ggtggacaag aaagttgagc ccaaatcttg tgacaaaact    1740

cacacatgcc caccgtgccc agcacctgaa ctcctggggg gaccgtcagt cttcctcttc    1800

cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg    1860

gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag    1920

gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc    1980

agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcagggtc  2040

tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc  2100

cgagaaccac aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc  2160

agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc  2220

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc  2280

ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc  2340

tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg  2400

tctccgggta aa                                                      2412

<210>92

<211>804

<212>PRT

<213>Artificial sequence

<400>92

Met Pro Ala Leu Ala Arg Asp Ala Gly Thr Val Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Met Pro Ala Leu Ala Arg Asp Ala Gly

            20                  25                  30

Thr Val Pro Leu Leu Val Val Phe Ser Ala Met Ile Phe Gly Thr Ile

        35                  40                  45

Thr Asn Gln Asp Leu Pro Val Ile Lys Cys Val Leu Ile Asn His Lys

    50                  55                  60

Asn Asn Asp Ser Ser Val Gly Lys Ser Ser Ser Tyr Pro Met Val Ser

65                  70                  75                  80

Glu Ser Pro Glu Asp Leu Gly Cys Ala Leu Arg Pro Gln Ser Ser Gly

                85                  90                  95

Thr Val Tyr Glu Ala Ala Ala Val Glu Val Asp Val Ser Ala Ser Ile

            100                 105                 110

Thr Leu Gln Val Leu Val Asp Ala Pro Gly Asn Ile Ser Cys Leu Trp

        115                 120                 125

Val Phe Lys His Ser Ser Leu Asn Cys Gln Pro His Phe Asp Leu Gln

    130                 135                 140

Asn Arg Gly Val Val Ser Met Val Ile Leu Lys Met Thr Glu Thr Gln

145                 150                 155                 160

Ala Gly Glu Tyr Leu Leu Phe Ile Gln Ser Glu Ala Thr Asn Tyr Thr

                165                 170                 175

Ile Leu Phe Thr Val Ser Ile Arg Asn Thr Leu Leu Tyr Thr Leu Arg

            180                 185                 190

Arg Pro Tyr Phe Arg Lys Met Glu Asn Gln Asp Ala Leu Val Cys Ile

        195                 200                 205

Ser Glu Ser Val Pro Glu Pro Ile Val Glu Trp Val Leu Cys Asp Ser

    210                 215                 220

Gln Gly Glu Ser Cys Lys Glu Glu Ser Pro Ala Val Val Lys Lys Glu

225                 230                 235                 240

Glu Lys Val Leu His Glu Leu Phe Gly Thr Asp Ile Arg Cys Cys Ala

                245                 250                 255

Arg Asn Glu Leu Gly Arg Glu Cys Thr Arg Leu Phe Thr Ile Asp Leu

            260                 265                 270

Asn Gln Thr Pro Gln Thr Thr Leu Pro Gln Leu Phe Leu Lys Val Gly

        275                 280                 285

Glu Pro Leu Trp Ile Arg Cys Lys Ala Val His Val Asn His Gly Phe

    290                 295                 300

Gly Leu Thr Trp Glu Leu Glu Asn Lys Ala Leu Glu Glu Gly Asn Tyr

305                 310                 315                 320

Phe Glu Met Ser Thr Tyr Ser Thr Asn Arg Thr Met Ile Arg Ile Leu

                325                 330                 335

Phe Ala Phe Val Ser Ser Val Ala Arg Asn Asp Thr Gly Tyr Tyr Thr

            340                 345                 350

Cys Ser Ser Ser Lys His Pro Ser Gln Ser Ala Leu Val Thr Ile Val

        355                 360                 365

Gly Lys Gly Phe Ile Asn Ala Thr Asn Ser Ser Glu Asp Tyr Glu Ile

    370                 375                 380

Asp Gln Tyr Glu Glu Phe Cys Phe Ser Val Arg Phe Lys Ala Tyr Pro

385                 390                 395                 400

Gln Ile Arg Cys Thr Trp Thr Phe Ser Arg Lys Ser Phe Pro Cys Glu

                405                 410                 415

Gln Lys Gly Leu Asp Asn Gly Tyr Ser Ile Ser Lys Phe Cys Asn His

            420                 425                 430

Lys His Gln Pro Gly Glu Tyr Ile Phe His Ala Glu Asn Asp Asp Ala

        435                 440                 445

Gln Phe Thr Lys Met Phe Thr Leu Asn Ile Arg Arg Lys Pro Gln Val

    450                 455                 460

Leu Ala Glu Ala Ser Ala Ser Gln Ala Ser Cys Phe Ser Asp Gly Tyr

465                 470                 475                 480

Pro Leu Pro Ser Trp Thr Trp Lys Lys Cys Ser Asp Lys Ser Pro Asn

                485                 490                 495

Cys Thr Glu Glu Ile Thr Glu Gly Val Trp Asn Arg Lys Ala Asn Arg

            500                 505                 510

Lys Val Phe Gly Gln Trp Val Ser Ser Ser Thr Leu Asn Met Ser Glu

        515                 520                 525

Ala Ile Lys Gly Phe Leu Val Lys Cys Cys Ala Tyr Asn Ser Leu Gly

    530                 535                 540

Thr Ser Cys Glu Thr Ile Leu Leu Asn Ser Pro Gly Pro Phe Pro Phe

545                 550                 555                 560

Ile Gln Asp Arg Ile Pro Lys Val Asp Lys Lys Val Glu Pro Lys Ser

                565                 570                 575

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

            580                 585                 590

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

        595                 600                 605

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

    610                 615                 620

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

625                 630                 635                 640

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

                645                 650                 655

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

            660                 665                 670

Gly Lys Glu Tyr Lys Cys Arg Val Ser Asn Lys Ala Leu Pro Ala Pro

        675                 680                 685

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

    690                 695                 700

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

705                 710                 715                 720

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

                725                 730                 735

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

            740                 745                 750

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

        755                 760                 765

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

    770                 775                 780

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

785                 790                 795                 800

Ser Pro Gly Lys

<210>93

<211>2412

<212>DNA

<213>Artificial sequence

<400>93

atgccggcgt tggcgcgcga cggcggccag ctgccgctgc tcgttgtttt ttctgcaatg  60

atatttggga tgccggcgtt ggcgcgcgac gcgggcaccg tgccgctgct cgttgttttt  120

tctgcaatga tatttgggac tattacaaat caagatctgc ctgtgatcaa gtgtgtttta  180

atcaatcata agaacaatga ttcatcagtg gggaagtcat catcatatcc catggtatca  240

gaatccccgg aagacctcgg gtgtgcgttg agaccccaga gctcagggac agtgtacgaa  300

gctgccgctg tggaagtgga tgtatctgct tccatcacac tgcaagtgct ggtcgatgcc  360

ccagggaaca tttcctgtct ctgggtcttt aagcacagct ccctgaattg ccagccacat  420

tttgatttac aaaacagagg agttgtttcc atggtcattt tgaaaatgac agaaacccaa  480

gctggagaat acctactttt tattcagagt gaagctacca attacacaat attgtttaca  540

gtgagtataa gaaataccct gctttacaca ttaagaagac cttactttag aaaaatggaa  600

aaccaggacg ccctggtctg catatctgag agcgttccag agccgatcgt ggaatgggtg    660

ctttgcgatt cacaggggga aagctgtaaa gaagaaagtc cagctgttgt taaaaaggag    720

gaaaaagtgc ttcatgaatt atttgggacg gacataaggt gctgtgccag aaatgaactg    780

ggcagggaat gcaccaggct gttcacaata gatctaaatc aaactcctca gaccacattg    840

ccacaattat ttcttaaagt aggggaaccc ttatggataa ggtgcaaagc tgttcatgtg    900

aaccatggat tcgggctcac ctgggaatta gaaaacaaag cactcgagga gggcaactac    960

tttgagatga gtacctattc aacaaacaga actatgatac ggattctgtt tgcttttgta    1020

tcatcagtgg caagaaacga caccggatac tacacttgtt cctcttcaaa gcatcccagt    1080

caatcagctt tggttaccat cgtaggaaag ggatttataa atgctaccaa ttcaagtgaa    1140

gattatgaaa ttgaccaata tgaagagttt tgtttttctg tcaggtttaa agcctaccca    1200

caaatcagat gtacgtggac cttctctcga aaatcatttc cttgtgagca aaagggtctt    1260

gataacggat acagcatatc caagttttgc aatcataagc accagccagg agaatatata    1320

ttccatgcag aaaatgatga tgcccaattt accaaaatgt tcacgctgaa tataagaagg    1380

aaacctcaag tgctcgcaga agcatcggca agtcaggcgt cctgtttctc ggatggatac    1440

ccattaccat cttggacctg gaagaagtgt tcagacaagt ctcccaactg cacagaagag    1500

atcacagaag gagtctggaa tagaaaggct aacagaaaag tgtttggaca gtgggtgtcg    1560

agcagtactc taaacatgag tgaagccata aaagggttcc tggtcaagtg ctgtgcatac    1620

aattcccttg gcacatcttg tgagacgatc cttttaaact ctccaggccc cttccctttc    1680

atccaagaca ggatccccaa ggtggacaag aaagttgagc ccaaatcttg tgacaaaact    1740

cacacatgcc caccgtgccc agcacctgaa ctcctggggg gaccgtcagt cttcctcttc    1800

cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg    1860

gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag    1920

gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc  1980

agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcagggtc  2040

tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc  2100

cgagaaccac aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc  2160

agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc  2220

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc  2280

ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc  2340

tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg  2400

tctccgggta aa                                                      2412

<210>94

<211>804

<212>PRT

<213>Artificial sequence

<400>94

Met Pro Ala Leu Ala Arg Asp Gly Gly Gln Leu Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Met Pro Ala Leu Ala Arg Asp Ala Gly

            20                  25                  30

Thr Val Pro Leu Leu Val Val Phe Ser Ala Met Ile Phe Gly Thr Ile

        35                  40                  45

Thr Asn Gln Asp Leu Pro Val Ile Lys Cys Val Leu Ile Asn His Lys

    50                  55                  60

Asn Asn Asp Ser Ser Val Gly Lys Ser Ser Ser Tyr Pro Met Val Ser

65                  70                  75                  80

Glu Ser Pro Glu Asp Leu Gly Cys Ala Leu Arg Pro Gln Ser Ser Gly

                85                  90                  95

Thr Val Tyr Glu Ala Ala Ala Val Glu Val Asp Val Ser Ala Ser Ile

            100                 105                 110

Thr Leu Gln Val Leu Val Asp Ala Pro Gly Asn Ile Ser Cys Leu Trp

        115                 120                 125

Val Phe Lys His Ser Ser Leu Asn Cys Gln Pro His Phe Asp Leu Gln

    130                 135                 140

Asn Arg Gly Val Val Ser Met Val Ile Leu Lys Met Thr Glu Thr Gln

145                 150                 155                 160

Ala Gly Glu Tyr Leu Leu Phe Ile Gln Ser Glu Ala Thr Asn Tyr Thr

                165                 170                 175

Ile Leu Phe Thr Val Ser Ile Arg Asn Thr Leu Leu Tyr Thr Leu Arg

            180                 185                 190

Arg Pro Tyr Phe Arg Lys Met Glu Asn Gln Asp Ala Leu Val Cys Ile

        195                 200                 205

Ser Glu Ser Val Pro Glu Pro Ile Val Glu Trp Val Leu Cys Asp Ser

    210                 215                 220

Gln Gly Glu Ser Cys Lys Glu Glu Ser Pro Ala Val Val Lys Lys Glu

225                 230                 235                 240

Glu Lys Val Leu His Glu Leu Phe Gly Thr Asp Ile Arg Cys Cys Ala

                245                 250                 255

Arg Asn Glu Leu Gly Arg Glu Cys Thr Arg Leu Phe Thr Ile Asp Leu

            260                 265                 270

Asn Gln Thr Pro Gln Thr Thr Leu Pro Gln Leu Phe Leu Lys Val Gly

        275                 280                 285

Glu Pro Leu Trp Ile Arg Cys Lys Ala Val His Val Asn His Gly Phe

    290                 295                 300

Gly Leu Thr Trp Glu Leu Glu Asn Lys Ala Leu Glu Glu Gly Asn Tyr

305                 310                 315                 320

Phe Glu Met Ser Thr Tyr Ser Thr Asn Arg Thr Met Ile Arg Ile Leu

                325                 330                 335

Phe Ala Phe Val Ser Ser Val Ala Arg Asn Asp Thr Gly Tyr Tyr Thr

            340                 345                 350

Cys Ser Ser Ser Lys His Pro Ser Gln Ser Ala Leu Val Thr Ile Val

        355                 360                 365

Gly Lys Gly Phe Ile Asn Ala Thr Asn Ser Ser Glu Asp Tyr Glu Ile

    370                 375                 380

Asp Gln Tyr Glu Glu Phe Cys Phe Ser Val Arg Phe Lys Ala Tyr Pro

385                 390                 395                 400

Gln Ile Arg Cys Thr Trp Thr Phe Ser Arg Lys Ser Phe Pro Cys Glu

                405                 410                 415

Gln Lys Gly Leu Asp Asn Gly Tyr Ser Ile Ser Lys Phe Cys Asn His

            420                 425                 430

Lys His Gln Pro Gly Glu Tyr Ile Phe His Ala Glu Asn Asp Asp Ala

        435                 440                 445

Gln Phe Thr Lys Met Phe Thr Leu Asn Ile Arg Arg Lys Pro Gln Val

    450                 455                 460

Leu Ala Glu Ala Ser Ala Ser Gln Ala Ser Cys Phe Ser Asp Gly Tyr

465                 470                 475                 480

Pro Leu Pro Ser Trp Thr Trp Lys Lys Cys Ser Asp Lys Ser Pro Asn

                485                 490                 495

Cys Thr Glu Glu Ile Thr Glu Gly Val Trp Asn Arg Lys Ala Asn Arg

            500                 505                 510

Lys Val Phe Gly Gln Trp Val Ser Ser Ser Thr Leu Asn Met Ser Glu

        515                 520                 525

Ala Ile Lys Gly Phe Leu Val Lys Cys Cys Ala Tyr Asn Ser Leu Gly

    530                 535                 540

Thr Ser Cys Glu Thr Ile Leu Leu Asn Ser Pro Gly Pro Phe Pro Phe

545                 550                 555                 560

Ile Gln Asp Arg Ile Pro Lys Val Asp Lys Lys Val Glu Pro Lys Ser

                565                 570                 575

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

            580                 585                 590

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

        595                 600                 605

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

    610                 615                 620

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

625                 630                 635                 640

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

                645                 650                 655

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

            660                 665                 670

Gly Lys Glu Tyr Lys Cys Arg Val Ser Asn Lys Ala Leu Pro Ala Pro

        675                 680                 685

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

    690                 695                 700

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

705                 710                 715                 720

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

                725                 730                 735

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

            740                 745                 750

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

        755                 760                 765

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

    770                 775                 780

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

785                 790                 795                 800

Ser Pro Gly Lys

<210>95

<211>2343

<212>DNA

<213>Artificial sequence

<400>95

atgccggcgt tggcgcgcga cgcgggcacc gtgccgctgc tcgttgtttt ttctgcaatg  60

atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat  120

aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg  180

gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct  240

gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac  300

atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta  360

caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa  420

tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata  480

agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac  540

gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat    600

tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg    660

cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa    720

tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta    780

tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga    840

ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg    900

agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg    960

gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct    1020

ttggttacca tcgtagaaaa gggatttata aatgctacca attcaagtga agattatgaa    1080

attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga    1140

tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga    1200

tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca    1260

gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa    1320

gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca    1380

tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa    1440

ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact    1500

ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt    1560

ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac    1620

aggatcccca aggtggacaa gaaagttgag cccaaatctt gtgacaaaac tcacacatgc    1680

ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttcctctt ccccccaaaa    1740

cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg    1800

agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat    1860

gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc  1920

accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcagggt ctccaacaaa  1980

gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca  2040

caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc  2100

tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag  2160

ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc  2220

tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc  2280

gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt  2340

aaa                                                                2343

<210>96

<211>781

<212>PRT

<213>Artificial sequence

<400>96

Met Pro Ala Leu Ala Arg Asp Ala Gly Thr Val Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val

            20                  25                  30

Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly

        35                  40                  45

Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly

    50                  55                  60

Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala

65                  70                  75                  80

Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp

                 85                  90                 95

Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu

            100                 105                 110

Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met

        115                 120                 125

Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe

    130                 135                 140

Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile

145                 150                 155                 160

Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met

                165                 170                 175

Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro

            180                 185                 190

Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu

        195                 200                 205

Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu

    210                 215                 220

Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu

225                 230                 235                 240

Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr

                245                 250                 255

Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys

            260                 265                 270

Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu

        275                 280                 285

Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser

    290                 295                 300

Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val

305                 310                 315                 320

Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro

                325                 330                 335

Ser Gln Ser Ala Leu Val Thr Ile Val Glu Lys Gly Phe Ile Asn Ala

            340                 345                 350

Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys

        355                 360                 365

Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr

    370                 375                 380

Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly

385                 390                 395                 400

Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr

                405                 410                 415

Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr

            420                 425                 430

Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser

        435                 440                 445

Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp

    450                 455                 460

Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu

465                 470                 475                 480

Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val

                485                 490                 495

Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val

            500                505                  510

Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu

        515                 520                 525

Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Arg Ile Pro Lys

    530                 535                 540

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

545                 550                 555                 560

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

                565                 570                 575

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

            580                 585                 590

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

        595                 600                 605

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

    610                 615                 620

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

625                 630                 635                 640

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Arg

                645                 650                 655

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

            660                 665                 670

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

        675                 680                 685

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

    690                 695                 700

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

705                 710                 715                 720

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

                725                 730                 735

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

            740                 745                 750

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

        755                 760                 765

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

    770                 775                 780

<210>97

<211>2343

<212>DNA

<213>Artificial sequence

<400>97

atgccggcgt tggcgcgcga cggcggccag ctgccgctgc tcgttgtttt ttctgcaatg  60

atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat  120

aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg  180

gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct  240

gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac  300

atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta  360

caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa  420

tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata  480

agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac  540

gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat  600

tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg  660

cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa  720

tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta  780

tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga    840

ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg    900

agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg    960

gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct    1020

ttggttacca tcgtagaaaa gggatttata aatgctacca attcaagtga agattatgaa    1080

attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga    1140

tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga    1200

tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca    1260

gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa    1320

gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca    1380

tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa    1440

ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact    1500

ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt    1560

ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac    1620

aggatcccca aggtggacaa gaaagttgag cccaaatctt gtgacaaaac tcacacatgc    1680

ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttcctctt ccccccaaaa    1740

cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg    1800

agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat    1860

gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc    1920

accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcagggt ctccaacaaa    1980

gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca    2040

caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc    2100

tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag  2160

ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc  2220

tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc  2280

gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt  2340

aaa                                                                2343

<210>98

<211>781

<212>PRT

<213>Artificial sequence

<400>98

Met Pro Ala Leu Ala Arg Asp Gly Gly Gln Leu Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val

            20                  25                  30

Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly

        35                  40                  45

Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly

    50                  55                  60

Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala

65                  70                  75                  80

Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp

                85                  90                  95

Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu

            100                 105                 110

Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met

        115                 120                 125

Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe

    130                 135                 140

Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile

145                 150                 155                 160

Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met

                165                 170                 175

Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro

            180                 185                 190

Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu

        195                 200                 205

Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu

    210                 215                 220

Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu

225                 230                 235                 240

Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr

                245                 250                 255

Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys

            260                 265                 270

Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu

        275                 280                 285

Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser

    290                 295                 300

Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val

305                 310                 315                 320

Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro

                325                 330                 335

Ser Gln Ser Ala Leu Val Thr Ile Val Glu Lys Gly Phe Ile Asn Ala

            340                 345                 350

Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys

        355                 360                 365

Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr

    370                 375                 380

Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly

385                 390                 395                 400

Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr

                405                 410                 415

Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr

            420                 425                 430

Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser

        435                 440                 445

Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp

    450                 455                 460

Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu

465                 470                 475                 480

Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val

                485                 490                 495

Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val

            500                 505                 510

Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu

        515                 520                 525

Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Arg Ile Pro Lys

    530                 535                 540

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

545                 550                 555                 560

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

                565                 570                 575

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

            580                 585                 590

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

        595                 600                 605

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

    610                 615                 620

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

625                 630                 635                 640

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Arg

                645                 650                 655

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

            660                 665                 670

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

        675                 680                 685

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

    690                 695                 700

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

705                 710                 715                 720

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

                725                 730                 735

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

            740                 745                 750

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

        755                 760                 765

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

    770                 775                 780

<210>99

<211>2349

<212>DNA

<213>Artificial sequence

<400>99

atgccggcgt tggcgcgcga cgcgggcacc gtgccgctgc tcgttgtttt ttctgcaatg    60

atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat    120

aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg    180

gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct    240

gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac    300

atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta    360

caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa    420

tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata    480

agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac    540

gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat    600

tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg    660

cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa    720

tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta    780

tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga    840

ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg    900

agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg    960

gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct    1020

ttggttacca tcgtaggaaa gggatttata aatgctacca attcaagtga agattatgaa    1080

attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga    1140

tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga    1200

tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca    1260

gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa    1320

gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca    1380

tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa    1440

ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact    1500

ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt    1560

ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac    1620

ggatccagca acaccaaggt ggacaagaaa gttgagccca aatcttgtga caaaactcac    1680

acatgcccac cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc    1740

ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg    1800

gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg    1860

cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc    1920

gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc    1980

aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga    2040

gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc    2100

ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat    2160

gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc    2220

ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca    2280

tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct    2340

ccgggtaaa                                                          2349

<210>100

<211>783

<212>PRT

<213>Artificial sequence

<400>100

Met Pro Ala Leu Ala Arg Asp Ala Gly Thr Val Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val

            20                  25                  30

Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly

        35                  40                  45

Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly

    50                  55                  60

Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala

65                  70                  75                  80

Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp

                85                  90                  95

Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu

            100                 105                 110

Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met

        115                 120                 125

Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe

    130                 135                 140

Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile

145                 150                 155                 160

Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met

                165                 170                 175

Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro

            180                 185                 190

Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu

        195                 200                 205

Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu

    210                 215                 220

Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu

225                 230                 235                 240

Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr

                245                 250                 255

Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys

            260                 265                 270

Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu

        275                 280                 285

Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser

    290                 295                 300

Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val

305                 310                 315                 320

Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro

                325                 330                 335

Ser Gln Ser Ala Leu Val Thr Ile Val Gly Lys Gly Phe Ile Asn Ala

            340                 345                 350

Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys

        355                 360                 365

Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr

    370                 375                 380

Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly

385                 390                 395                 400

Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr

                405                 410                 415

Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr

            420                 425                 430

Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser

        435                 440                 445

Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp

    450                 455                 460

Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu

465                 470                 475                 480

Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val

                485                 490                 495

Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val

            500                 505                 510

Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu

        515                 520                 525

Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Gly Ser Ser Asn

    530                 535                 540

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

545                 550                 555                 560

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

                565                 570                 575

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

            580                 585                 590

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

        595                 600                 605

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

    610                 615                 620

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

625                 630                 635                 640

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

                645                 650                 655

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

            660                 665                 670

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

        675                 680                 685

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

    690                 695                 700

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

705                 710                 715                 720

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

                725                 730                 735

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

            740                 745                 750

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

        755                 760                 765

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

    770                 775                 780

<210>101

<211>2349

<212>DNA

<213>Artificial sequence

<400>101

atgccggcgt tggcgcgcga cggcggccag ctgccgctgc tcgttgtttt ttctgcaatg    60

atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat    120

aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg    180

gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct    240

gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac    300

atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta    360

caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa    420

tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata    480

agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac    540

gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat    600

tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg    660

cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa    720

tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta    780

tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga    840

ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg    900

agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg    960

gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct    1020

ttggttacca tcgtaggaaa gggatttata aatgctacca attcaagtga agattatgaa    1080

attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga    1140

tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga    1200

tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca    1260

gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa    1320

gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca    1380

tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa    1440

ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact    1500

ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt    1560

ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac    1620

ggatccagca acaccaaggt ggacaagaaa gttgagccca aatcttgtga caaaactcac    1680

acatgcccac cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc    1740

ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg    1800

gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg    1860

cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc    1920

gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc    1980

aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga    2040

gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc    2100

ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat    2160

gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc    2220

ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca    2280

tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct    2340

ccgggtaaa                                                            2349

<210>102

<211>783

<212>PRT

<213>Artificial sequence

<400>102

Met Pro Ala Leu Ala Arg Asp Gly Gly Gln Leu Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val

            20                  25                  30

Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly

        35                  40                  45

Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly

    50                  55                  60

Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala

65                  70                  75                  80

Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp

                85                  90                  95

Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu

            100                 105                 110

Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met

        115                 120                 125

Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe

    130                 135                 140

Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile

145                 150                 155                 160

Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met

                165                 170                 175

Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro

            180                 185                 190

Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu

        195                 200                 205

Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu

    210                 215                 220

Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu

225                 230                 235                 240

Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr

                245                 250                 255

Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys

            260                 265                 270

Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu

        275                 280                 285

Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser

    290                 295                 300

Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val

305                 310                 315                 320

Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro

                325                 330                 335

Ser Gln Ser Ala Leu Val Thr Ile Val Gly Lys Gly Phe Ile Asn Ala

            340                 345                 350

Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys

        355                 360                 365

Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr

    370                 375                 380

Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly

385                 390                 395                 400

Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr

                405                 410                 415

Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr

            420                 425                 430

Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser

        435                 440                 445

Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp

    450                 455                 460

Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu

465                 470                 475                 480

Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val

                485                 490                 495

Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val

            500                 505                 510

Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu

        515                 520                 525

Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Gly Ser Ser Asn

    530                 535                 540

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

545                 550                 555                 560

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

                565                 570                 575

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

            580                 585                 590

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

        595                 600                 605

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

    610                 615                 620

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

625                 630                 635                 640

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

                645                 650                 655

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

            660                 665                 670

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

        675                 680                 685

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

    690                 695                 700

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

705                 710                 715                 720

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

                725                 730                 735

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

            740                 745                 750

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

        755                 760                 765

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

    770                 775                 780

<210>103

<211>2349

<212>DNA

<213>Artificial sequence

<400>103

atgccggcgt tggcgcgcga cgcgggcacc gtgccgctgc tcgttgtttt ttctgcaatg    60

atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat    120

aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg    180

gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct    240

gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac    300

atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta    360

caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa    420

tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata    480

agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac    540

gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat    600

tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg    660

cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa    720

tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta    780

tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga    840

ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg    900

agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg    960

gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct    1020

ttggttacca tcgtagaaaa gggatttata aatgctacca attcaagtga agattatgaa    1080

attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga    1140

tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga    1200

tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca    1260

gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa    1320

gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca    1380

tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa    1440

ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact    1500

ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt    1560

ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac    1620

ggatccagca acaccaaggt ggacaagaaa gttgagccca aatcttgtga caaaactcac    1680

acatgcccac cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc    1740

ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg    1800

gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg    1860

cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc    1920

gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc    1980

aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga    2040

gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc    2100

ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat    2160

gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc    2220

ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca    2280

tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct    2340

ccgggtaaa                                                            2349

<210>104

<211>783

<212>PRT

<213>Artificial sequence

<400>104

Met Pro Ala Leu Ala Arg Asp Ala Gly Thr Val Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val

            20                  25                  30

Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly

        35                  40                  45

Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly

    50                  55                  60

Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala

65                  70                  75                  80

Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp

                85                  90                  95

Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu

            100                 105                 110

Asn Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met

        115                 120                 125

Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe

    130                 135                 140

Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile

145                 150                 155                 160

Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met

                165                 170                 175

Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro

            180                 185                 190

Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu

        195                 200                 205

Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu

    210                 215                 220

Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu

225                 230                 235                 240

Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr

                245                 250                 255

Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys

            260                 265                 270

Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu

        275                 280                 285

Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser

    290                 295                 300

Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val

305                 310                 315                 320

Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro

                325                 330                 335

Ser Gln Ser Ala Leu Val Thr Ile Val Glu Lys Gly Phe Ile Asn Ala

            340                 345                 350

Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys

        355                 360                 365

Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr

    370                 375                 380

Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly

385                 390                 395                 400

Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr

                405                 410                 415

Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr

            420                 425                 430

Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser

        435                 440                 445

Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp

    450                 455                 460

Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu

465                 470                 475                 480

Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val

                485                 490                 495

Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val

            500                 505                 510

Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu

        515                 520                 525

Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Gly Ser Ser Asn

    530                 535                 540

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

545                 550                 555                 560

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

                565                 570                 575

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

            580                 585                 590

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

        595                 600                 605

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

    610                 615                 620

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

625                 630                 635                 640

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

                645                 650                 655

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

            660                 665                 670

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

        675                 680                 685

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

    690                 695                 700

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

705                 710                 715                 720

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

                725                 730                 735

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

            740                 745                 750

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

        755                 760                 765

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

    770                 775                 780

<210>105

<211>2349

<212>DNA

<213>Artificial sequence

<400>105

atgccggcgt tggcgcgcga cggcggccag ctgccgctgc tcgttgtttt ttctgcaatg    60

atatttggga ctattacaaa tcaagatctg cctgtgatca agtgtgtttt aatcaatcat    120

aagaacaatg attcatcagt ggggaagtca tcatcatatc ccatggtatc agaatccccg    180

gaagacctcg ggtgtgcgtt gagaccccag agctcaggga cagtgtacga agctgccgct    240

gtggaagtgg atgtatctgc ttccatcaca ctgcaagtgc tggtcgatgc cccagggaac    300

atttcctgtc tctgggtctt taagcacagc tccctgaatt gccagccaca ttttgattta    360

caaaacagag gagttgtttc catggtcatt ttgaaaatga cagaaaccca agctggagaa    420

tacctacttt ttattcagag tgaagctacc aattacacaa tattgtttac agtgagtata    480

agaaataccc tgctttacac attaagaaga ccttacttta gaaaaatgga aaaccaggac    540

gccctggtct gcatatctga gagcgttcca gagccgatcg tggaatgggt gctttgcgat    600

tcacaggggg aaagctgtaa agaagaaagt ccagctgttg ttaaaaagga ggaaaaagtg    660

cttcatgaat tatttgggac ggacataagg tgctgtgcca gaaatgaact gggcagggaa    720

tgcaccaggc tgttcacaat agatctaaat caaactcctc agaccacatt gccacaatta    780

tttcttaaag taggggaacc cttatggata aggtgcaaag ctgttcatgt gaaccatgga    840

ttcgggctca cctgggaatt agaaaacaaa gcactcgagg agggcaacta ctttgagatg    900

agtacctatt caacaaacag aactatgata cggattctgt ttgcttttgt atcatcagtg    960

gcaagaaacg acaccggata ctacacttgt tcctcttcaa agcatcccag tcaatcagct    1020

ttggttacca tcgtagaaaa gggatttata aatgctacca attcaagtga agattatgaa    1080

attgaccaat atgaagagtt ttgtttttct gtcaggttta aagcctaccc acaaatcaga    1140

tgtacgtgga ccttctctcg aaaatcattt ccttgtgagc aaaagggtct tgataacgga    1200

tacagcatat ccaagttttg caatcataag caccagccag gagaatatat attccatgca    1260

gaaaatgatg atgcccaatt taccaaaatg ttcacgctga atataagaag gaaacctcaa    1320

gtgctcgcag aagcatcggc aagtcaggcg tcctgtttct cggatggata cccattacca    1380

tcttggacct ggaagaagtg ttcagacaag tctcccaact gcacagaaga gatcacagaa    1440

ggagtctgga atagaaaggc taacagaaaa gtgtttggac agtgggtgtc gagcagtact    1500

ctaaacatga gtgaagccat aaaagggttc ctggtcaagt gctgtgcata caattccctt    1560

ggcacatctt gtgagacgat ccttttaaac tctccaggcc ccttcccttt catccaagac    1620

ggatccagca acaccaaggt ggacaagaaa gttgagccca aatcttgtga caaaactcac    1680

acatgcccac cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc    1740

ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg    1800

gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg    1860

cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc    1920

gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc    1980

aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga    2040

gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc    2100

ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat    2160

gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc    2220

ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca    2280

tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct    2340

ccgggtaaa                                                            2349

<210>106

<211>783

<212>PRT

<213>Artificial sequence

<400>106

Met Pro Ala Leu Ala Arg Asp Gly Gly Gln Leu Pro Leu Leu Val Val

1               5                   10                  15

Phe Ser Ala Met Ile Phe Gly Thr Ile Thr Asn Gln Asp Leu Pro Val

            20                  25                  30

Ile Lys Cys Val Leu Ile Asn His Lys Asn Asn Asp Ser Ser Val Gly

        35                  40                  45

Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly

    50                  55                  60

Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala

65                  70                  75                  80

Val Glu Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp

                85                  90                  95

Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu

            100                 105                 110

Asn Cys Gln Pro Hi s Phe Asp Leu Gln Asn Arg Gly Val Val Ser Met

        115                  120                 125

Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe

    130                 135                 140

Ile Gln Ser Glu Ala Thr Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile

145                 150                 155                 160

Arg Asn Thr Leu Leu Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Met

                165                 170                 175

Glu Asn Gln Asp Ala Leu Val Cys Ile Ser Glu Ser Val Pro Glu Pro

            180                 185                 190

Ile Val Glu Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cys Lys Glu

        195                 200                 205

Glu Ser Pro Ala Val Val Lys Lys Glu Glu Lys Val Leu His Glu Leu

    210                 215                 220

Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Glu Leu Gly Arg Glu

225                 230                 235                 240

Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gln Thr Pro Gln Thr Thr

                245                 250                 255

Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg Cys

            260                 265                 270

Lys Ala Val His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu

        275                 280                 285

Asn Lys Ala Leu Glu Glu Gly Asn Tyr Phe Glu Met Ser Thr Tyr Ser

    290                 295                 300

Thr Asn Arg Thr Met Ile Arg Ile Leu Phe Ala Phe Val Ser Ser Val

305                 310                 315                 320

Ala Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro

                325                 330                 335

Ser Gln Ser Ala Leu Val Thr Ile Val Glu Lys Gly Phe Ile Asn Ala

            340                 345                 350

Thr Asn Ser Ser Glu Asp Tyr Glu Ile Asp Gln Tyr Glu Glu Phe Cys

        355                 360                 365

Phe Ser Val Arg Phe Lys Ala Tyr Pro Gln Ile Arg Cys Thr Trp Thr

    370                 375                 380

Phe Ser Arg Lys Ser Phe Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly

385                 390                 395                 400

Tyr Ser Ile Ser Lys Phe Cys Asn His Lys His Gln Pro Gly Glu Tyr

                405                 410                 415

Ile Phe His Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr

            420                 425                 430

Leu Asn Ile Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Ser Ala Ser

        435                 440                 445

Gln Ala Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp

    450                 455                 460

Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Thr Glu

465                 470                 475                 480

Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val

                485                 490                 495

Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Ile Lys Gly Phe Leu Val

            500                 505                 510

Lys Cys Cys Ala Tyr Asn Ser Leu Gly Thr Ser Cys Glu Thr Ile Leu

        515                 520                 525

Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile Gln Asp Gly Ser Ser Asn

    530                 535                 540

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

545                 550                 555                 560

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

                565                 570                 575

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

            580                 585                 590

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

        595                 600                 605

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

    610                 615                 620

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

625                 630                 635                 640

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

                645                 650                 655

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

            660                 665                 670

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

        675                 680                 685

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

    690                 695                 700

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

705                 710                 715                 720

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

                725                 730                 735

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

            740                 745                 750

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

        755                 760                 765

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

    770                 775                 780

<210>107

<211>27

<212>DNA

<213>Artificial sequence

<400>107

ggggatatcc cggagtcggc atgaatc                                        27

<210>108

<211>27

<212>DNA

<213>Artificial sequence

<400>108

ccctgcgaat tcactctcct gccgatg                                        27

<210>109

<211>60

<212>DNA

<213>People

<400>109

atgaatcgct gctgggcgct cttcctgtct ctctgctgct acctgcgtct ggtcagcgcc    60

<210>110

<211>20

<212>PRT

<213>People

<400>110

Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg

1               5                   10                  15

Leu Val Ser Ala

            20

<210>111

<211>183

<212>DNA

<213>People

<400>111

gagggggacc ccattcccga ggagctttat gagatgctga gtgaccactc gatccgctcc    60

tttgatgatc tccaacgcct gctgcacgga gaccccggag aggaagatgg ggccgagttg    120

gacctgaaca tgacccgctc ccactctgga ggcgagctgg agagcttggc tcgtggaaga    180

agg                                                                  183

<210>112

<211>61

<212>PRT

<213>People

<400>112

Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu Ser Asp His

1               5                   10                  15

Ser Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu Leu His Gly Asp Pro

            20                  25                  30

Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu Asn Met Thr Arg Ser His

        35                  40                  45

Ser Gly Gly Glu Leu Glu Ser Leu Ala Arg Gly Arg Arg

    50                  55                  60

<210>113

<211>327

<212>DNA

<213>People

<400>113

agcctgggtt ccctgaccat tgctgagccg gccatgatcg ccgagtgcaa gacgcgcacc    60

gaggtgttcg agatctcccg gcgcctcata gaccgcacca acgccaactt cctggtgtgg    120

ccgccctgtg tggaggtgca gcgctgctcc ggctgctgca acaaccgcaa cgtgcagtgc    180

cgccccaccc aggtgcagct gcgacctgtc caggtgagaa agatcgagat tgtgcggaag    240

aagccaatct ttaagaaggc cacggtgacg ctggaagacc acctggcatg caagtgtgag    300

acagtggcag ctgcacggcc tgtgacc                                        327

<210>114

<211>109

<212>PRT

<213>People

<400>114

Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu Cys

1               5                   10                  15

Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp Arg

            20                  25                  30

Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln Arg

        35                  40                  45

Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr Gln

    50                  55                  60

Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg Lys

65                  70                  75                  80

Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu Ala

                85                  90                  95

Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr

            100                 105

<210>115

<211>153

<212>DNA

<213>People

<400>115

cgaagcccgg ggggttccca ggagcagcga gccaaaacgc cccaaactcg ggtgaccatt    60

cggacggtgc gagtccgccg gccccccaag ggcaagcacc ggaaattcaa gcacacgcat    120

gacaagacgg cactgaagga gacccttgga gcc                                 153

<210>116

<211>51

<212>PRT

<213>People

<400>116

Arg Ser Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr

1               5                   10                  15

Arg Val Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys

            20                  25                  30

His Arg Lys Phe Lys His Thr His Asp Lys Thr Ala Leu Lys Glu Thr

        35                  40                  45

Leu Gly Ala

    50

<210>117

<211>723

<212>DNA

<213>Artificial sequence

<400>117

atgaatcgct gctgggcgct cttcctgtct ctctgctgct acctgcgtct ggtcagcgcc    60

gagggggacc ccattcccga ggagctttat gagatgctga gtgaccactc gatccgctcc    120

tttgatgatc tccaacgcct gctgcacgga gaccccggag aggaagatgg ggccgagttg    180

gacctgaaca tgacccgctc ccactctgga ggcgagctgg agagcttggc tcgtggaaga    240

aggagcctgg gttccctgac cattgctgag ccggccatga tcgccgagtg caagacgcgc    300

accgaggtgt tcgagatctc ccggcgcctc atagaccgca ccaacgccaa cttcctggtg    360

tggccgccct gtgtggaggt gcagcgctgc tccggctgct gcaacaaccg caacgtgcag    420

tgccgcccca cccaggtgca gctgcgacct gtccaggtga gaaagatcga gattgtgcgg    480

aagaagccaa tctttaagaa ggccacggtg acgctggaag accacctggc atgcaagtgt    540

gagacagtgg cagctgcacg gcctgtgacc cgaagcccgg ggggttccca ggagcagcga    600

gccaaaacgc cccaaactcg ggtgaccatt cggacggtgc gagtccgccg gccccccaag    660

ggcaagcacc ggaaattcaa gcacacgcat gacaagacgg cactgaagga gacccttgga    720

gcc                                                                  723

<210>118

<211>241

<212>PRT

<213>Artificial sequence

<400>118

Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg

1               5                   10                  15

Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met

            20                  25                  30

Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu Leu

        35                  40                  45

His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu Asn Met

    50                  55                  60

Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala Arg Gly Arg

65                  70                  75                  80

Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu

                85                  90                  95

Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp

            100                 105                 110

Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln

        115                 120                 125

Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr

    130                 135                 140

Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg

145                 150                 155                 160

Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu

                165                 170                 175

Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser

            180                 185                 190

Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr Arg Val

        195                 200                 205

Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys His Arg

    210                 215                 220

Lys Phe Lys His Thr His Asp Lys Thr Ala Leu Lys Glu Thr Leu Gly

225                 230                 235                 240

Ala

<210>119

<211>1299

<212>DNA

<213>Artificial sequence

<400>119

atgaatcgct gctgggcgct cttcctgtct ctctgctgct acctgcgtct ggtcagcgcc    60

gagggggacc ccattcccga ggagctttat gagatgctga gtgaccactc gatccgctcc    120

tttgatgatc tccaacgcct gctgcacgga gaccccggag aggaagatgg ggccgagttg    180

gacctgaaca tgacccgctc ccactctgga ggcgagctgg agagcttggc tcgtggaaga    240

aggagcctgg gttccctgac cattgctgag ccggccatga tcgccgagtg caagacgcgc    300

accgaggtgt tcgagatctc ccggcgcctc atagaccgca ccaacgccaa cttcctggtg    360

tggccgccct gtgtggaggt gcagcgctgc tccggctgct gcaacaaccg caacgtgcag    420

tgccgcccca cccaggtgca gctgcgacct gtccaggtga gaaagatcga gattgtgcgg    480

aagaagccaa tctttaagaa ggccacggtg acgctggaag accacctggc atgcaagtgt    540

gagacagtgg cagctgcacg gcctgtgacc 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>120

<211>433

<212>PRT

<213>Artificial sequence

<400>120

Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg

1               5                   10                  15

Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met

            20                  25                  30

Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu Leu

        35                  40                  45

His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu Asn Met

    50                  55                  60

Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala Arg Gly Arg

65                  70                  75                  80

Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu

                85                  90                  95

Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp

            100                 105                 110

Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln

        115                 120                 125

Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr

    130                 135                 140

Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg

145                 150                 155                 160

Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu

                165                 170                 175

Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr 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>121

<211>28

<212>DNA

<213>Artificial sequence

<400>121

ggtgatatcg ccatcatgaa ctttctgc                                        28

<210>122

<211>28

<212>DNA

<213>Artificial sequence

<400>122

ttatcaggat ccgctagctc accgcctc                                        28

<210>123

<211>78

<212>DNA

<213>People

<400>123

atgaactttc tgctgtcttg ggtgcattgg agccttgcct tgctgctcta cctccaccat    60

gccaagtggt cccaggct                                                  78

<210>124

<211>26

<212>PRT

<213>People

<400>124

Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu

1               5                   10                  15

Tyr Leu His His Ala Lys Trp Ser Gln Ala

            20                  25

<210>125

<211>495

<212>DNA

<213>People

<400>125

gcacccatgg cagaaggagg ggggcagaat catcacgaag tggtgaagtt catggatgtc    60

tatcagcgca gctactgcca tccaatcgag accctggtgg acatcttcca ggagtaccct    120

gatgagatcg agtacatctt caagccatcc tgtgtgcccc tgatgcgatg cgggggctgc    180

tgcaatgacg agggcctgga gtgtgtgccc actgaggagt ccaacatcac catgcagatt    240

atgcggatca aacctcacca aggccagcac ataggagaga tgagcttcct acagcacaac    300

aaatgtgaat gcagaccaaa gaaagataga gcaagacaag aaaatccctg tgggccttgc    360

tcagagcgga gaaagcattt gtttgtacaa gatccgcaga cgtgtaaatg ttcctgcaaa    420

aacacagact cgcgttgcaa ggcgaggcag cttgagttaa acgaacgtac ttgcagatgt    480

gacaagccga ggcgg                                                     495

<210>126

<211>495

<212>DNA

<213>Artificial sequence

<400>126

gcacccatgg cagaaggagg agggcagaat catcacgaag tggtgaagtt catggatgtc    60

tatcagcgca gctactgcca tccaatcgag accctggtgg acatcttcca ggagtaccct    120

gatgagatcg agtacatctt caagccatcc tgtgtgcccc tgatgcgatg cgggggctgc    180

tgcaatgacg agggcctgga gtgtgtgccc actgaggagt ccaacatcac catgcagatt    240

atgcggatca aacctcacca aggccagcac ataggagaga tgagcttcct acagcacaac    300

aaatgtgaat gcagaccaaa gaaagataga gcaagacaag aaaatccctg tgggccttgc    360

tcagagcgga gaaagcattt gtttgtacaa gatccgcaga cgtgtaaatg ttcctgcaaa    420

aacacagact cgcgttgcaa ggcgaggcag cttgagttaa acgaacgtac ttgcagatgt    480

gacaagccga ggcgg                                                     495

<210>127

<211>165

<212>PRT

<213>People

<400>127

Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys

1               5                   10                  15

Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu

            20                  25                  30

Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys

        35                  40                  45

Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu

    50                  55                  60

Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile

65                  70                  75                  80

Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe

                85                  90                  95

Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg

            100                 105                 110

Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe

        115                 120                 125

Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser

    130                 135                 140

Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys

145                 150                 155                 160

Asp Lys Pro Arg Arg

                165

<210>128

<211>573

<212>DNA

<213>Artificial sequence

<400>128

atgaactttc tgctgtcttg ggtgcattgg agccttgcct tgctgctcta cctccaccat  60

gccaagtggt cccaggctgc acccatggca gaaggagggg ggcagaatca tcacgaagtg  120

gtgaagttca tggatgtcta tcagcgcagc tactgccatc caatcgagac cctggtggac  180

atcttccagg agtaccctga tgagatcgag tacatcttca agccatcctg tgtgcccctg  240

atgcgatgcg ggggctgctg caatgacgag ggcctggagt gtgtgcccac tgaggagtcc  300

aacatcacca tgcagattat gcggatcaaa cctcaccaag gccagcacat aggagagatg  360

agcttcctac agcacaacaa atgtgaatgc agaccaaaga aagatagagc aagacaagaa  420

aatccctgtg ggccttgctc agagcggaga aagcatttgt ttgtacaaga tccgcagacg  480

tgtaaatgtt cctgcaaaaa cacagactcg cgttgcaagg cgaggcagct tgagttaaac  540

gaacgtactt gcagatgtga caagccgagg cgg                               573

<210>129

<211>573

<212>DNA

<213>Artificial sequence

<400>129

atgaactttc tgctgtcttg ggtgcattgg agccttgcct tgctgctcta cctccaccat    60

gccaagtggt cccaggctgc acccatggca gaaggaggag ggcagaatca tcacgaagtg    120

gtgaagttca tggatgtcta tcagcgcagc tactgccatc caatcgagac cctggtggac    180

atcttccagg agtaccctga tgagatcgag tacatcttca agccatcctg tgtgcccctg    240

atgcgatgcg ggggctgctg caatgacgag ggcctggagt gtgtgcccac tgaggagtcc    300

aacatcacca tgcagattat gcggatcaaa cctcaccaag gccagcacat aggagagatg    360

agcttcctac agcacaacaa atgtgaatgc agaccaaaga aagatagagc aagacaagaa    420

aatccctgtg ggccttgctc agagcggaga aagcatttgt ttgtacaaga tccgcagacg    480

tgtaaatgtt cctgcaaaaa cacagactcg cgttgcaagg cgaggcagct tgagttaaac    540

gaacgtactt gcagatgtga caagccgagg cgg                                 573

<210>130

<211>191

<212>PRT

<213>Artificial sequence

<400>130

Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu

1               5                   10                  15

Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly

            20                  25                  30

Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln

        35                  40                  45

Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu

    50                  55                  60

Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu

65                  70                  75                  80

Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro

                85                  90                  95

Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His

            100                 105                 110

Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys

        115                 120                 125

Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly

    130                 135                 140

Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr

145                 150                 155                 160

Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln

                165                 170                 175

Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg

            180                 185                 190

<210>131

<211>1302

<212>DNA

<213>Artificial sequence

<400>131

atgaactttc tgctgtcttg ggtgcattgg agccttgcct tgctgctcta cctccaccat  60

gccaagtggt cccaggctgc acccatggca gaaggagggg ggcagaatca tcacgaagtg  120

gtgaagttca tggatgtcta tcagcgcagc tactgccatc caatcgagac cctggtggac  180

atcttccagg agtaccctga tgagatcgag tacatcttca agccatcctg tgtgcccctg  240

atgcgatgcg ggggctgctg caatgacgag ggcctggagt gtgtgcccac tgaggagtcc  300

aacatcacca tgcagattat gcggatcaaa cctcaccaag gccagcacat aggagagatg  360

agcttcctac agcacaacaa atgtgaatgc agaccaaaga aagatagagc aagacaagaa  420

aatccctgtg ggccttgctc agagcggaga aagcatttgt ttgtacaaga tccgcagacg  480

tgtaaatgtt cctgcaaaaa cacagactcg cgttgcaagg cgaggcagct tgagttaaac  540

gaacgtactt gcagatgtga caagccgagg cggggatcca gcaacaccaa ggtggacaag  600

aaagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa  660

ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc  720

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc  780

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag  840

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg  900

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag  960

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca  1020

tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat  1080

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc  1140

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac  1200

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac  1260

aaccactaca cgcagaagag cctctccctg tctccgggta aa                     1302

<210>132

<211>1302

<212>DNA

<213>Artificial sequence

<400>132

atgaactttc tgctgtcttg ggtgcattgg agccttgcct tgctgctcta cctccaccat    60

gccaagtggt cccaggctgc acccatggca gaaggaggag ggcagaatca tcacgaagtg    120

gtgaagttca tggatgtcta tcagcgcagc tactgccatc caatcgagac cctggtggac    180

atcttccagg agtaccctga tgagatcgag tacatcttca agccatcctg tgtgcccctg    240

atgcgatgcg ggggctgctg caatgacgag ggcctggagt gtgtgcccac tgaggagtcc    300

aacatcacca tgcagattat gcggatcaaa cctcaccaag gccagcacat aggagagatg    360

agcttcctac agcacaacaa atgtgaatgc agaccaaaga aagatagagc aagacaagaa    420

aatccctgtg ggccttgctc agagcggaga aagcatttgt ttgtacaaga tccgcagacg    480

tgtaaatgtt cctgcaaaaa cacagactcg cgttgcaagg cgaggcagct tgagttaaac    540

gaacgtactt gcagatgtga caagccgagg cggggatcca gcaacaccaa ggtggacaag    600

aaagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa    660

ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc    720

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc    780

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag    840

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg  900

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag  960

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca  1020

tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat  1080

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc  1140

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac  1200

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac  1260

aaccactaca cgcagaagag cctctccctg tctccgggta aa                     1302

<210>133

<211>434

<212>PRT

<213>Artificial sequence

<400>133

Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu

1               5                   10                  15

Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly

            20                  25                  30

Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln

        35                  40                  45

Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu

    50                  55                  60

Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu

65                  70                  75                  80

Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro

                85                  90                  95

Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His

            100                 105                 110

Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys

        115                 120                 125

Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly

    130                 135                 140

Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr

145                 150                 155                 160

Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln

                165                 170                 175

Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg Gly

            180                 185                 190

Ser Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

        195                 200                 205

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

    210                 215                 220

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

225                 230                 235                 240

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

                245                 250                 255

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

            260                 265                 270

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

        275                 280                 285

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

    290                 295                 300

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

305                 310                 315                 320

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

                325                 330                 335

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

            340                 345                 350

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

        355                 360                 365

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

    370                 375                 380

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

385                 390                 395                 400

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

                405                 410                 415

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

            420                 425                 430

Gly Lys

<210>134

<211>2145

<212>DNA

<213>People

<400>134

atgggggtgc acggtgagta ctcgcgggct gggcgctccc gccgcccggg tccctgtttg    60

agcggggatt tagcgccccg gctattggcc aggaggtggc tgggttcaag gaccggcgac    120

ttgtcaagga ccccggaagg gggagggggg tggggcagcc tccacgtgcc agcggggact    180

tgggggagtc cttggggatg gcaaaaacct gacctgtgaa ggggacacag tttgggggtt    240

gaggggaaga aggtttgggg gttctgctgt gccagtggag aggaagctga taagctgata    300

acctgggcgc tggagccacc acttatctgc cagaggggaa gcctctgtca caccaggatt    360

gaagtttggc cggagaagtg gatgctggta gctgggggtg gggtgtgcac acggcagcag    420

gattgaatga aggccaggga ggcagcacct gagtgcttgc atggttgggg acaggaagga    480

cgagctgggg cagagacgtg gggatgaagg aagctgtcct tccacagcca cccttctccc    540

tccccgcctg actctcagcc tggctatctg ttctagaatg tcctgcctgg ctgtggcttc    600

tcctgtccct gctgtcgctc cctctgggcc tcccagtcct gggcgcccca ccacgcctca    660

tctgtgacag ccgagtcctg gagaggtacc tcttggaggc caaggaggcc gagaatatca    720

cggtgagacc ccttccccag cacattccac agaactcacg ctcagggctt cagggaactc    780

ctcccagatc caggaacctg gcacttggtt tggggtggag ttgggaagct agacactgcc    840

cccctacata agaataagtc tggtggcccc aaaccatacc tggaaactag gcaaggagca    900

aagccagcag atcctacggc ctgtgggcca gggccagagc cttcagggac ccttgactcc    960

ccgggctgtg tgcatttcag acgggctgtg ctgaacactg cagcttgaat gagaatatca    1020

ctgtcccaga caccaaagtt aatttctatg cctggaagag gatggaggtg agttcctttt    1080

tttttttttt tcctttcttt tggagaatct catttgcgag cctgattttg gatgaaaggg    1140

agaatgatcg ggggaaaggt aaaatggagc agcagagatg aggctgcctg ggcgcagagg    1200

ctcacgtcta taatcccagg ctgagatggc cgagatggga gaattgcttg agccctggag    1260

tttcagacca acctaggcag catagtgaga tcccccatct ctacaaacat ttaaaaaaat    1320

tagtcaggtg aagtggtgca tggtggtagt cccagatatt tggaaggctg aggcgggagg    1380

atcgcttgag cccaggaatt tgaggctgca gtgagctgtg atcacaccac tgcactccag    1440

cctcagtgac agagtgaggc cctgtctcaa aaaagaaaag aaaaaagaaa aataatgagg    1500

gctgtatgga atacattcat tattcattca ctcactcact cactcattca ttcattcatt    1560

cattcaacaa gtcttattgc ataccttctg tttgctcagc ttggtgcttg gggctgctga    1620

ggggcaggag ggagagggtg acatgggtca gctgactccc agagtccact ccctgtaggt    1680

cgggcagcag gccgtagaag tctggcaggg cctggccctg ctgtcggaag ctgtcctgcg    1740

gggccaggcc ctgttggtca actcttccca gccgtgggag cccctgcagc tgcatgtgga    1800

taaagccgtc agtggccttc gcagcctcac cactctgctt cgggctctgg gagcccaggt    1860

gagtaggagc ggacacttct gcttgccctt tctgtaagaa ggggagaagg gtcttgctaa    1920

ggagtacagg aactgtccgt attccttccc tttctgtggc actgcagcga cctcctgttt    1980

tctccttggc agaaggaagc catctcccct ccagatgcgg cctcagctgc tccactccga    2040

acaatcactg ctgacacttt ccgcaaactc ttccgagtct actccaattt cctccgggga    2100

aagctgaagc tgtacacagg ggaggcctgc aggacagggg acaga                    2145

<210>135

<211>18238

<212>DNA

<213>People

<400>135

atgaatcgct gctgggcgct cttcctgtct ctctgctgct acctgcgtct ggtcagcgcc    60

gaggtgagtt gccacggcgg ctggggctgg ttcttcattc attaccttcg cccccccctt    120

ctgaccgccc cctcctctcc ctgcagtgaa ctttggaccc ttgcagcccg cgagcctgac    180

gccgggcgct gggtgacctc ttcgggctgg gagcgaggtc cgggggtgac aggctctaag    240

ggaaggcaac agcggtggct ttctttccaa ccggcgggcg aatctggctc cctaagccgt    300

tccgtgtcgg gggagggtgt gtgtggccct gtcccccacc ctttgggaac ccgagaacaa    360

gccccttccc ggccggggga gagggggtgg ggtggtgccc agggtgcaga aggcagcgcg    420

tcctcccgag cccacttcgg cgccagcctc ggcttaggct ctgtcctgcc atcggcttgc    480

ccaggaggtg caagcttgcg caccggtgcc atccacgccg acctttgacg gggctcgggt    540

cccaccctgg ctcccggtcg gggtccgggt ggacgccctc cggcacgcgc agggcatgcc    600

cggccccaga gcacggtctg gaagcagctt gtgtctcggg cgcggggttt ggggtgggag    660

taacagggag agagagagag agagagagaa agagagagag agagagagag acggagagag    720

agacggagag agagagggag agaaagatag agacgcaggt aggtttcggg cactcgggta    780

gggggaggac taggggctct gaaggaatcc agcccccggg gcgctgtgtg cctgaggcct    840

tgccgtacct gtgcccgcgc cctgcaggag gaagcacctc tctcccccgc atgcagggtg    900

cggagcgcgc cgcctgcaaa agcctagctg cacgggagat ggggtataaa aacatctgag    960

ggagcgctgg ggtcggagcc gtccccaccc ctcccagcct cacgtagccg tgagttggca    1020

agtctacccg gaatccaggt gggaaagggg gcggggcagg acgacgcgcg cggtggaggg    1080

gaggagaggg cagccgggag cgcggcgtga ctgggtaccg tcccacctcc gcccggctgc    1140

ggccgggagt ccgcacctcg cccgcacctc gccggctccc ggggagggtc ggcccctgcg    1200

gcctccagcc aggcgcagga aggaggcagc ggcggcgaag gggttaaggc ggagggctcg    1260

gaggccgcgg cccggccttg ggctgccgcc agcgcaggtt gttttgacca cggaggagcc    1320

gtcgccgtct ccttttgttc tcggggctcc tcgagggccg caggccgccc gcccgggggc    1380

cccgcccttc cgcggccgcc ccccgcggcc cgcacccggg agggaggacg cggggctcgg    1440

ccgggccgcc ttcaggcccc tcccgacgcc ccctctccgc ctctcccgca gccccccggg    1500

ccgcggccag ctgttgggga gggggtggcc ggccggcccc agctgccgcc tcgcctcgcc    1560

gtgggacctg ggggctgggc ccggtgccag ggcgtcctgg gaacggcggc gccccgtggc    1620

tgctctccgc agcccacccc gcccgggccc ccgactcgct cactcacccc acgcatgcac    1680

actcctggcc ggaggcgatg ctgcgctccg gcgggcgggc gcgcagagcg acgggcacgc    1740

actaccgcgg ccaggtcgcg cgcccgcctc tgccacgccc gtgcacacgc gggacacacg    1800

cgcgcgcacc gcacacaaac gcacgcacgg cccctgcacg cgcggcctga gcacacgtgc    1860

gcgcacaccc acgcacgtac acaggcacgc acgggccccc gtgcacccag cgcctggtgc    1920

tcgcccccgc gcaacaggtg ggccctccgt gggccctggc acctcaccac ctctgtagcg    1980

gccccatttc cttcctggcg tcctgtgagg gagggagaac ctcccatcag caccacagca    2040

cctacttttt tttttgcctc gtcagcccga cgcccctcaa accttaccca tctgtgactc    2100

ctttttttca accacctccg cgtggtggaa aatggtggtg atgtgactct gaggggcact    2160

gagctgtcca gagcgattcc cccttcacat aagccttcat ttgaacctgc aagactggag    2220

ggacctggcg tgtgcaaccg cggagggggc tcccacccct ggctgttgca ttctcttggc    2280

tgatcccagc gtgccccggg gaggccgctg acagctggat gtttccccag cctcccctta    2340

ccatttccag cttcgtccag cacctcctcc ttctttccca cagctccacg ggctcgtgta    2400

tctggggtgg aggctgtggc acagaaactg cctttctcct cactttagtc acagcattct    2460

tgaacacatg gccacaggcg cgatgtatgt ggcactttgc agtttatgaa gcactttgct    2520

gctaagcctg agtgagcctc aggctggccc tgggggaggg gacctgcatg gggatggaac    2580

cacgcagggg tcagtccagg aaggagctgt aatggccagt gctgggagag tcagggcagg    2640

cctgctggtg gaggtggcct tggagctgtc cacgtcctgg tcgtgctcgg actaatcttt    2700

cagcagacgg caggcagccg tgaggcaggg ctgggtggag ggcctgccga ggcctctgag    2760

gtgccatctc caccagctga gctggcttcc aggagggcga gtcccactgt cacgtgacgc    2820

gtctggcctc agcacacttc ttccgggaaa gagtgaaggg ccccactgcc ctttgccatc    2880

cagcttcctc tggctttgct aatggcccta gggggcagga gaccaactgc tggaatccca    2940

gagccctgga ggtgtgcaag ggcaggtcaa acagaatttg gaggatctgg tgcaagagcc    3000

aggaagagag agagagagag agtgtgtgtg tgtgtgtgtg tgcgcatctg agagagagag    3060

agagagagac tgactgagca ggaatggtga gatgtttatc atgggcctcg taagtacctc    3120

tccacgtctt gtcttcccct ccccacattg aggagcctct tctgtgacaa ctcttcctat    3180

gttctggttt atttcattgt ttattacctg ctttctctac tggagtgtca accccattag    3240

agagctttcc tcctggtccc cacttttaga acagtgccca gagccctgtg gacaatcagt    3300

aactattagt gcaaagaaca gttagtgctg ggttggtggg gagggcagga cctggccccc    3360

tgttgctcca tggccccact gagtcctcgc tttgctctgc tccgagggca gccccccttt    3420

tcagccctct ggtaggcatc agtctttcaa agccccccat cttgagagag aagcatgtct    3480

gtctctcctc tgtcttggga cttgggctac caggattaga caggacagag gagaagaaac    3540

agaaagaaaa tcagactgca aaggttctgt gtatctttag ctggggctaa gtttctgcca    3600

cctaaaaaaa aaaagagaga gagaagattg gattagtgca gagttcccaa agaatggcag    3660

aatcccctgg ccccgtcccc tggctgggat ggggatgggc agtgattgcc gtggttgcca    3720

gtattataaa tggctggact ggttcctctc ttcctgcttt aattttcctc tgctcctgca    3780

agaggtgggg aggccagggt tctgggccca cgtgaaggag cagaggctga gcgggagggt    3840

cccaagagca ataaagaagg ggaatgctag gtgggaaaca ctggttctaa tggcttctgt    3900

ggtttgcccc gagagggctt cttcaaaggg gttggttggc tttggcattc gatctaaata    3960

aggcctgcga ctctcaggca ggcaggctct gggaggcctc atcagcttct tcctctgcca    4020

gccacagaca acgcccctgg ttgcttgggc ctgtgtgtcc cttggtggga atggcaggcg    4080

ggccggggag tgtgtatctg tgtgtgtgtg ctgggcccag gaggagggtg gggttcaagc    4140

ccctttgatc tgccagcctg gttgggagca gatcactcac ctggcctcac gctcgctcgt    4200

gcccttccta cctgctgcag ctggcgctgg gggcggggtc ggaggaggct gtttaccttg    4260

gctcccacgg ctggctttgc cccagctgcc tcctcgccac gccctcactc tgccagaaac    4320

cccgggcctg agatcttggg acagcttctt cagggtgcca ggcctccttt cccatctctg    4380

aagtgagctg tccacctgga ggcctgcgga acctgtgccc aggaaaacca ggctccgggc    4440

ggctcacctt cccataccaa gaagagcctg tgactcccaa caggtgctca tgctcgtcat    4500

ccccagagca ttgcatcctg gagctgagca cgtgctgagt gtccccccac cctcacccac    4560

ccccagcccc ggaagggcct tgtaagccca cacggcccag gctctgccag tgtggaggta    4620

gggtaccatt tcctgtggcc cagcacaagg gataatgcaa agtcacgcac tctttcatgg    4680

gcaggcagct ctccacccac tccttgtcat cctcaaaaat gtcctgtgct gctggccctg    4740

agcacgtgtg ccactcgctg ctgcccacaa aggagccatc cggaaagaat taatgatgat    4800

gacagtgaca actcattaag ctcccattat ctgctggcag ctctgctctg tgcaattatg    4860

cctcccagta accccgggcg aggtattatt agccccattt aaaagatgca gaagccgagg    4920

ttttagattg tgacaagatt ccaagaatcc aggttgccct ggcatctgga gcccttgctc    4980

tttcctccgc tacagggttt ctcagcctct gcactcctga tgtttgggaa ggccgtcctg    5040

tgccctgaag ggtggtgagc ggcatccccg gcctccccca acttaagatg cagtgcacac    5100

cccaagttgt ggccaccagc gatgccttca tacattgcca ctgtcccctg ggggcccact    5160

gggccctggt tgagagcctc tgacgtggga catgctgcag agtaaagagc caacacgaac    5220

cttgtgtgat gctgagggcc agtggcctta gaacacagga gagggaggct gagacgcccc    5280

atggtgggct ggaacaggtg ggcagtctgg agcctgggaa cgacccagct ctgccacttg    5340

ctaattgtct ggacctgttg ctgcttgttt aattttttct aagcttcagt gttctcatct    5400

ggaggatggg gatacaaata ctgccagcct cttaggattg gtagtttata cggtgttttt    5460

ctcagcgtgt ggcttgtagc tggcattcat agatgttagt tgtggtatta ttaccaaatc    5520

cgggggctta tgggtagttc tgggcatcta gtggccacat taccaaaagc atcttcaggg    5580

acggtctccc tgctcagatt aaaagggact ctagttgctc atttgcctag ggttggggaa    5640

gaatatccca ggtggccctc ctggtggagg aggcagaaga atcatactgg acagaacctg    5700

gaaaatggta gactccttca tgcatttgac cagtatttac cgaggaccta ctatgtgcct    5760

ctctctcctc ctcagaccct atcctgggct ctttcacact cagccttgga ggggcatctc    5820

ctatgtgtcg tgggcctgga tctggggtgt ccactcggcc tgttcagggg tctgcctcct    5880

ctgccgtggc tggcagctgc agccggctct ccagtgggca gggagatctt ggcacaagca    5940

ctaaatgcca tttgctacta ggcctttggg cccctcccct ggggggttaa ttgaatcttt    6000

actccacaaa cgcttcttgt tctccttctg ccagtgatcc agggaggcct ggctaggcac    6060

ccggctttgg cgttttctct gccccttcta acgcctcctg gcagaattag aattgaatcg    6120

aatgttcttc aagcccctcg attcacagat gagagccagg accatagggg cagtgaccag    6180

ccgaggtcac acggcagttg gtccaaaggc gtgggattca gctctctggc ctgccagttc    6240

tgggtttgac agaactctca ggggaagcgg ctttcgctgg tgttcactgg agaccaagcc    6300

ctctgttctg tgggttgggt actttgtcaa catttggaga gcaccggagg cgtggtctct    6360

cttctgaggt ggaaagaatg aacttgcttg agggcgggag gaggcagagc ccggaggaga    6420

tggggccgaa agtgaggggc agcggggccc ataccctcct ctgtcttgca cgtgcagccc    6480

ctggctgccc ggatattgaa ggaaagaccc tagttctcag ccaagccctg ccaccttttt    6540

ttcctcaagt tgcccagcag cccttagaca ttgggcggag ttgtaagctc catgttcaaa    6600

caaagacatg gagactcagg ggtttcaggg aatgatgtag ctttaaaagt ggtcaccccc    6660

accccccgcc ctgagtcttc tggctcctgg atcgatgtgg gtggccagag ttggtcatgg    6720

atgaacccct ggaggtgttg ctagacctca tgataagctg gaggaaacag agctttcggg    6780

gagagccccc tgaggctggc cacagggctg agacgacctc ttctcctctc cctcgctccc    6840

cctccgccgt caggcagcga gaaagcagca tcccttttgt gaaggtagcc tgcaccgacc    6900

ccctctgagc cccccgacct tcggctccgg gaatgtggag cgaaccaaga agggaagtcc    6960

tcccttctta atccagggaa actgaggcag aaaatagttt aacccaaggt tctatagtaa    7020

tgtgccaagc ctattgatgc cctctccagg actgtctgtc cagaccgtgc agcccgagtg    7080

cctgtgcctg cacgtgtgtg tttgcacacg tggaagtccg gttccagcct ttgaacgtgc    7140

agcgacatct taggaatttt ctttgcttct cagtggctgg tggtctggat tgtgattctc    7200

agtctcccat ctgccctcgc ctctttcttg ggatctctgg catccgcccc ttgggtctcc    7260

tgggactgtg ggggacgtgg ctgtgacctt cctacggggc tgctcttgtg tcattgtggg    7320

acctgcggtt tccctggagg aagcccagct ccgaatgagg cctgttgaag tgttatcagg    7380

tttcgagtat gtttggcaac aggccaaagg ggctctaaaa atagggtttg gttgggcaca    7440

gggcacgggt aagctttcag gtttcccggc cgtgtccgag gattcccttt tcgcctcttt    7500

cgagtgagag acttcataga taccgctagg acctggggcc tcctgaggcc atcgcactct    7560

gcacgcgttt attaagcacc tcttgtaccc aggagcctgg gagtacaggc tctgtcatcc    7620

caaaggcatg ggtttgaatg cagcttttgc caataattga gcctctcttt gaacgtttgt    7680

tttcccctct gtaaaatggg ggttagattg gccatgggag atgctggctg tcttcagctg    7740

tcaggtgtgt tgtgagactc agggagaaat cagattttgg tgcattccca gaacatgtag    7800

tttggtgggg gagacagaca tagagacaag caggtccaac tcaaagcaag ctggggttcc    7860

tgttgggggt tgagggtgca gggactgagc tgggtctcag aggcttcggc aggtccaggc    7920

cccgaggcct ttgtgctcct gatcatcagg cctggatcct gcctgtccgt ctccctgtga    7980

ccttggagct ttccacagga gaaagcgaga aagcgtgtgg tgggggagac agccatgctg    8040

gaaagccccc actcccagct cactcagcct tttggtgtct gcccggcagg gggaccccat    8100

tcccgaggag ctttatgaga tgctgagtga ccactcgatc cgctcctttg atgatctcca    8160

acgcctgctg cacggagacc ccggaggtaa atggaatcct cgccccgcgc tccggccctc    8220

cgaggagact ttaagagatc tgggaggggc aggacaggag gcatccctcc ttcttgacgt    8280

ctggagaact agaggcccat ggcagcccag agagagcgtg gccacaccca tccagggcag    8340

ggccgagtca gcaggcgggt tggtacctgg gacttggggt gtggcaggag aagcacccac    8400

gtgtggctcc ggcttggtgc caagggtggg gtgccaggag gagggagggc ggggagaagg    8460

tgcccagggc atctgccgag tatgtctcca ggtgtgggcc cagccaggga gggtggcact    8520

ggaggagtca tctccccgcc cctctggcta ggggcccggt ctgtgaggtc ccctggggac    8580

acctggcctc gaccagcagg cagccctgcc cgtgccccag agctcactca gcgaatgggc    8640

acgtgctcgg tggcacacac gtggcggggc tggcgggctg gattgacaat gtatttataa    8700

acgctgtctt cagagcaaat tccattctat tctaacctct ggcctgttcc ctggagccct    8760

ggtcagcagc ccccctgcac ccccaggtcc ccttccctct ggggttctgt ctctttgtca    8820

ctttgtaatc cttgcccaga cgctatctac gggggaacag catttcctgc ctttgtttcc    8880

tctccccgtt gggcccctgg ctccctctca aaagcattcc cgggcccttt caatcccgcc    8940

tgtgctgggg ggcggtgagg caggcaggag ggggccccag ctgggcccac ctattgttcg    9000

ccagggcccc cacccgatgt ctcccacaac ccccacccta tgcccgactg gccgcccctg    9060

gccacacaca atggggcaac ttccaaattt agcctttcta ccatttcttt ccaagccttt    9120

acacccccac cctcagacgg cccctccaca cccctggtgg gggtcgggat cttggagatg    9180

aggtttcaat agcaggcctg tttcaaggca accatgtggc tattttttcc taatcaactt    9240

aacctttcca caaagcgcat cttttcccca tctcctcccc accagggaca ttccagagat    9300

ggcagggaga aaggaaagga gccagaggga cagataggct cgttgacgta ggggctgtca    9360

ggctggacgg aagcggacta gtcatcagac tccctgcagt acccgggacg gacaggcggc    9420

aggcgccttt gtcttggatg gggccagtgg gtcggtgcca gaactggagc catggggtgc    9480

tgtgtggagg tggcaggagc gtgttcccaa tgccccttag tttatttgtg gctcctcacc    9540

catgagagag acagaagggt ctgtccacgc cttaacatcc agctcagagg cctccctcag    9600

ccctcacttc ccagcagcca acctctggcc atgggctctg gaaaggaccg atgtctccaa    9660

ggagggctga tgggactgag atggtgccca ggctagaaaa gggagctgtc atttgtgacc    9720

agaggaatca acagacggct gggggcagag gtggggcaac agtttagaga aggggaggga    9780

gacagacaca gatacagata cggtgtgagg gccccggagc tagagagacc aggccctaga    9840

agggtgtgtg gcagggactc cccaccccca gcaggcccaa ctcccacccc tcttctcctc    9900

ttctctgtct tcagctccca cccccatccc tctccctccc tccctccctc cgtggctccc    9960

gcctgcccag cgcggccatt gtccggggct ccagaggggc tgtgttcagg gcatgcggtc    10020

cccctggggc ttctgggaat ttctccattc agcacttcct atgggaacgc tgggtggagg    10080

ggcactggaa actggccttc agtgccctag gtcccggccc cgccctggag gccgaggagg  10140

gttcacttac agtagcaaag gggaagggtt atttttaact ccactgacat gggttctggc  10200

caaaaatgtt ggctaaagag ccagggatgc agtcaggatc ctgggcctgc agctgtcctg  10260

ggggctgctt ggagggtccc tgctcagggc caggacccgt gcatttatgg gggctggaag  10320

gagggactgt tctctggagt gggggagagg caaacctccc gcctgcccag acaccccggc  10380

ggagggagcc cctgcccaag gtggggccgc ctggtgtctg actgtgactt ctcctgcaga  10440

ggaagatggg gccgagttgg acctgaacat gacccgctcc cactctggag gcgagctgga  10500

gagcttggct cgtggaagaa ggagcctggg taagactgag acacccaaca agggtccttc  10560

aaattagcat gggggccagg gaaagagaac gggggcgggc agccagtcgg agggccccga  10620

gggtcctctt acaactcccg ttccccccac attcaggact gagcgaactg agagagtaaa  10680

accatcctta aatgcctgtg ttcagtcatc acagcccctc gcccccacac tctctggttt  10740

ctaaatatag tgctgcaaag ctcattcgtt ccttatttca atcctggagg tagatattat  10800

taaccccatt tcacagagtt ggaaactggg gcccagagag ggtaaagaac taggcgaggt  10860

cacactgcta gggagcggta gggctgagat cccaacctgc tcccctcact tgaacccagc  10920

gtggtccctg atgggctatg ggtatctcct gtggcaaagg gccgcggcct aggctggtag  10980

acctcgagca aagcctggaa cttcctcctg ctcccttgca gagacctaaa ggagaccgta  11040

gatcattagg tcccagagaa accctcccag gtgaggcaaa tgtccactca aacccctcct  11100

cctctttgac acctggagtc cccaaaacac tgcagttaga ggctggggga acgatgaata  11160

agagtcaggt ggcctgggcc gggcatggtg gctcacgcct gtaatcctag cactttggga  11220

ggctgaggca ggcagatcac gaggtcaaga gatggagatc atcctgaccg acatggtgaa  11280

accctgtctc tattaaaaat acaaaaatta gctgggcgtg gtggcaggtt cctgtagtcc  11340

atgctacttg ggaggctgag gcaggagaat tgcttgaacc agggaggcgg aggttgcagt  11400

tagccgagac cacgccactg cactccagcc tggcgacaga gcaagactcc gtctcaaaaa  11460

aaaagaaaaa agtcaggtgg cctgagttcc acccccaatc tatgaatgtg ggcggacggg  11520

atattcagct ctcagagcct cctgtcctct gtacatggat agcatcggta acctcagtgc  11580

ttgctgtagg caaggcttac tcactttaca ccttagatcc acatgggagg gaggtgctgt  11640

tattatccca tttcatagag gaggaagctg aggctttgaa agcagcagtg atgcaggaat  11700

ccaaggcacc gtgagacagc agtgcacgcg tggtttcgag ctaatatggt acctgcataa  11760

aaaccacatg ttgggcaagc ccataaatgt gtaaaatcat aaagaagaat aaaatagtca  11820

tgtgccagca gagggaagcg gacatttact aagcatttac aacatgcccc acactctgaa  11880

ttttctcact tgaaactaca gtggttcttg ggtgtagatc tcatggtccc attttcagat  11940

ggggaaacac aggcttagag tggctcagcc atctgcccaa gtcccatggc taatgacagc  12000

caggacttga aacctgaacc ctggggttga agggcgtgag aaagagcagt cccggaggca  12060

cccgctgagc tcccctgcca agtgggggga ctctggacag gcctacgctc ctgacccagg  12120

tttcgtctcc tcccaggttc cctgaccatt gctgagccgg ccatgatcgc cgagtgcaag  12180

acgcgcaccg aggtgttcga gatctcccgg cgcctcatag accgcaccaa cgccaacttc  12240

ctggtgtggc cgccctgtgt ggaggtgcag cgctgctccg gctgctgcaa caaccgcaac  12300

gtgcagtgcc gccccaccca ggtgcagctg cgacctgtcc aggtgcgtag gctccgcggc  12360

cccccgaggc tggtcccggt ggtgggtgga gaccaccctt ctggggctca tacctgacca  12420

ggcttccttg actgggcaga gtggtgtgtc gaaggacatt cctgatttgt tcctgggctc  12480

tgtccagaga ggcccactct tgggaagctc ttcctaggca ggtggactga ggccgaccat  12540

gagtcctctg aggtggctca ggccactgta gccaaaagcc acagacagac cccccgggct  12600

gggcgaggtg tggagccaac gtgaagtagg gatgtcacct cctttacaag gacaaaatcg  12660

caaaacgtcc cttgaagagc cgtatccgca aacatttatc tgacactcag aatgttttgg  12720

ggagctactc aggttccccc aaagctctcc ctattcaggg ctgtcttgag aaaggctcag  12780

atgggtggcc ctgcatcttt gacacctgct tgtctctctt tgacaagctg tgtctttaat  12840

aaagagagtg cagagaggcc caccaggtga tagacactgc ggagctgggg caggtcccct  12900

gacaacagtg gaatgctgat cctgcttttc ttcggaggga cgggggatac atacactgga  12960

ctcagggaaa attctagagt agaggctgtc tcagggagca ccctggtctc accctagtcg  13020

gggcagagtc agagaagctg ctgcttctgc ttcttgtgga aagattcctg cacgccccag  13080

gccagcctcg cccacaggct gggcagtgcg catttctcta gctcagcacc ggcccttgcc  13140

cgatgctctg cctgcttagc cgcacttcct tctcctgcca ccagcccgtg gcctctcctc  13200

ctgcccacct tccctccctg gcttgggccc cacgcctgag actacgaatg tcactccacc  13260

ccactgccca ctctcgtggt atctcctgtc ctcgatgctt ccagctctat ggatggacac  13320

ctgacatctg acctccccgt tcccacctcc ctcctagatg aagcctcccc ccacttcctt  13380

ccagacaacc atctcccgcc tctgccgcag ccctgacctt ggctggcgct ccgggaatga  13440

ggataccgca ggccccagct caacccggaa atgcctttct ggctgtccct ctctgggggc  13500

actgaggggg gccccaggtc gggagggctt gttttgatgg aaaagctacg agaagggcag  13560

aggtcaaggt cctgctattg tttgggccaa agcttttgcc ccacagcctg ctctccgggc  13620

tttcgaggaa agattgctgc aggggacacc ggggtgggaa gcgcaagcca ttcgggcttc  13680

ccgggctcct ggctgtgtgg tctctacaga ggcattttgt ggctctgtcc tccaggtgag  13740

aaagatcgag attgtgcgga agaagccaat ctttaagaag gccacggtga cgctggaaga  13800

ccacctggca tgcaagtgtg agacagtggc agctgcacgg cctgtgaccc gaagcccggg  13860

gggttcccag gagcagcgag gtaaccacct ttccaggctc agccctcagc ccccttcccc  13920

tgcagcagct gggggcaggg gccggtctta aaaatggaaa tgggatcagg cccgcccccg  13980

gttaaattca cgggaggctt tcttttcatc ttgctgagac acaagccagg ttcctccctg  14040

gcctgagagc cctacatgag gattagcccc tggctggctc tctggtctct gccaaccagt    14100

acccttcacc tgtgccaccc ccgccgggca ggcctctttc agtaactaat ggagtttctg    14160

cctcaaggct ttagcacaca ctgctgtggc tgaagaacac ttctcccagc tctccacaag    14220

cctagctcct tctcttcatt agagtctcag ctcagaagtc acctcctcct agaagtctgc    14280

cctgaccacc ccatccagag cagcgccatt tccagtcttg ctaccatagc cccctgcttt    14340

gttggatgtg gggatgggat ggtggaagga atctgggcct ggatgagaga aagctccgcg    14400

cacatcaact tccgatagtg agcagcttgt cctggggatg agaaatcaaa cgaatttctc    14460

gtatccttga gcgggggaac caggaccagt ggggagaagc cactggagag agatttcagc    14520

tccatatgca gaacagctag ctggtgatca gtcaggggtg caaggtggaa ttggcagcat    14580

tgtgaggcat ctctacaaaa aatacaaaaa ttagccaggc atagcggcac acgcctgtag    14640

tctcagctac ttaggaggct gaggtgggag gatcgcttga ggctgggatg tcgaggctac    14700

agtgagccgt gatggcacca ctgcactcca gcctgggcaa cagagcaaga ccctgcctca    14760

aaaaaaaaat gattaatacg attagatgag cgtttaaaaa aatcacgtaa gtgatagttc    14820

tttttcagta actttctatt gttagggaca ggacaatctt ttcattctca tttcttatta    14880

acaagaccct gaagacggag ccatttcttg gcatctgtgt cttattccca gagctgagct    14940

gagtcgctgg atgccatgga aaggctgatc atatgacagc ttcaaactga catgttgtaa    15000

attattgagt taatatggag ggtgatttct ctattaaggg aaaattatat atatctatat    15060

atatatatat tttttttgag gcagtctcac cctgtcacgc agtctggagt acagtggcac    15120

aatcttggct cactgcaacc tctacctcct gggttcaagc aattctcccg ccttagcctc    15180

ccgagtagct ggaattacag acacatgcca ctacacccgg ctaatttttg tatttttagt    15240

agaaacgggg tttcaccatg ttggccaggc tggtcttgaa ctcctgacct caagtgatcc    15300

acctgcctcg gcctcccaaa gtgctgggat tacaggcgtg agccaccacg cccagcctct    15360

ttctttttta agagacaggg tctctctcca tcacccaggc tggagtgcaa tggcatgatc    15420

atagctcact gcagccttga actccttcct gggctcaagc gagcctccct cctcagcctc    15480

ctgagtagct gggattacag gcatgtgcca ccacgtccag ctaatatttt aagtttttgt    15540

agagatgagg tcttgattcg ttgctcaggc tggtctctaa ctcctgggtt caagtgatcc    15600

tcccacctca gcctcccaag gtgctgggat tacaggcatt gagccaccac gcctggcctt    15660

ctttatgttt agagtgagtt acttcatgta tttgtttgac gtaattttct aaaccagttt    15720

cttgggaggc gcattatacc agtgaaacgt agatgatgta aagtcccatc attttagttc    15780

cggtattttg gttgagtatg acggtgtttc tgtcacctgt ccatgggcct ggctaaggct    15840

tggtagtgtg gctctctgtg ataatgtggt cagtgtcaga cctcctgggc tcagatctgc    15900

gtgttgtcac ttgagctgtg tggtgacctt gtgcttgcct gggcttggta tagcccagtg    15960

tcctgctgat ggcccaggct ctggggaaga aactccctgg gcccttcagt ggccctcctg    16020

gtgtcttctg tctgagggga tctgaagccc attcaagtcc ctgaactgct agaaaatctg    16080

cgcaattgca gagatgggag agctgcctcg tgggagctgg tgtctgtaag gtgggaggtc    16140

aggtgtcaga tcccggtagg ctgggagagc tcaaaaggct cccaggaaat ggctggcctt    16200

gagctcagca gacacagctg tccctgccag ggtggccacc tgggaatcca ggacggagag    16260

aagtgtataa gagctcccct tgactgaggc tcacaggcac tgtcgtttgt gctgagtggt    16320

tggaggtgtg cactctagag gcagatccct gagttcaaat cccagtctct ctgcttcagt    16380

ttcctcatct gtaaagtgtg ggtaacagca tctgccggta acagcacctg ccgtgaaggg    16440

aaatgagcat agaacagcac caggtacatg gtaaacacta gctggggggt ttctggccat    16500

ttccatgtat tgtctcctaa cgctaggagg ccagggctgc tgtcttctct gctttaaaga    16560

tgtggaaatc ggctgggcgt ggtggctcac gcctgtaatc ccagcacttt gggaggccaa    16620

ggcaggtgga tcacttgagg tcaggagttc aagaccagcc tggccaacat ggtgaaaccc    16680

catctctact aaaaatacaa aaattaacca ggtgtcatgg cgtatgcctg taatcccagc    16740

actttgggag gccaaggcag gtggatcact tgaggtcagg agttcaagac cagcctggcc    16800

aacatggtga aaccccatct ctactaaaaa tacaaaaatt aaccaggtgt catggtgcat    16860

gcctgtaatc ccagctactc gggaggctga ggcaggagaa tcacttgaac ctgcgaggtg    16920

gaggttgcag tgagctgaga tcgcgccact gcactccaac ctgggcaaca gagtgagcct    16980

ccatcttaaa aacaaataaa taaagatgcg gaaatcgagg cacagggcag ttaagtgact    17040

tgctccaatg tccacgatac gtgggagagc ccagatttga gcccaggtgg gtcagatcct    17100

cagcctgagt tgtggcccat cccactctac catttctcag agtggcaagg ggcaggtagg    17160

tgacaggcac aactgacccg aaggcccagc ttcatggtgg gagtatgatt ctggcccaga    17220

gcccagtggc ctccacacca cgtgatcgga cggggctgag aggttgggag gaaacctcaa    17280

tagtaggtca agctaaggaa aataagaaaa aaaagtgagt cgtagcagct gccatgtgct    17340

gaccacttca tacttggagc tttttattat ctttcccagg tctgggggtt gactgggctc    17400

agctgggcag ttctattggc ccctctattg gttgcagtca gctggtggcc tgggctgtgg    17460

tcatctggag actcaacggg gacagctggg cctcttttcc tctccctgta gtccagggcc    17520

tctggctcta cggggagagc cagtccccac caggcctctc catgtggcct ctccagcaag    17580

gtagccagac ctgttatgtg gcagcccagg gtcccccaaa gcatgaaggt gagaactgcc    17640

agcccttctt aagacctctg cctggaactg gcacggcacc acgtctgcct cttgttggtt    17700

aaccagatcc aactccaggc cagattcaag gggcagggaa ataaactcta ttggtcagtg    17760

ggcaatgaca aagaacttga agccatattt aatctacccg gctctgtgtc cagtactgtg    17820

caaagagctt tgccagcata ctgtcattta ttcctcccac cagttctatg aggtgggcag    17880

tcttattacc cccattccac agatggaggc gccaaggctc agagagtttc agtcattgtc    17940

cccaaggcac atacagctgg tgagcagtgg agttgggatc caaacccagg accctggtcc    18000

agattcccct cctgtccctt tcctgagccc agaaggtcca tggcaggcct tggtcagtgg  18060

ggagagacct ccccaatggt ccacatgctg acgaggtctt tctttttctt gtgcagccaa  18120

aacgccccaa actcgggtga ccattcggac ggtgcgagtc cgccggcccc ccaagggcaa  18180

gcaccggaaa ttcaagcaca cgcatgacaa gacggcactg aaggagaccc ttggagcc    18238

<210>136

<211>15442

<212>DNA

<213>People

<400>136

tctgcggaga agatccatct aacccctttc tggccccaga gtccagggaa aggatgatca    60

ctgtcagaag tcgtggcgcg ggagcccact gggcgctttg tcacattcca ccgaaagtcc    120

cgacttggtg acagtgtgct tcccttccct cgccaacagt tccgagtgag ctgtgcttta    180

gctctcgtgg gggtgggtca agggaggatt tgaagagtca ttgccccact ttaccctttt    240

ggagaaatgg cttgaaattt gctgtgacac gggcagcatg ggaatagtcc ttcctgaacc    300

ctggaaagga gctcctgcca gccttgcaca cactttgtcc tggtgaaagg cagccctgga    360

gcaggtgttt ttttggaact ccaaacctgc ccacccaact tgcttctgaa agggactcta    420

aagggtccct ttccgctcct ctctgacgcc ttccctcagc cagaattccc ttggagagga    480

ggcaagagga aagccatgga caggggtcgc tgctaacacc gcaagttcct cagaccctgg    540

cacaaaggcc ttggctacag gcctccaagt agggaggagg gggaggagtg gctgcctggc    600

cacagtgtga ccttcagagg cccccagaga aggacacctg gcccctgcct gcctagaacc    660

gcccctcctg tgccccctgg ccttggaagg ggtatgaaat ttccgtcccc tttcctcctt    720

ggggcccagg aggagtggag ggtcccggga gaatattgtc agggggaagg cagggggtgt    780

catgggaatg ggtgaggggg ctgaggtgca gaatccaggg ggtccctgca ggagccgcag    840

tggtaagctg tccagctgga agcctggtaa ctgttgtttt ctcttgagag gggcttcctg    900

tgaccttggc tgtctctggg agcagggctg gggtacctga gtggggtgca tttggggtgt    960

gtgggaagga gagggaaaga aagatggaca gtgggactct cccctagcag ggtctggtgt    1020

tccgtaggct agagtgcccc tctgctctgc gagtgctggg cgggagggga gttggtgaga    1080

gctggagacc cccaggaagg gctggcagaa gcctttcctt ttgggtgctg tcaggtccgc    1140

atgtcttggc gtgttgacct tcacagcttc tggcgagggg aggaatgatc tgatgcgggt    1200

ggggagggtt agaggaggcc tcaggcctaa ggtggtgcag ggggccccct aggggctggg    1260

cagtgccaag gcataaaagc cttccctggt ccctggtggc atttgaaggt gcccaggtga    1320

gaggggcttg gcacctcctc accctgggag ggagaagaaa ccagggaaca ggtaggagtg    1380

ggagacaggt gaggctttgg aaatctattg aggctctgga gagatttgtg tagagaggaa    1440

aatgtggttc tcccccaggg tctcctcctg ggtttttacc ctctaagcaa cctgtgggca    1500

tgctgggtta ttcctaagga ctagaagagc ttggatgggg gagggtggtt ggtgcccttc    1560

ggtcctcggc acccccctcc gtctccaaca ccagctcacc ctggtatttg tcatgtcagc    1620

aggagaaggt caccatgttg tttttctcgc ccctagtcct tccttcctgc cccagtccaa    1680

atttgtcctc ctatttgacc ttaatactta ccatggcttt ggaccaggga actaggggga    1740

tagtgagagc agggagaggg aagtgtgggg aaggtacagg ggacctcgac agtgaagcat    1800

tctggggttt tcctcctgca tttcgagctc cccagccccc aacatctggt tagtctttaa    1860

cttcctcggg ttcataacca tagcagtcca ggagtggtgg gcatattctg tgcccgtggg    1920

gacccccggt tgtgtcctgt tcgactcaga agacttggag aagccagagg ctgttggtgg    1980

gagggaagtg aggagggagg aggggctggg tggctgggcc tgtgcacccc agcccctgcc    2040

catgcccatg ccttgctctc tttctgtcct cagtggtccc aggctgcacc catggcagaa    2100

ggaggagggc agaatcatca cgaaggtgag tccccctggc tgttggatgg ggttccctgt    2160

cctctcaggg gatgggtgga tggcctaatt cctttttctt cagaactgtg gggaggaagg    2220

ggaaggggca caggaatata aggatcaaga aagaaagagc tgggcaccac gaggttcacc    2280

ctcagtttcg tgaggactct ccgctgttca ggtctctgct agaagtagga cttgttgcct    2340

ttttcttctg ctctttccag taaaatttta tttggagaag gagtcgtgcg cacagagcag    2400

gaagacagtg ttcacggatc ctaggtgttg ggggaagtgt cccttgtttc ccctagctcc    2460

caggggagag tggacattta gtgtcatttc ctatatagac atgtcccatt tgtgggaact    2520

gtgacccttc ctgtgtgagc tggaggcaca gagggctcag cctaatggga tctcccctcc    2580

cttccctggt ttgcattcct ttgggggtgg agaaaacccc acttgactat gttcgggtgc    2640

tgtgaacttc cctcccaggc cagcagaggg ctggctgtag ctcccaggcg ccccgccccc    2700

ctgcccaacc ccgagtccgc ctgccttttg ttccgttgtg gtttggatcc tcccatttct    2760

ctggggacac cctggctctc cccaccactg actgtggcct gtgctctcca cctctgggga    2820

gggaaggccc tggggtcttc cttcccgcga gtttccctga cctaaatctg gcgtggctgg    2880

gtagtggcca gcagtggtga tgcccagcct gttctgcctc ctccttcccc accccaggag    2940

ccctttcctt ggcctaggac ctggcttctc agccactgac cggccccctg cttccagtgc    3000

gccacttacc ccttccagct tcccagtggt ctctggtctg ggagaggcag gacaaatggt    3060

ctttgtttgc tggagaaaag gttgtctgcg ataaataagg aaaaccacga aagcctggtt    3120

gttggagtgt acgtgtgtgc tcccccaggc agtggaggcc agccctcctt ggaggggcgg    3180

ctgcctgatg aaggatgcgg gtgaggttcc ccgcctccac ctcccatggg acttggggat    3240

tcattccaag gggaagcttt ttgggggaat tcctacccca ggtcttttta ccctcagtta    3300

ccaacccctt gcccaggcca gaccttcctg ctatcccctc ctgggccaca agcctggccc    3360

tcctctgtcc caattgtgat gaaggggcag ttcaaaactt cttgattagt catcttctcc    3420

cctatcgact tggctttaaa aaatgacctt ttcagacttc tagtctcgtt cactcttttt    3480

gatgatgctt tgccgtaacc cttcgtgggt agagaaggat tctgtgccca ttggtggtct    3540

ggataaaaga aatagagacc tcacaggaag cagtggactg gcctgtttcc ccactgttct    3600

ttctgttttc acacctgtgg ccttctcccc accttcttcc caatcaacct attgtgtaca    3660

tagcccccct cattgtcctt tattcttctg gaaagcagac cttggaggga ggagtgaggg    3720

ggaggctcag ctgtggtctc tggggggtgg gggttgggag ctggggtgga agtccacgaa    3780

gcatacactt aagatgcttt ggtgaagttc taaacttcat attacccagg ctgaaaaaag    3840

agcacttgtt cctagggctg gaaatggaag ccaaaacacc acctttttca gcctgtttca    3900

gcatctttag agatcagccc aacccactta cacagttgag cagagttgga ggcctagaga    3960

ggggagggac tggcccaagg tcataccaac tcatggccag agcctgggcc tcctcactgg    4020

ccaggtgtta tttcttccct ctgggtaggg aacctatttc agggacagga ttgctatgtg    4080

gtagtggtgg tggggtgcga taggcgtggc aggctgggcc acaatttgga gtagtcatgc    4140

cagagtcctg catttattta ttctcaaggg ccccgcctct gtggcccaga attacccctt    4200

catgctccag tgcaccccag gcttcgtggc cagcctggga aactgtctct accctggtct    4260

cccttcagat cagcttctag aaatgtttcg tggctacagt ggcagcactg ttttttccat    4320

gatgcaagca gtttgccctc ttgggcgggg ttatcagtgg ctggcagggc tggcacagcg    4380

tgtccgccca ctgccacctg tgggttccag gagggcccag cccctgtgct gatgcccacc    4440

accttctcag ctcatgtctg gggaagagga ctggcagggg gaaaggtgcc tcctcctgaa    4500

aggtgcctcc tctgtttttg cctaatatag gcttgggaac actttgatgt cagctaattc    4560

tgactccttt acttactagc tgtgcggcct tggggcaact tacttagcct ctttgagcct    4620

cctgttcccc atctgtaaaa tggaatctca atagtgtcta atagtaccat gtggagaaac    4680

ttgtgtgaaa tgatagctgt ggactactgt acacagtact caggatgtag taagtgctca    4740

ataaacagct gttggtatgg ttgacgttat ggtagtggtt gtggggagga cgtaggaaac    4800

tggagactag cttggcaaag ctggctcttc ctccttttag ggaaagctta gagcatcccc    4860

atggggtata cccatactca gactgtcctc tggcatcgag gttggcccag gattcagttc    4920

agctgtcaca gtgaggtggc gggatcagat gtggcaggcc atgtcccttg gaacttgagt    4980

acatcgtgtg atctctggaa tgaaaacagg ccttcaccag tgttgatggt ggaaagctta    5040

gggaagtgct tcaaacacag taggagggac ttacgttaga ttttggaagg acttgcctga    5100

ttcggaagct ccaaagagtg gcattacaga gctgggtgga gagaggggct agccatcttt    5160

tgtgtcgccc accgggctca tgtgtcatcg cctctcatgc agtggtgaag ttcatggatg    5220

tctatcagcg cagctactgc catccaatcg agaccctggt ggacatcttc caggagtacc    5280

ctgatgagat cgagtacatc ttcaagccat cctgtgtgcc cctgatgcga tgcgggggct    5340

gctgcaatga cgagggcctg gagtgtgtgc ccactgagga gtccaacatc accatgcagg    5400

tgggcatctt tgggaagtgg ggcaaggggg ggatagggag gggggtaaca ctttgggaac    5460

aggtggtccc aggtcgtttc ctggctagat ttgccttgtc tggctcctgc ccctgagttg    5520

cacaggggag gtatggtggg gtcttgcctt ctgtggagaa gatgcttcat tcccagccca    5580

ggttcccagc aagccccaac catctccttc tccctgatgg ttgcccatgg gctcaggagg    5640

ggacagatgg atgcctgtgt caggagcccc tctctccctc tcttggagag agtcctgagt    5700

gccccccctt cttgggggct ttgtttggga agctggatga gcctggtcca tggagagttt    5760

aaaaagtctt ttggtgttac ctggtaatgg ggcacatctc agcccagata gggtgggagg    5820

gagctgtgaa acacagggag ggggttgctt tcgggtatct actaggagtc agggtgaagc    5880

ctagagagga tgaaagaagg ggaggggatg gggagtggta agaacctagg atttgaattc    5940

ccagcctggc caacccttgc agccatgtct tggcctcaag tggaacaagg gctccttgag    6000

gccagcaggg ttgggggagt tggggtgggc ctgagcctct ttcctgctag agctcttggt    6060

cctccctgcc tccaccaccc atccctgctc tgcagaaccc ctgggtgctg agtggcagga    6120

gccccagggt tgtcccatct gggtatggct ggctgggtca ctaacctctg tgatctgctt    6180

ccttcctttc cagattatgc ggatcaaacc tcaccaaggc cagcacatag gagagatgag    6240

cttcctacag cacaacaaat gtgaatgcag gtgaggatgt agtcacggat tcattatcag    6300

caagtggctg cagggtgcct gatctgtgcc agggttaagc atgctgtact ttttggcccc    6360

cgtccagctt cccgctatgt gacctttggc attttacttc aatgtgcctc agtttctaca    6420

tctgtaaaat gggcacaata gtagtatact tcatagcatt gttataatga ttaaacaagt    6480

tatatatgaa aagattaaaa cagtgttgct ccataataaa tgctgttttt actgtgatta    6540

ttattgttgt tatccctatc attatcatca ccatcttaac ccttccctgt tttgctcttt    6600

tctctctccc tacccattgc agaccaaaga aagatagagc aagacaagaa aagtaagtgg    6660

ccctgacttt agcacttctc cctctccatg gccggttgtc ttggtttggg gctcttggct    6720

acctctgttg ggggctccca tagcctccct gggtcaggga cttggtcttg tgggggactt    6780

gtggtggcag caacaatggg atggagccaa ctccaggatg atggctctag ggctagtgag    6840

aaaacatagc caggagcctg gcacttcctt tggaagggac aatgccttct gggtctccag    6900

atcattcctg accaggactt gctgtttcgg tgtgtcaggg ggcactgtgg acactggctc    6960

actggcttgc tctaggacac ccacagtggg gagagggagt gggtggcaga gaggccagct    7020

tttgtgtgtc agaggaaatg gcctcttttg gtggctgctg tgacggtgca gttggatgcg    7080

aggccggctg gagggtggtt tctcagtgca tgccctcctg taggcggcag gcggcagaca    7140

cacagccctc ttggccaggg agaaaaagtt gaatgttggt cattttcaga ggcttgtgag    7200

tgctccgtgt taaggggcag gtaggatggg gtgggggaca aggtttggcg gcagtaaccc    7260

ttcaagacag ggtgggcggc tggcatcagc aagagcttgc agggaaagag agactgagag    7320

agagcacctg tgccctgccc tttcccccac accatcttgt ctgcctccag tgctgtgcgg    7380

acattgaagc ccccaccagg cctcaacccc ttgcctcttc cctcagctcc cagcttccag    7440

agcgagggga tgcggaaacc ttccttccac cctttggtgc tttctcctaa gggggacaga    7500

cttgccctct ctggtccctt ctccccctcc tttcttccct gtgacagaca tcctgaggtg    7560

tgttctcttg ggcttggcag gcatggagag ctctggttct cttgaagggg acaggctaca    7620

gcctgccccc cttcctgttt ccccaaatga ctgctctgcc atggggagag tagggggctc    7680

gcctgggctc ggaagagtgt ctggtgagat ggtgtagcag gctttgacag gctggggaga    7740

gaactccctg ccaagtaccg cccaagcctc tcctccccag acctccttaa ctcccacccc    7800

atcctgctgc ctgcccaggg ctccaggaca cccagccctg cctcccagtc caggtcgtgc    7860

tgagcaggct ggtgttgctc ttggttccgt gccagctccc aaggtagccg cttcccccac    7920

accgggattc ccagaggttc tgtcgcagtt gcaaatgaag gcacaaggtg cctgatacac    7980

agccctccct cccactcctg ctccccatcc aggcaggtct ctgaccttct ccccaaagtc    8040

tggcctacct tttatcaccc ccggaccttc agggtcagac ttggacaggg ctgctgggca    8100

aagagccttc cctcaggctt tgccccctgc cggggactgg gagccactgt gagtgtggag    8160

acctttgggt cctgtgccct ccacccagtc tcggcttccc accaaagcct tgtcaggggc    8220

tgggtttgcc atcccatggt gggcagcgtg aggagaagaa agagccatcg agtgcttgct    8280

gcccagacac gcctgtgtgc gcccgcgcat gcctccccag agaccacctg cctcctgaca    8340

cttcctccgg gaagcggccc tgtgtggctt tgctttggtc gttcccccat ccctgcccac    8400

cttaccactt cttttactcc ccccaccgcc cccgctctct ctctgtctct gtttttttat    8460

tttccagaaa atcagttcga ggaaagggaa aggggcaaaa acgaaagcgc aagaaatccc    8520

ggtataagtc ctggagcgtg tacgttggtg cccgctgctg tctaatgccc tggagcctcc    8580

ctggccccca gtacaacctc cgcctgccat tccctgtaac cctgcctccc tcccctggtc    8640

cctccctggc tctcatcctc ctggcccgtg tctctctctc actctctcac tccactaatt    8700

ggcaccaacg ggtagatttg gtggtggcat tgctggtcca gggttggggt gaatgggggt    8760

gccgacttgg cctggaggat taagggaggg gaccctggct tggctgggca ccgattttct    8820

ctcacccact gggcactggt ggcgggccca tgttggcaca ggtgcctgct cacccaactg    8880

gtttccattg ctctaggctt ctgcactcgt ctggaagctg agggtggtgg ggagggcaga    8940

catggcccaa gaagggctgt gaatgactgg aggcagcttg ctgaatgact ccttggctga    9000

aggaggagct tgggtgggat cagacaccat gtggcggcct cccttcatct ggtggaagtg    9060

ccctggctcc tcacggaggt ggggcctctg gaggggagcc ccctattccg gcccaaccca    9120

tggcacccac agaggcctcc ttgcagggca gcctcttcct ccgggtcgga ggctgtggtg    9180

ggccctgccc tgggccctct ggccaccagc ggcctggcct ggggacactg cctccgggct    9240

tagcctccca tcacacccta ctttagccca ccttggtgga agggcctgga catgagcctt    9300

gcacggggag aaggtggccc ctgattgcca tccccagcag gtgaagagtc aaggcgtgct    9360

ccgatggggg caacagcagt tgggtccctg tggcctgaga ctcacccttg tctcccagag    9420

acacagcatt gccccttatg gcagcctctc cctgcactct ctgcccgtct gtgcccgcct    9480

cttcctgcgg caggtgtcct agccagtgct gcctctttcc gccgctctct ctgtcttttg    9540

ctgtagcgct cggatccttc cagggcctgg gggctgaccg gctgggtggg ggtgcagctg    9600

cggacatgtt agggggtgtt gcatggtgat ttttttctct ctctctgctg atgctctagc    9660

ttagatgtct ttccttttgc ctttttgcag tccctgtggg ccttgctcag agcggagaaa    9720

gcatttgttt gtacaagatc cgcagacgtg taaatgttcc tgcaaaaaca cagactcgcg    9780

ttgcaaggcg aggcagcttg agttaaacga acgtacttgc aggttggttc ccagagggca    9840

agcaagtcag agaggggcat cacacagaga tggggagaga gagagagaaa gagagtgagc    9900

gagcgagcga gcgggagagc gcctgagagg ggccagctgc ttgctcagtt tctagctgcc    9960

tgcctggtga ctgctgcctt ctctgctttt aaggcccctg tggtgggctg caggcactgg  10020

tccagcctgg cggggcctgt tccgaggttg ccctggttgc ctgagtggta ggctggtgtg  10080

gcttagtgta gtggtgtgga cgcaagctgt gtgttgtgtc ctgtggtcct tctgctcata  10140

gtggctgttg gtcctgatgt tattactacc tctggtagta atgctgagaa gctgaaagcc  10200

gattccaggt gtggacaatg tcaacaaagc acagatgctc tcgctggggc cttgcctcgg  10260

ccctttgaag tctgcatggc tgggcttctc actcactcag tgtttcttgc tgggggaagg  10320

aattgagtct cccacttcag actgggcctc cctgaggaaa gggttgtgtc tccccactca  10380

gactgaggtt ccctgagggt agggctgtgt ctctcccctc cgacctgggc tccctgatag  10440

ggctgtctcc ccgctcagac tgaggctccc tcaggccagg gctatgtctc cctcctcaga  10500

ctggggctct gagggcaagg ggtctggctg ttcgtttagg atggggcagt tttgcctaca  10560

cactgaagga gctgtagcat ccaagaatac tagatacctt taatcctcca ccagtcatgg  10620

tgacaacccc aagcagccca cacattttca agtgccccca ggatgcgtgg agggaggggt  10680

ctgtgcccat tctcctgaca ttagcctgtg agctccgtaa gcccgggcct cgtttacgta  10740

cctttgtgag ccccgggcat ctgtacctct ttcctttgcc catactgggg accaaggaag  10800

tgtcaagtgc atgagtgaat gtgtgactca gttcagaggg tgaggtcagg agcacagggt  10860

cgggacaggt ggctggcatc ttttaatgcc ttagcttatg ttctttatac caacttggcc  10920

tgtgctcaga gtgagggagg ccctgggggt cagggtaagc gtcagtcagg gaggcaagac  10980

tttgtgggga tttcctagac agggccaagg cacccccagc tcaccccgag gctgcgttag  11040

ggaagtcctt ggagtgtctc ccctccccca gcaatgttct tgtggcttgt gtgtgctcag  11100

gggatgctgg gaaccaggcc tgggtagttg gtgtggggtg ctgtctgtct tggccctatg  11160

tgaaaccaag agggcgtata ttagtgctgg ggtgggggct ctgcctaact tcagggctgg  11220

atgaggggag tctcagttcc ccaggggtcc ttgggaaaga taagggactt gacattttag  11280

ggtttttagg tgattattct gctgatgggg gtttgtgtga agtgacctgg gagctaactg  11340

aagttactct aacctcccaa tacctttacc caacccccaa gctggctgta tctgggaata  11400

tcagtttcca aaattggagg cttaggactc cgtttcgggg ctccccagaa gggtagggcc  11460

tgttctgcct ccttctcaca atcacccagg ggcaggggca tgctgagaaa gttcttggag  11520

gccccctttg cttcagctgg agtagtgaag ccgccgaatt gtctctcccc atcctaagtg  11580

aagcagcata tttgaaagga aagacaacct gttacctggg cctgcaacct ccaggcagct  11640

caagagagat gaggcctaca gccacactgg gaggggacat ggggaatgga gatggtccct  11700

caccttcctg gggcctcctg ctctacgcta ccccctcggg agcctcctgt ccccagggca  11760

ggcccttgcc attgttggtc acccggccaa gcctctctgc ctcaggcgtt ctcccagaag  11820

atctgcccac tctcttcccc acaccagccc ctagagactg aactgaaaac cctcctcagc  11880

agggagcctc ttctgattaa cttcatccag ctctggtcac ccatcagctc ttaaaatgtc  11940

aagtggggac tgttctttgg tatccgttca tttgttgctt tgtaaagtgt tcccatgtcc  12000

ttgtcttgtc tcaagtagat tgcaagctca ggagggtaga ctgggagccc ctgagtggag  12060

ctgctgctca ggccggggct ccctgagggc agggctgggg ctgttctcat actggggctt  12120

tctgccccag gaccacacct tcctgtcctc tctgctctta tggtgccgga ggctgcagtg  12180

acccaggggc ccccaggaat ggggaggccg cctgcctcat cgccaggcct cctcacttgg  12240

ccctaacccc agcctttgtt ttccatttcc ctcagatgtg acaagccgag gcggtgagcc  12300

gggcaggagg aaggagcctc cctcagggtt tcgggaacca gatctctcac caggaaagac  12360

tgatacagaa cgatcgatac agaaaccacg ctgccgccac cacaccatca ccatcgacag  12420

aacagtcctt aatccagaaa cctgaaatga aggaagagga gactctgcgc agagcacttt  12480

gggtccggag ggcgagactc cggcggaagc attcccgggc gggtgaccca gcacggtccc  12540

tcttggaatt ggattcgcca ttttattttt cttgctgcta aatcaccgag cccggaagat  12600

tagagagttt tatttctggg attcctgtag acacacccac ccacatacat acatttatat  12660

atatatatat tatatatata taaaaataaa tatctctatt ttatatatat aaaatatata  12720

tattcttttt ttaaattaac agtgctaatg ttattggtgt cttcactgga tgtatttgac  12780

tgctgtggac ttgagttggg aggggaatgt tcccactcag atcctgacag ggaagaggag  12840

gagatgagag actctggcat gatctttttt ttgtcccact tggtggggcc agggtcctct  12900

cccctgccca ggaatgtgca aggccagggc atgggggcaa atatgaccca gttttgggaa  12960

caccgacaaa cccagccctg gcgctgagcc tctctacccc aggtcagacg gacagaaaga  13020

cagatcacag gtacagggat gaggacaccg gctctgacca ggagtttggg gagcttcagg  13080

acattgctgt gctttgggga ttccctccac atgctgcacg cgcatctcgc ccccaggggc  13140

actgcctgga agattcagga gcctgggcgg ccttcgctta ctctcacctg cttctgagtt  13200

gcccaggagg ccactggcag atgtcccggc gaagagaaga gacacattgt tggaagaagc  13260

agcccatgac agctcccctt cctgggactc gccctcatcc tcttcctgct ccccttcctg  13320

gggtgcagcc taaaaggacc tatgtcctca caccattgaa accactagtt ctgtcccccc  13380

aggagacctg gttgtgtgtg tgtgagtggt tgaccttcct ccatcccctg gtccttccct  13440

tcccttcccg aggcacagag agacagggca ggatccacgt gcccattgtg gaggcagaga  13500

aaagagaaag tgttttatat acggtactta tttaatatcc ctttttaatt agaaattaaa  13560

acagttaatt taattaaaga gtagggtttt ttttcagtat tcttggttaa tatttaattt  13620

caactattta tgagatgtat cttttgctct ctcttgctct cttatttgta ccggtttttg  13680

tatataaaat tcatgtttcc aatctctctc tccctgatcg gtgacagtca ctagcttatc  13740

ttgaacagat atttaatttt gctaacactc agctctgccc tccccgatcc cctggctccc  13800

cagcacacat tcctttgaaa taaggtttca atatacatct acatactata tatatatttg  13860

gcaacttgta tttgtgtgta tatatatata tatatgttta tgtatatatg tgattctgat  13920

aaaatagaca ttgctattct gttttttata tgtaaaaaca aaacaagaaa aaatagagaa  13980

ttctacatac taaatctctc tcctttttta attttaatat ttgttatcat ttatttattg  14040

gtgctactgt ttatccgtaa taattgtggg gaaaagatat taacatcacg tctttgtctc  14100

tagtgcagtt tttcgagata ttccgtagta catatttatt tttaaacaac gacaaagaaa  14160

tacagatata tcttaaaaaa aaaaaagcat tttgtattaa agaatttaat tctgatctca  14220

aagctcctct tggtttctcc ttctccattg aatccttgct ctagacttcc tcccgccccc  14280

tttccctctt cctctgggga acatggcatt tgtcttgggt ctgggaaagg tacgtctaat  14340

gtgtaggata tggggtgacc caccttgttg tgctgggggc aaagtccttc cattttggct  14400

gagctggtcc tgggggaacc catccatcct gtcttgatat agaaggtggg aagctctggg  14460

aatgggtgga gggggagaaa cagcttagga gccaagggcc cttgcaattg gtagtgctgc  14520

cttcaggaat tagatgatcc aggcccctgt cccttagcca gggaaagaac tggccatgtc  14580

tccaagcttg ctgcccagga gacaggagag agctgttttt gtctgtgggg gtcttgttgg  14640

ctgcaacagg ctgggagtgg gagggggatg ctgctggagg gctgtgactc cagggtgtaa  14700

cttaatttta catagtttta caccctggag ttccttgagc ctctggaaga tggacccata  14760

ggtttggtca ccactgatgg gactccctgc cccagcttgc cataagctct tctctcacgt  14820

cctgctcctg ggtaaggtgg cacctatcca ggtctttgac actgaagggc agtgcttcca  14880

aattcacttc ctctagcctc tcatttattc ttgcaaacca tagatatggc ttgaataaat  14940

attgagccag tcattgtgct gctatgaata agacacaatt ccacccctca aggatctggt  15000

gaggatgggt gggtggggag acccaaaact ataaatccat gagcagaaaa atacataaaa  15060

tgtgctgggg gcatctgatc tagcttggga gtgggagttg tattgggggt ggtggctggg  15120

gggtggtgtc ttatgacatt atctctaggc tgccacttaa agtatggttt gaagacaggg  15180

agaacggggc ggcggagtga aagggttgag gacatcccag gcagaaggga tagtgtgagc  15240

aaggcatgaa ggtggcactt gggcaggggc agggagtggg tgggcagggt ggaggctgga  15300

gacacgggca agggttagct ccaagggagc ctgcagcacc atgccaagag cctagggcct  15360

gtcagggatg agcagcagcc gaggggtttt aaacactgag cggctcagtc agatagactt  15420

ttccataggt gcctggcaac ag                                           15442

<210>137

<211>5915

<212>DNA

<213>People

<400>137

atgacagtgc tggcgccagc ctggagccca acagtgcgta aaccccaggg acaagatcag    60

gggagagggg aggcacaatg tcaggatggg gcagagatga ggggagatgg acgggagaac    120

agatggacag atgacgagga aataggaggg gagatggaca gatgtgaggg gagatggata    180

ggagaggaga cggacagagg agggggagat ggacagagga tggggagatg gacagaggag    240

ggggagatag aggagaggga gattgagagg agtgggagat ggacaggagg ggggagattg    300

acagaggagg gggagatgga cagaggagag ggcagatgaa cagaggagag ggagataaag    360

aggagggagg tggacaggag gagggagatg gacagagaag ggggagatgg acagaggtgg    420

gggagatgga caggagggaa gatggagagg agggggtatg gacagaggag aggggagatg    480

gactggaggg ggagatggtc agaggagagg gagatgaaga ggagggggag gtggacagga    540

ggagggagac ggacagagga gaggaagaga gacagaggtg gggggagatg gacagaggag    600

ggggagatgg acagaaaagg ggggagatgg acagaggaga gggaaggtgg acagagccaa    660

gaacaaatga agaggacgtg gaccaagatc aagagagaag caggcaacag tggtgtagaa    720

ggcagaggga gggacagagc tggaggaacc cgggcgagga aaccagacgt gaagatgagg    780

tggttggaga gagaccagca gagtggggga gatggggaga gagaggggtg gggcagaggg    840

ggatgcaaac tggacagcat tggaccagag gcagagagaa accgggaaag acaggcagag    900

atgggcccat gtctccagaa agtgtggaag aggcagaagg acacccaggg aaggaggagc    960

ggtgaagaca gaacagtaca ggtgggaagc ccggaggagg gggctgtgtg tggaacagca    1020

gagggctccc ccagcacccg ctcccctgca gacctatctc ctcctgctgc tgctgctgag    1080

ctcgggactc agtgggaccc aggactgctc cttccaacac agccccatct cctccgactt    1140

cgctgtcaaa atccgtgagc tggtgagcgg cgctgccccg gaccccctca tgtgatcccc    1200

cttcccccca cttttttttt ttaagtagag atggggtctc tctccctgtg tttcccaggg    1260

tgggcttgaa ctcctgggat caagagatcc acccaccttg gtctcccaaa gtgctggaat    1320

tacaggagtg agacactgtg cttaggggtg tcatcccttt ttaagggcaa ggttctgtgg    1380

cttcttctgg gctccccctc tcttggtctt gtccctctct ctctggatct ctgctgccac    1440

ctctgggtcc ccacagttct gtttctcgct gttttcagcc aggcctgatc ctgttttctc    1500

ccgcagtctg actacctgct tcaagattac ccagtcaccg tggcctccaa cctgcaggac    1560

gtaagtcatg ttgggaggga cctgggatgg aggtggggac cacagactca agatgctcca    1620

ccgaggcgag tggataacca ggccctcccc tccccaaacc caggaatcag agtcctcagc    1680

ccctcctccc tcagacccag gagccccggc ccagcccctc ctccctcaga cccagcagcc    1740

ccgtgcccag ctcctccctc agacccgtgg gttctcccct ctaggaggag ctctgcgggg    1800

gcctctggcg gctggtcctg gcacagcgct ggatggagcg gctcaagact gtcgctgggt    1860

ccaagatgca aggcttgctg gagcgcgtga acacggagat acactttgtc accaaatgtg    1920

cctttcaggt cagccctcaa cttaggggac aagtgagggg agggagatgt cttcctacga    1980

attagaagta aagctccact aggccttatt ggcgatttgg accatagcca cccaacgaag    2040

gtagagcgag aagtgccacc ctggagagcc ctgttcctac agaacaacac gtccccaggc    2100

accggtgatg gggagcagtc tggtcccatt ctggggcccc ggtttcctag gccatgatga    2160

agggtgccac tgaggggttc ttcccccaaa aaaaaacagg gcagagaagg ggtctctaaa    2220

ctgaggaggc cgggcgtggt ggctcactcc tgtaatccta gcactttggg aggctgaggt    2280

gggcggatca cttgaggtca ggagtttgag accagcctgg ccaacatgat gaaatcccgg    2340

ctgtactaaa aatacaaaaa ttagccgggc atggtggctc aggaggctga ggcacaggaa    2400

tcgcttgaac ccgggagcca gaggctgcag tgagccgaga tcatgccact gcactccagc    2460

ctgggagaca gagtgaaact gtctcaaaaa caaaacaaac aaacaaaaac ctctctctga    2520

gggctgggtg cagtggctca cacctgtaat cccagcactt tcggaggcca aggtgggagg    2580

attatttgag ccctggtgtt caagatcagt ccgggtatca cagtgaaacc tcatctctga    2640

acaaaaataa aaataaatta gccaggatgg tggtgcccac ctgtggtccc agactactca    2700

gaaggctgag gtgggaggga tcacttgaga cctggaggtc gacgctgcta tgagttctga    2760

ttgggacact gcactccagc ttgagcgaca gagcaagacc tgtctcaaaa catagaatag    2820

gccgtgtatg gtggctcacg actgtaatcc cagcactttg ggaggctgag gcgggtggat    2880

tgcctgagct caggagttcg agaccagcct cggcaacgtg atgaaaacca tctctactaa    2940

aatacaaaaa caaaattagc caggcatggt ggtgggcacc tgtagtccca gctacttggg    3000

agactgaggc aggagaattg cttgaaccca ggaggcagag gttgcagtga gccgagatca    3060

caccactgcc ctccagcctg ggcgacagag caagactcca tctccaaaaa aataaaataa    3120

aataaaaggc tgggtacagt ggctcacgcc tgtaatccca gcactttggg agcccgaggc    3180

gggcagatca cgaggtcagg agtttgagac caccctggcc aatgtggtga aaccccgtct    3240

ctactaaaaa tacaaaaatt agctgggcat ggtggcgcgc gcctgtagtc ccagctactc    3300

aggaggctga ggcagaattc cttgaacccg ggaggtagaa gttgcagtaa gccgagatcg    3360

tgccactgta ctccagccag ggtgacagag caagactctg tccccaaaaa ataaataaat    3420

aataaagtaa ctttgggagg ccgaggcggg cgactcacct gaggtcagag tccgagacca    3480

gcctggacaa catagagaaa ccccgtttct actaaaaata taaataaata aataaataag    3540

taagtaggct ggggacggag gttcacgcct gttttcccac actttggggg gccgaggcgg    3600

gcggatcaca aggtcaggag atcaagacca tcctgggtaa cacagtgaaa ccccatctct    3660

actaaaaata caaacaatta gccgggggtg gtggcagggg cctgtagtcc caactacctg    3720

ggaggttgag gcaggtggtt ggggcaaccc aggaggtgga gcttgcagag agccgcgatt    3780

gcgccactgt cctccagcct gggcaacaga gcgacacggt ggctcatgcc tacaatccca    3840

gcactttggg aggccaaggt gggcagatca cctgaggtca ggagttcgag accagcctgg    3900

ccaacatgga gaaaccccgt ctctactaca aatacaaaaa atagctgggc atggtggtgg    3960

gcacctatag tcccaaccac tgaggaggct gaggcaggag aatcacttga acctgggagg    4020

cagaggttgc atttcaccac tccagcctgg gcaacagagt gagactctat ctcaaaaaaa    4080

aattaattaa ttaaataaat aaactctgag agccagagct cactgggccc tgttgctggg    4140

catcgccagc agggaagggc ctttggcctg cggaggggcg gtggggggat gacgtggtgg    4200

tgacgtctcc ctcccctgct cccagccccc ccccagctgt cttcgcttcg tccagaccaa    4260

catctcccgc ctcctgcagg agacctccga gcagctggtg gcgctgaagc cctggatcac    4320

tcgccagaac ttctcccggt gcctggagct gcagtgtcag cccggtaaag gttccaggca    4380

cccccactcc ttcccctcct gtcctcacgg ccgctcctcc tctctgcaca gtgcatccca    4440

gaccccatct ttctcatatt ggttgtgaca agggcaagct tattcctctt tctggagctc    4500

agtttaccaa tttttttttt ttttttttga gacggagtct cgcactgtcg cccaggctgg    4560

agtgcagtgg cgtgatcttg gctcactgca agctccgcct ctcgggttca tgccattctc    4620

ctgcctcagc ctcccaagta gctgggacca caggcgcccg ccaccacacc cggctaaatt    4680

tttgtatttt tagtagagac ggggttttac cgagttaaac caggatggtc tcgatctcct    4740

gaccttgtga tccacctgcc ttggcctccc aaagtgctgg gattacaggc gtgagccaca    4800

gtgcccagcc tacctttttt tttttttttt ttttgagatg gagtcttgct ctgaccccca    4860

ggctggagtg caggggtgct atctcggctc actgcaagct ctgcctcctg ggttcacgcc    4920

attctcttgc ctcagcctcc ccagcagctg ggactacagg cgcctgccac ctcaacgcgg    4980

ctaatttttt tttttgtatt tttagtagag acggtgtttc accgtgtcag ccaggatggt    5040

ctcgatctcc tgacctcgtg atctgcccgc ctcggcctcc caaagtgcta ggattacaga    5100

tgtgagccac cgcgcccagc ctattgtttt ttttttctaa gtcggagtct tgctctgtcg    5160

cccaggctgg agtgcagtgg cgcaatcttg gctcgctgca acctccatct ccagggttca    5220

agcgaatctt ctgcctcagc cttccgagta cctgggatta cagatgcgca taccatgcct    5280

ggctagtttt tgtgttttta gtagagatgg gttttcacca tgttggccag gctggtcttg    5340

aactcctgac ctcaagtgat ccacccacct tggcctccca aagtgctggg attataggcg    5400

tgagccaccg cgctcagcct attcactcat ttaatttgtg acagtctgat gaggtaggta    5460

caatgattat cctagtttta cagatgacca aactgaggca cagagaggcc aagcagccca    5520

tccaaggtca cacagccagt ggcagccagg cctctctttc cttccttacc ccagcccttc    5580

tccttggtca cccagcctcc tctttctccc cagactcctc aaccctgcca cccccatgga    5640

gtccccggcc cctggaggcc acagccccga cagccccgca gccccctctg ctcctcctac    5700

tgctgctgcc cgtgggcctc ctgctgctgg ccgctgcctg gtgcctgcac tggcagagga    5760

cgcggcggag gacaccccgc cctggggagc aggtgagcag gctgggaaga gggggtgagg    5820

gggccgagag ggtggcccac ttgtggctga cactttgggg tccacaggtg ccccccgtcc    5880

ccagtcccca ggacctgctg cttgtggagc actga                               5915

Claims (16)

1. a kind of protein of separation, it has measurable biochemical parameter feature, wherein described character representation, the basis for the pharmacological characteristics for associating or forming one or more characteristics, wherein the protein of described separation, which has, includes the biochemical character { [P of multiple measurable biochemical parameters_x]₁、[P_x]₂、...[P_x]_n, wherein P_xRepresent measurable biochemical parameter and " n " be >=1 integer, wherein [P_x]₁To [P_x]_nIn each be respectively different measurable biochemical parameters, the value of the measurable biochemical characteristic of any of which or a series of value of more than one measurable biochemical characteristic represent, associate or formed the pharmacological characteristics T of characteristic_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 be selected from EPO, Flt3- part, Flt3-Fc, PDGF-B and VEGF-165.

2. the protein of the separation of claim 1, wherein described protein has measurable biochemical 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, its EPO, Flt3- part, PDGF-B or VEGF-165 comprising claim 1 or its segment, it is fused to one or more peptides, polypeptide or 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 are selected from EPO-Fc, Flt3- part-Fc, PDGF-B-Fc and VEGF-165-Fc.

7. a kind of pharmaceutical composition, the protein or chimeric molecule of the separation containing any one of claim 1 to 6.

8. a kind of method that 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 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.

9. a kind of nucleic acid molecules of separation, including selected from SEQ ID NO：31st, 41,51,59,63,65 and 67 nucleotide sequence, or 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 condition with the above-mentioned any sequence enumerated.

10. the protein or chimeric molecule of a kind of separation, by selected from SEQ ID NO：31st, coded by 35,37,39,47,51,53,55,63,65,71,73,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,119,126,129,131 and 132 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 condition with the above-mentioned any sequence enumerated.

11. a kind of nucleic acid molecules of separation, encoding proteins matter or chimeric molecule or its funtion part, including with SEQ ID NO：31st, 35,37,39,47,51,53,55,63,65,71,73,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,119,126,129,131 and 132 have at least 90% similitude nucleotide sequence, or after optimal arrangement and/or can be with SEQ ID NO：31st, 35,37,39,47,51,53,55,63,65,71,73,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,119,126,129,131 and 132, or their complementary type one or more nucleotide sequences hybridized under high high stringency conditions.

12. a kind of nucleic acid molecules of separation, including coding have substantially such as SEQ ID NO：32nd, the protein or the nucleotide sequence of chimeric molecule of 36,38,40,48,52,54,56,64,66,72,74,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,120,127,130 and 133 one or more shown amino acid sequences, or coding and SEQ ID NO：32nd, one or more nucleotide sequences with least about protein of the amino acid sequence of 90% similitude or chimeric molecule after 36,38,40,48,52,54,56,64,66,72,74,78,80,82,84,86,88,90,92,94,96,98,100,102,104,106,120,127,130 and 133 optimal arrangements.

13. a kind of kit, for determining the level that the human protein or chimeric molecule of people's cell expression are present in biological products, 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.

14. the kit of claim 13, 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.

15. the kit of claim 13 or 14, 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.

16. the human protein of the kit of claim 13 to 15, wherein people's cell expression is naturally occurring people EPO, Flt3- part, Flt3, PDGF-B, VEGF-165.

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