CN101146825A - A molecule 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 that comprises a dimeric 4-helix bundle, such as IFN-a2B, IFN-b1, IFN-g, IL-10 or its receptor, such as IFNAR2, IL-10Ra or chimeric molecules thereof comprising at least a portion of the protein molecule, such as IFN-a2B-Fc, IFN-bl -Fc, IFN-g-Fc, IFNAR2-Fc, IL-IO-Fc, IL-10Ra-Fc; wherein the protein or chimeric molecule thereof has a profile of measurable physiochemical parameters, wherein the profile is indicative of, associated with or forms the basis of one or more pharmacological traits. The present invention further contemplates the use of the isolated protein or chimeric molecule thereof in a range of diagnostic, prophylactic, therapeutic, nutritional and/or research applications.

Description

Molecule and chimeric molecule thereof

Technical field

The present invention briefly relates to proteinology, diagnostics, therapeutics and trophology.Especially, the invention provides a kind of isolating protein molecule, its structure comprises dimerization 4-helical bundle, IFN-a2B for example, IFN-b1, IFN-g, IL-10 or its acceptor, IFNAR2 for example, IL-10Ra, or its chimeric molecule that comprises a protein molecule part at least, IFN-a2B-Fc for example, IFN-b1-Fc, IFN-g-Fc, IFNAR2-Fc, IL-10-Fc, IL-10Ra-Fc; Wherein this protein or its chimeric molecule have measurable parameter attribute, wherein character representation, be associated with one or more pharmacological characteristics or form the basis of one or more pharmacological characteristics.The present invention further imagination is used for isolating protein or its chimeric molecule in diagnosis, prevention, treatment, nutrition and/or the research range of application.

Background technology

Any prior art that relates in this specification sheets not by, should not be understood that it is a kind of approval or any type of prompting that prior art is formed the part of general general knowledge yet.

Human interferon (IFN) is the pleiotropy cytokine, can promote natural immunity reaction and the acquired immune response.IFN not only plays a role in antitumor, also regulates the immune response that virus and infectation of bacteria cause simultaneously.These albumen are brought into play growth, differentiation and the propagation of immunoregulation effect and adjusting cell by a lot of modes in addition.These molecules comprise IFNa-2b, IFN-b1, IFN-g and acceptor IFN-aR2.IFN-aR2 belongs to the II type cytokines as the IL-10 acceptor from structure.Interferon, rabbit (such as: IFN-a2b, IFN-b1 and IFN-g) have obviously identical structure with IL-10, in other words the both has dimerization 4-helical bundle structure.

IFN-a comprises the same family protein of structurally associated, by 14 non-allelic genes IFN-a genes encodings, and one of them IFN-a2 that encodes.IFN-a2 has α-Luo Xuanjiegou and the glycosylated monomer of 0-.The characteristics of IFN-a2b polymorphism show as the K → R of precursor 46 amino acids.IFN-a is mainly produced by monocyte/macrophage, and other is produced by natural killer cell (NK), dendritic cell, plasmoid dendritic cell.Virus or infectation of bacteria induce and the ratio of component of pathogenic agent as: LPS, DNA of bacteria and double-stranded RNA have been induced the expression of IFN-a.The major function of IFN-a is to regulate the microbial reaction of antiviral and antitumor reaction and non-virus.IFN-a is in conjunction with the general IFN cell surface receptor that is present in target cell, scavenger cell or DCs, thus the above effect of performance.The maturation and the incremental adjustments of IFN-a treatment can the promotion costimulatory molecules of DCs are such as CD40 CD80 CD86 and MHC class II.

People IFN-b1 forms precursor protein by 187 amino acid and produces, and in the glycosylation of the N of 80 Asn end, is the dimer of zinc mediation.Although epithelial cell and lymphoid stem cells in the reaction that virus infection causes and after contacting double-stranded RNA, can both detect the expression of IFN-b1, IFN-b1 is still mainly expressed by inoblast.IFN-b1 is the pleiotropy cytokine, and there is multiple effect in it for immune various cells, has not only influenced natural immunity reaction, has also influenced the acquired immune response simultaneously.IFN-b1 has shown can induce MHC II expression and angtigen presentation effect, can improve cell immunocompetent thus.In addition, IFN-b1 has antiproliferative properties, can suppress growth of tumour cell and the apoptosis of inducing lymphoid cell line.Antiviral and the antiproliferative activity of IFN-b1 makes it become the treatment means of very attractive virus infection and multiple malignant tumour.

The polypeptide that people IFN-g is made up of 166 amino acid is non-covalent relevant homodimer.IFN-g is the pleiotropy cytokine, has not only promoted natural immunity reaction, has also promoted the acquired immune response simultaneously.Proinflammatory cytokine IL-12 and THF-α have induced IFG-g, IFG-g has mediated the activation of scavenger cell, the activation of scavenger cell can nonspecificly be killed in the various born of the same parents and the outer pathogenic agent of born of the same parents, such as bacterium, virus, fungi, protozoon, parasite and tumour cell.The IFN-g dependency of IL-12 is expressed and has been promoted TH ₁Cell-mediated immune response can be removed the reaction of virus infection and microorganism and tumor rejection thus.

IFN-g has shown the antigen propagation that can improve the B cytositimulation, promotes IgM to change the Ig type conversion of the same clan of IgG2 into simultaneously, has therefore promoted antibody dependent cellular cytotoxicity by NK IgG acceptor.

IFNAR2 synthesizes the transmembrane protein that 515 amino acid are formed, and signal peptide sequence and 5 possible N of forming comprising one section 26 amino acid hold glycosylation sites.The short type IFNAR2 that contains the outer ligand binding domain of born of the same parents can be as I class IFN agonist, also as the synergistic agent of blocker or IFN effect.If as the synergistic agent of IFN effect, IFN α, β, ω, τ, κ and ζ are also simultaneously in the extracellular domain of IFN acceptor, and the circulating half-life significance of IFN α, β, ω, τ, κ and ζ prolongs.The IFNAR2 that shears can be by the membrane-bound IFN acceptor of active cells or is interacted with it and as I interferoid agonist.And whether this exists irrelevant with the IFNAR2 part that combines shearing.Based on above reason, IFNAR2 may be the effective medicine that replaces the I interferoid, perhaps unites 1 interferoid medication.In addition, the IFNAR2 of shearing is by bringing into play the effect of blocker or inhibitor, in order to the unusual disease that raises and cause of treatment I interferoid in conjunction with the I interferoid.Such as, often can find that in autoimmune disorder IFN-α raises unusually.Treatment through secular IFN-α can be found this type of disease, comprising autoimmune hepatitis, lupus, systemic scleroderma and diabetes.IFN-α has brought into play effect in the allograft rejection pathogenesis in bone marrow transplantation, and IFN κ antagonist can be used in treatment psoriasis and atopic dermatitis and prevention type 1 diabetes.

Interleukin-11 0 (IL-10) molecular weight is 37kDa, and mainly the homodimer of being expressed by scavenger cell still also has part to express at activated T cell, B cell, mastocyte, monocyte and keratinocyte.IL-10 is the pleiotropy cytokine, by acting on T cell, B cell, monocytes/macrophages and antigen presenting cell (APC) thus regulate multinomial immune response, IL-10 is generally with T _H1Immunne response change T into _H2Immunne response.IL-10 suppresses cytokine IL-1 α, IL-1 β, IL-6, IL-8, TNF α, GM-CSF and the G-CSF in activatory monocyte and the scavenger cell.IL-10 also can suppress the generation of IFN γ in the NK cell.The immature dendritic cell that IL-10 handles can not be reached maturity, and shows as the ability drop that stimulates the CD4+T cell.IL-10 has suppressed the Langerhan cell antigen and has offered function.This shows that IL-10 has preserved the antigen uptake ability by suppressing antigen presenting cell (APCs), and has suppressed the migration to draining lymph node.The important effect of having brought into play is replied in this natural immunity that has shown that simultaneously this process causes for pathogenic agent.IL-10 overexpression in the various malignant disease of bibliographical information (comprising melanoma, cancer and lymphoma) is arranged.On the contrary, the some diseases characteristics that comprise psoriasis and inflammatory bowel are the I cytokines pattern relevant with the horizontal relative deficiency of IL-10.

IL-10 by in conjunction with IL-10 acceptor (IL-10R) thus mixture performance biological effect.This mixture is a heterodimer, comprises interleukin-11 0 receptor alpha chain (IL-10R α) and interleukin-11 0 acceptor β chain (IL-10R β), and the cytokine receptor of IL-22, IL-28 and IL-29 all comprises this structure.IL-10R α is the polypeptide that contains 6 possibility glycosylation sites, the site glycosylation of one of them at least.IL-10R mainly expresses in granulocyte, comprising T cell, NK cell, scavenger cell, monocyte, B cell, neutrophil leucocyte and dendritic cell.

The activity of proteinic biological effect device is by reaching with its corresponding acceptor interaction, and the acceptor that this means Interferon, rabbit and associated protein thereof (for example IL-10) and its correspondence has the important potential as the therapeutant of regulating the physiology process.Yet, the little variation of molecule such as one-level, secondary, three grades or quaternary structure and translation or posttranslational modification form can produce great influence to proteic activity, secretion and antigenicity and removing altogether.Therefore, albumen may follow special one-level, secondary, three grades or quaternary structure or altogether translation or translation back structure or for replenish the uniqueness of being given or useful especially characteristic produce.So, needs assessment under difference generation condition proteic physicochemical property to determine whether they have the pharmacological characteristics of useful physicochemical property and other.

Present problem is that commercial available proteic generation is by coming from the last cell realization from the far species of the mankind such as bacterium, yeast, fungi and insect of evolving, these cells or shortage glycosylation, perhaps its glycosylation and human cell are completely different, and these have all influenced their clinical efficacy considerably.For example, expressed proteins contains highdensity seminose in yeast or fungal systems such as aspergillus tubigensis, makes albumen not possess result of treatment (.FASEB J7:540-550 such as Herscovics, 1993).

Even in inhuman Mammals expression system such as Chinese hamster ovary (CHO) cell, confirmed glycosylated form also has marked difference with the human cell.For example, most of Mammalss comprise rodent α 1, and the 3-galactotransferase can produce semi-lactosi (α 1,3)-semi-lactosi (β 1,4)-G1cNAc on glycoprotein.Yet in the monkey of people, ape and old generation, the expression of this kind of enzyme is the inactivation (Larsen etc., J BiolChem 265:7055-7061,1990) because of the phase shift mutation of gene.Though most of Chinese hamster ovaries (CHO) cell strain is used for the synthetic of recombinant protein such as Dux-B11; (α 1 in deactivation; 3)-the galactotransferase expression of gene; they still lack, and functional (α 2; 6) the sialyl based transferase synthesizes the sialic acid that (α 2,6) connection end is arranged that the human cell expresses.And the expressed glycoprotein sialic acid of this Chinese hamster ovary celI is easily by the endogenic sialidase degraded of Chinese hamster ovary celI .Biotechnology 13 (7): 692-8 such as (, 1995) Gramer.

As a result, show the biochemistry different and pharmacological characteristics for example transformation period, antigenicity, stability and functional effect by the albumen of these inhuman expression system productions with the cell-derived protein of people.

The progress of nearest stem cells technology has strengthened in fact and utilizes stem cell to be applied to for example potentiality of transplantation treatment, drug screening, toxicologic study and functional genomics.Yet stem cell routine in the substratum that comprises non-human protein's matter is kept, because therefore inhuman infectious substance contamination of heavy is not suitable for clinical application.Further, the stem cell in the inhuman medium of deriving is cultivated and may cause introducing inhuman carbohydrate part so endanger graft application.(Martin etal Nature Medicine11(2)：228-232，2005)。Therefore, special people's derived protein stem cell keep and/or break up in use will improve the proteinic introducing of recessive allele and increase the clinical application of stem cell.

Correspondingly, be necessary to develop protein and their acceptor, it has special desired biochemistry and pharmacological characteristic uses to be used for diagnosis, prevention, treatment, nutrition and/or research, and the invention provides the albumen that contains dimer 4 helical bundles and acceptor thereof and be used for clinical, commercial and research is used.

Summary of the invention

In whole specification sheets, unless stated otherwise, term " contains " or its variant for example " has " or " comprising ", will be understood that to hint comprises described key element or all or the group of key element or whole group, but does not get rid of any other key element or all.

Nucleotide and aminoacid sequence are expressed as sequence identification number (SEQ ID NO :).These SEQ IDNO: corresponding in number sequence identification number＜400〉1 (SEQ ID NO:1),＜400〉2 (SEQ IDNO:2), the rest may be inferred.Table 1 shows is the summary info of recognition sequence number.Sequence list provides after claim.

The present invention briefly relates to the protein isolate that comprises dimer 4 helical bundles or its acceptor or its chimeric molecule with physical and chemical parameter feature, wherein said character representation, association or form the basis of one or more distinctive pharmacological characteristics.Isolating protein particularly provided by the invention or its chimeric molecule are selected from IFN-a2B, IFN-a2B-Fc, IFN-b1, IFN-b1-Fc, IFN-g, IFN-g-Fc, IFNAR2, IFNAR2-Fc, IL-10, IL-10-Fc, IL-10Ra, IL-10Ra-Fc, it has the biochemical character { [P that comprises a plurality of measurable physical and chemical parameters _x] ₁, [P _x] ₂... [P _x] _n,, P wherein _xRepresent that measurable physical and chemical parameter and " n " are 〉=1 integer, wherein between and be included in [P _x] ₁To [P _x] _nBetween the parameter different measurable physical and chemical parameter of respectively doing for oneself, wherein the value representation of any or more than one measurable biochemical characteristic, be associated with a distinctive pharmacological characteristics T _yPerhaps the pharmacological characteristics { [T of series of features _y] ₁, [T _y] ₂... .[T _y] _m, or form a distinctive pharmacological characteristics T, perhaps the pharmacological characteristics { [T of series of features _y] ₁, [T _y] ₂... .[T _y] _mThe basis, wherein Ty represents that a distinctive pharmacological characteristics and m are 〉=1 integer, wherein [T _y] ₁To [T _y] _mBe different pharmacological characteristics.

The term here " distinctive " relates to the pharmacological characteristics of protein or its chimeric molecule, and one or more that is meant protein or its chimeric molecule in the present invention is different from the pharmacological characteristics of the biochemical characteristic of further feature.Specifically, one or more pharmacological characteristics of protein isolate or its chimeric molecule is with different in the characteristic of prokaryotic cell prokaryocyte or the low same protein that waits eukaryotic cell even inhuman higher eucaryotic cells generation or relative difference arranged.In other specific embodiments, help to realize a certain function as the isolating albumen of target or the pharmacological characteristic of its chimeric molecule.Term here " measurable physical and chemical parameter " or P _xOne or more characteristics of measuring of expression protein isolate or its chimeric molecule.In a specific embodiments of the present invention, as measurable physicochemical property of the isolating albumen of target or its chimeric molecule or help or produced pharmacological characteristics T _y

Isolating protein of the present invention or chimeric molecule have physical and chemical parameter (P _x), this parameter is used for intactly defining protein molecule protein molecule or chimeric molecule.This physical and chemical parameter can be selected from: apparent molecular weight (P ₁), iso-electric point (PI) (P ₂), isoform number (P ₃), the relative intensity (P of different isoform numbers ₄), carbohydrate weight percentage (P ₅), N-connects the actual measurement molecular weight (P after the oligosaccharides de-glycosylation ₆), the actual measurement molecular weight (P after the oligosaccharides de-glycosylation that is connected with O-that connects of N- ₇), the per-cent (P of acid contents of monosaccharides ₈), contents of monosaccharides (P ₉), sialic acid content (P ₁₀), vitriol and phosphate content (P ₁₁), Ser/Thr:GalNAc ratio (P ₁₂), N-connects the neutral per-cent (P of oligosaccharides ₁₃), N-connects the acid per-cent (P of oligosaccharides ₁₄), O-connects the neutral per-cent (P of oligosaccharides ₁₅), O-connects the acid per-cent (P of oligosaccharides ₁₆), N-connects the ratio (P of oligosaccharides ₁₇), O-connects the ratio (P of oligosaccharides ₁₈), N-connects the structure (P of oligosaccharides composition ₁₉), O-connects the structure (P of oligosaccharides composition ₂₀), N-connects the position and the formation (P of oligosaccharides ₂₁), O-connects the position and the formation (P of oligosaccharides ₂₂), altogether (P is modified in translation ₂₃), posttranslational modification (P ₂₄), acidylate (P ₂₅), acetylize (P ₂₆), amidation (P ₂₇), deacylated tRNA amine (P ₂₈), biotinylation (P ₂₉), carbamylation (P ₃₀), carboxylation (P ₃₁), decarboxylation (P ₃₂), disulfide linkage forms (P ₃₃), lipid acid acidylate (P ₃₄), myristoylation (P ₃₅), palmitoylation (P ₃₆), octadecane acidylate (P ₃₇), formylation (P ₃₈), saccharification (P ₃₉), glycosylation (P ₄₀), glycophosphatidyl inositol grappling (P ₄₁), hydroxylation (P ₄₂), the combination (P of seleno-cysteine ₄₃), lipid (P ₄₄), the addition (P of Thioctic Acid ₄₅), (P methylates ₄₆), N or C end sealing (P ₄₇), N or C end removes (P ₄₈), nitrated (P ₄₉), methionine(Met) oxidation (P ₅₀), phosphorylation (P ₅₁), the proteolytic enzyme enzyme cuts (P ₅₂), prenylation (P ₅₃), farnesylation (P ₅₄), geranylization (P ₅₅), pyridoxal phosphate addition (P ₅₆), sialylated (P ₅₇), asialoglycoproteinization (P ₅₈), sulphating (P ₅₉), ubiquitinization (P ₆₀), the addition (P of ubiquitin sample molecule ₆₁), primary structure (P ₆₂), secondary structure (P ₆₃), tertiary structure (P ₆₄), quaternary structure (P ₆₅), chemical stability (P ₆₆), thermostability (P ₆₇).The feature of these parameters provides in table 2.

-in a specific embodiment, with physical and chemical parameter (P _x) and pharmacological characteristics (T _y) feature characterize IFN-a2b of the present invention, it comprises

Apparent molecular weight (the P of-1-250kD ₁), as: 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,5O, 51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,9O, 91,92,93,94,95,96,97,98,99,100,110,12O, 13O, 140,15O, 160,17O, 180,190,200,210;

22O, 23O, 24O, 25O kDa are 13-24kDa in a specific embodiments.

Iso-electric point pI (the P of IL-2 of the present invention ₂) be 2-14, as 2,3,4,5,6,7,8,9,10,11,12,13,14, and in a specific embodiments, be 4.5-7;

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

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

Contents of monosaccharides (P ₉) and sialic acid content (P ₁₀) be standard with galn (GalNAc), it is 1: the 0-3 trehalose, 1: 0-3 n acetylglucosamine n (GlcNAc), 1: the 0-6 semi-lactosi, 1: 0-3 seminose and 1: 0-5N-n acetylneuraminic acid n (NouNAc), occurrence are 1: 0-1 trehalose, 1: 0-1 n acetylglucosamine n (GlcNAc), 1: 1-4 semi-lactosi, 1: 0-1 seminose and 1: 0-2N-n acetylneuraminic acid n (NouNAc).

Sialic acid (P ₁₀) be expressed as the per-cent of the contents of monosaccharides among the IFN α 2b of the present invention, be 0 to 50%, 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 embodiments, be 0-10%.

O-connects the neutral per-cent (P of oligosaccharides ₁₅) approximately be 60%-100%, such as: 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 specific embodiments, P ₁₅Be 80%～100%.

O-connects the acid per-cent (P of oligosaccharides ₁₆) approximately be 0～40%, 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%, in specific embodiments, P ₁₆Be O～20%.

With the different biological activity of people IFN-a2b that the non-human cell system is expressed, in specific embodiments, GM-CSF induces propagation (T in IFN of the present invention-a2b inhibition TF-1 cell ₃₂) ability be 250～600 times of the people IFN-a2b that expresses in the E.coli cell.

In a specific embodiment, with physical and chemical parameter feature (P _x) and pharmacological characteristics (T _y) characterizing IFN-b1 of the present invention, it has

Apparent molecular weight (the P of 1-250 ₁), 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, l40,150,160,170,180,190,200,210,220,230,240,250kDa is 15-40kDa in a specific embodiments.

Iso-electric point pI (P ₂) be 2-14, as 2,3,4,5,6,7,8,9,10,11,12,13,14, have about 2-100 isoform (P ₃), as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100 isoforms, in a specific embodiments, isoform number (P ₃) be 1-50.

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

In a specific embodiment, with characterizing physical and chemical parameter feature (P _y) and pharmacological characteristics (T _y) IFN-g of the present invention, it has

Apparent molecular weight (the P of 1-250 ₁), 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 embodiments, be 15 to 30kDa.

Iso-electric point pI (P ₂) at 2-14,, in a specific embodiments, be 4～14 as 2,3,4,5,6,7,8,9,10,11,12,13,14,

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

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

N-connects the actual measurement molecular weight (P after the oligosaccharides de-glycosylation ₆) be approximately 10～25kDa, in a specific embodiments, be 12～20kDa.

Actual measurement molecular weight (P after the oligosaccharides de-glycosylation that is connected with O-that N-connects ₇) be approximately 10～25kDa, in a specific embodiments, be 12～20kDa.

PNGase handles the site (P that the N-that identifies by PMF the back connects oligosaccharides ₂₁), comprise N-48 and N-120 (from the section start open numbering of signal sequence);

With the different biological activity of people IFN-g that the non-human cell system is expressed, in a specific embodiments, IFN-g of the present invention suppresses TNF-a and has HT-29 cell proliferation (T down ₃₂) ability be 11～17 times of the people IFN-g that expresses in the E.coli cell.

In a specific embodiment, with physical and chemical parameter feature (P _y) and pharmacological characteristics (T _y) characterizing IFNAR2-Fc of the present invention, it has

Apparent molecular weight (the P of 1-250 ₁), 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 is 50-105kDa in a specific embodiments.

Iso-electric point pI (P ₂) be 2-14, as 2,3,4,5,6,7,8,9,10,11,12,13,14, in a specific embodiments, be 4-7,

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

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

N-connects the actual measurement molecular weight (P after the oligosaccharides de-glycosylation ₆) between 40-100kDa, in a specific embodiments, be 45-95kDa.

Actual measurement molecular weight (P after the oligosaccharides de-glycosylation that is connected with O-that N-connects ₇) between 40-90kDa, in a specific embodiments, be 45-80kDa.

In a specific embodiment, with physical and chemical parameter feature (P _x) and pharmacological characteristics (T _y) characterizing IL-10 of the present invention, it has

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

Iso-electric point pI (P ₂) be 2-14, as 2,3,4,5,6,7,8,9,10,11,12,13,14, in a specific embodiments, be 6-10,

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

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

N-connects the actual measurement molecular weight (P after the oligosaccharides de-glycosylation ₆) between 8-23kDa, in a specific embodiments, be 10-23kDa.

Actual measurement molecular weight (P after the oligosaccharides de-glycosylation that is connected with O-that N-connects ₇) between 8-23kDa, in a specific embodiments, be 10-23kDa.

Immune response feature (T ₁₃) obviously different with the expressed people IL-10 of inhuman cell system, especially, the protein concentration of IL-10 of the present invention is over-evaluated when detecting with the standardized ELISA test kit of the expressed soluble human IL-10 of inhuman cell system; With the different biological activity of people IL-10 that the non-human cell system is expressed, in a specific embodiments, IL-10 of the present invention suppresses IL-4 and has MC/9 cell proliferation (T down ₃₂) ability be the people IL-10 that expresses in the E.coli cell 10-25 doubly.

In a specific embodiment, with physical and chemical parameter feature (P _x) and pharmacological characteristics (T _y) characterizing IL-10Ra-Fc of the present invention, it has

Apparent molecular weight (the P of 1-250 ₁), 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 76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,110,120,130,140,150,160,17O, 180,190,200,210,220,230,240,250kDa is 50-100kDa in a specific embodiments.

Iso-electric point pI (P ₂) be 2-14, as 2,3,4,5,6,7,8,9,10,11,12,13,14, in a specific embodiments, be 4.5-9.5,

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

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

N-connects the actual measurement molecular weight (P after the oligosaccharides de-glycosylation ₆) between 35-95kDa, in a specific embodiments, be 40-85kDa.

Actual measurement molecular weight (P after the oligosaccharides de-glycosylation that is connected with O-that N-connects ₇) between 30-95kDa, in a specific embodiments, be 36-85kDa.-contents of monosaccharides (P ₉) and sialic acid content (P ₁₀) be standard with galn (GalNAc), be respectively 1: 0.1-4 trehalose, 1: 2-34GlcNAc, 1: 0.5-8 semi-lactosi, 1: 1-13 seminose and 1: 0-3 N-n acetylneuraminic acid n, it when being standard, is 3: 0.1-2 trehalose, 3: 0.01-3 GalNAc, 3: 1-30 GlcNAc, 3: 0.1-4 semi-lactosi and 3: 0-3 N-n acetylneuraminic acid n with 3 times of galns.

-sialic acid (P ₁₀) be expressed as the per-cent of the contents of monosaccharides among the IL-10R alpha-Fc of the present invention, be 0 to 50%, 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 embodiments, be 0-10%.Vitriol (sulfate) content (P ₁₁) be standard with GalNac, be 1: the vitriol of 0-3 in a specific embodiments, is 1: the vitriol of 0-1.5 when being standard with 3 times of seminoses, is 3: 0-1 vitriol in a specific embodiments, is 3: 0-0.6 vitriol;

Sulphating (sulfation) (P ₅₉) be expressed as the per-cent of the monose in the molecule, be 0-50%, for example 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 embodiments, be 0-3%;

N-connects the neutral percentage ratio (P of oligosaccharides ₁₃) be 40-85%, for example 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%, in a specific embodiments, be 55-75%;

N-connects the acid percentage ratio (P of oligosaccharides ₁₄) be 15 to 60%, for example 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%, in a specific embodiments, be 25-45%;

PNGase handles the back and connects glycan site (P by the N-that PMF identifies ₁₁) comprise N-110, N-154, N-177 and N-323 (from the section start open numbering of signal sequence); And the different biological activity of people IL-10Ra-Fc with the expression of non-human cell system, in a specific embodiments, among the IL-10Ra-Fc of the present invention and IL-4 exist the ability of IL-10 inductive Mc/9 cell proliferation (T32) down be the soluble human IL-10Ra molecule of expressing in the E.coli cell 18-150 doubly.

In a specific embodiments, the present invention has imagined a kind of unpack format albumen that comprises dimer 4-helical bundle, for example IFN-a2B, IFN-b1, IFN-g, IL-10 and acceptor thereof, for example IFNAR2, IL-10Ra or its chimeric molecule, IFN-a2B-Fc for example, IFN-b1-Fc, IFN-g-Fc, IFNAR2-Fc, IL10-Fc and IL-10Ra-Fc.Protein among the present invention or its chimeric molecule have distinctive pharmacological characteristics, comprise the following characteristic of part or are made up of following characteristic: result of treatment (T ₁), dose therapeutically effective (TCID ₅₀) (T ₂), bioavailability (T ₃), from being administered into the time (T that keeps treatment level ₄), uptake rate (T ₅), discharge rate (T ₆), special activity (T ₇), thermostability (T ₈), freeze-drying stability (T ₉), blood serum stability (T ₁₀), serum half-life (T ₁₁), the solubleness (T in the blood flow ₁₂), immune response feature (T ₁₃), immunogenicity (T ₁₄), neutralizing antibody suppresses (T _14A), side effect (T ₁₅), receptor/ligand avidity (T ₁₆), receptor/ligand activation (T ₁₇), tissue or cell category specificity (T ₁₈), penetrativity (for example intestines, lung, hemato encephalic barrier, skin etc. the) (T of microbial film or barrier ₁₉), generate the ability (T of blood vessel _19A), tissue absorbs (T ₂₀), the degraded stability (T ₂₁), freeze-thaw stability (T ₂₂), proteolytic enzyme stability (T ₂₃), ubiquitin stability (T ₂₄), administration reduces (T ₂₅), administering mode (T ₂₆), with the consistency (T of other pharmacy auxiliary materials or carrier ₂₇), the residual (T in organism or environment ₂₈), the stability (T in the preservation process ₂₉), (T such as toxicity in organism or environment ₃₀).

In addition, protein of the present invention or chimeric molecule can have different biological effect (T in different cell categories ₃₁), include but not limited to people's primary cell, lymphocyte for example, red corpuscle, the retina cell, liver cell, neuron, keratinocyte, endotheliocyte, the endoderm cell, ectoderm cell, mesoblastema, epithelial cell, nephrocyte, liver cell, osteocyte, medullary cell, lymph-node cell, dermal cell, inoblast, the T cell, the B cell, plasmocyte, natural killer cell, scavenger cell, bite neutrophil leucocyte, the grain cell of Langerhan, dendritic cell, bite sour granulocyte, bite the alkali granulocyte, mammary cell, little leaf cell, prostatic cell, pneumonocyte, the oesophagus cell, pancreatic cell, Beta cell (insulin secretory cell), angioblast, the myocyte, elliptocyte (liver cell), mesenchymal cell, the brain micro blood vessel endothelium cell, astroglia cell, spongiocyte, multiple stem cell comprises adult and embryonic stem cell, multiple progenitor cell; Permanent, conversion or the cancer cell system with other people.

Biological effect in the cell comprises multiplication effect (T ₃₂), the differentiation (T ₃₃), apoptosis (T ₃₄), the growth (T of cell size ₃₅), cytokine adhesion (T ₃₆), cell adhesion (T ₃₇), cell amplification (T ₃₈), cell mobility (T ₃₉), migration and invade (T ₄₀), chemotaxis (T ₄₁), cytophagy (T ₄₂), signal transduction (T ₄₃), rich protein is to receptor/ligand (T ₄₄), the activation (T of JAK/STAT approach ₄₅), the activation (T of Ras-erk approach ₄₆), the activation (T of AKT approach ₄₇), the activation (T of PKC approach ₄₈), the activation (T of PKA approach ₄₉), src activates (T ₅₀), fas activates (T ₅₁), TNFR activates (T ₅₂), NFkB activates (T ₅₃), p38MAPK activates (T ₅₄), c-fos activates (T ₅₅), the secretion (T ₅₆), the acceptor (T that caves in ₅₇), acceptor interaction (T ₅₈), the rise or the downward modulation (T of surface markers ₅₉), before the FACS/change (T of other scattering signatures ₆₀), the change (T of subgroup ratio ₆₁), differential gene expression (T ₆₂), necrocytosis (T ₆₃), cell agglutination (T ₆₄), cellular rejection (T ₆₅), with (the T that combines of heparin sulfate ₆₆), with (the T that combines of glycosylation structure ₆₇), with (the T that combines of chondroitin sulfate ₆₈), with extracellular matrix combine (for example collagen, Zeta protein) (T ₆₉), with artificial material combine (for example support) (T ₇₀), with (the T that combines of carrier ₇₁), with (the T that combines of cofactor ₇₂), independent or in containing other proteinic mixtures to the effect (T of stem cells hyperplasia, differentiation and/or self ₇₃) etc.The feature of these characteristics provides in table 3.

The present invention further provides and comprised isolating albumen or its pulsating chimeric molecule, such as the outer functional domain of the born of the same parents of a membrane bound protein by constant region (Fc) or the framework region of one or more protein connexons in conjunction with human normal immunoglobulin.Such chimeric molecule also is expressed as albumen-Fc here.The present invention has imagined the chimeric example of such albumen-Fc and has comprised IFN-a2B-Fc, IFN～b1-Fc, IFN-g-Fc, IFNAR2-Fc, IL-10-Fc, IL-10Ra-Fc.

Such protein-Fc has the characteristic of measurable physical and chemical parameter, and these characteristics are represented, the one or more specific pharmacological characteristics of related isolating albumen-Fc.Other chimeric molecules of the present invention's imagination comprise albumen or an albumen-Fc or its segment, link to each other with a fat base as the polyunsaturated fatty acid molecule.The fat base here in can the binding molecule skeleton amino-acid residue or an amino-acid residue in the side chain.

The present invention further provides and comprised protein isolate or its pulsating chimeric molecule, such as the outer functional domain of the born of the same parents of an embrane-associated protein by constant region (Fc) or the framework region of one or more protein connexons in conjunction with human normal immunoglobulin.In other respects, the Fc of mammalian immune sphaeroprotein or framework region derive from and comprise primates, comprise the mankind, suede, chimpanzee and gorilla, domestic animal (ox for example, sheep, pig, horse, donkey), laboratory animal is (as mouse, rat, cavy, hamster, rabbit), pet (as cat, dog) and the wildlife that catches are (as rodent, fox, deer, kangaroo).In other embodiments, Fc or framework region are human normal immunoglobulins.Mammals is human in specific embodiments.Such chimeric molecule also is expressed as albumen-Fc here.The present invention has imagined other chimeric molecules and has comprised albumen or an albumen-Fc or its segment, links to each other with a fat base as the polyunsaturated fatty acid molecule.The fat base here in may the binding molecule skeleton amino-acid residue or an amino-acid residue in the side chain.Chimeric molecule among the present invention, comprise IFN-a2B-Fc, IFN-b1-Fc, IFN-g-Fc, IFNAR2-Fc, IL-10-Fc, IL-10Ra-Fc, it has the characteristic of measurable physical and chemical parameter, and these characteristics are represented, the one or more specific pharmacological characteristics of related isolating albumen-Fc.

Therefore, the invention provides by nucleotide sequence coded isolated polypeptide, described sequence is selected from SEQ ID NO:27,29,31,33,35,39.41,43,45,47,49,51,53,55,59,6l, 63,65,67,69,7l, 73,75,79,8l, 83,85,87,89,91,93,95,99,101,103,105,107,111,113,115,116,118,119,121,122,124,125,127,128,130,131,133,134,136,137,139,140,142,143,145,146,148,149, perhaps wherein any one has at least 65% conforming nucleotide sequence with above-mentioned sequence, perhaps can with the sequence of any hybridize under low stringency condition in above-mentioned sequence or its complementary type.

Another aspect of the present invention provides isolated polypeptide, and described polypeptide is by coded through the sequence of cell processing montage corresponding to following sequence mRNA separately separately, and described sequence is made of SEQ IDNO:151,152,153,154.

Another aspect of the present invention provides isolated polypeptide, its aminoacid sequence that comprises is selected from SEQ ID NO:28,30,32,34,36,40,42,44,46,48,50,52,54,56,60,62,64,66,68,70,72,74,76,80,82,84,86,88,90,92,94,96,100,102,104,106,108,112,114,117,12,123,126,129,132,135,138,141,144,147,150, and perhaps wherein any one has the aminoacid sequence of at least 65% similarity with above-mentioned sequence.

The present invention has further imagined and has comprised acceptable carrier, cofactor and/or thinner bonded pharmaceutical composition at least a portion protein or its mosaic, the pharmacology.

Aspect primary structure, the invention provides isolating protein or its mosaic or its segment, its nucleotide coding sequence is selected from SEQ ID NO:27,29,31,33,35,39,41,43,45,47,49,51,53,55,59,61,63,65,67,69,71,73,75,79,81,83,85,87,89,91,93,95,99,101,103,105,107,111,113,115,116,118,119,121,122,124,125,127,128,130,131,133,134,136,137,139,140,142,143,145,146,148,149, perhaps wherein any one has at least 60% conforming nucleotide sequence with above-mentioned sequence, perhaps can with any the sequence in above-mentioned sequence or its complementary type in low stringency condition hybridization.

In addition, other aspects of the present invention provide the isolated nucleic acid molecule of coded protein or its chimeric molecule or its functional area, comprise NO:27 with SEQ ID, 29,3l, 33,35,39.41,43,45,47,49,51,53,55,59,61,63,65,67,69,71,73,75,79,81,83,85,87,89,91,93,95,99,101,103,105,107,111,113,115,116,118,119,121,122,124,125,127,128,130,131,133,134,136,137,139,140,142,143,145,146,148,149, or after the best pairing and/or can with SEQ ID NO:27,29,31,33,35,39,41,43,45,47,49,51,53,55,59,61,63,65,67,69,71,73,75,79,81,83,85,87,89,91,93,95,99,101,103,105,107,111,113,115,116,118,119,121,122,124,125,127,128,130,131,133,134,136,137,139,140,142,143,145,146,148,149 or their nucleotide sequence of complementary type hybridize under low stringency condition.

In a specific embodiments, the present invention points to isolated nucleic acid molecule, it has the nucleotide sequence of the following molecule of coding, affiliated molecule is the protein that comprises dimer 4-helical bundle, it is selected from: IFN-a2B, IFN-b1, IFN-g, IL-10 or its acceptor, IFNAR2 for example, IL-10Ra, or its fragment, basically has SEQ ID NO:28,30,32,34,36,40,42,44,46,48,50,52,54,56,60,62,64,66,68,70,72,74,76,8O, 82,84,86,88,90,92,94,96,100,102,104,106,108,112,114,117,120,123,126,129,132,135,138,141,144,147, one or more aminoacid sequence shown in 150, or with SEQ ID NO:28,30,32,34,36,40,42,44,46,48,50,52,54,56,60,62,64,66,68,70,72,74,76,80,82,84,86,88,90,92,94,96,100,102,104,106,108,112,114,117,120,123,126,129,132,135,138,141,144,147, one or more sequences after 150 pairings have the aminoacid sequence of at least 60% similarity.

In other respects, the invention provides isolated nucleic acid molecule, coding comprises the protein of dimer 4-helical bundle, I FN-a2B for example, IFN-b1, IFN-g, IL-10 or its acceptor, IFNAR2 for example, IL-10Ra, or its chimeric molecule, for example IFN-a2B-Fc, IFN-b1-Fc, IFN-g-Fc, IFNAR2-Fc, IL-10-Fc, IL-10Ra-Fc, or its fragment, comprise to be selected from and have SEQ ID NO:29,31,41,43,45,47,61,63,65,67,81,83,101,103,115,116,118,119,121,122 nucleotide sequence, it is directly or the nucleotide sequence by the coded protein connexon known on one or more this areas, link to each other with constant (Fc) of coding human normal immunoglobulin or the nucleotide sequence of framework region, constant (Fc) of described coding human normal immunoglobulin or the nucleotides sequence of framework region are basically as SEQID NO:1,3,5,7,9,11,13,15,17 or 19 one or more shown in.In a specific embodiments, the nucleotide sequence of coded protein connexon comprises IP, the nucleotide sequence of GSSNT, TRA or VDGIQwIP.

In other respects, the invention provides the isolating protein that comprises dimer 4-helical bundle, IFN-a2B for example, IFN-b1, IFN-g, IL-10 or its acceptor, IFNAR2 for example, IL-10Ra, or its chimeric molecule, IFN-a2B-Fc for example, IFN-b1-Fc, IFN-g-Fc, IFNAR2-Fc, IL-10-Fc, IL-10Ra-Fc, or its fragment, comprise to be selected from and have SEQ IDNO:30,32,42,44,46,48,62,64,66,68,82,84,102,104,117,120,123 aminoacid sequence, its direct or protein connexon by knowing on one or more this areas links to each other with constant (Fc) of human normal immunoglobulin or the nucleotide sequence of framework region, and constant (Fc) of described coding human normal immunoglobulin or the nucleotide sequence of framework region are basically as SEQ ID NO:2,4,6,8,10,12,14,16,18 or 20 one or more shown in.

In specific embodiments, IFN-a2B of the present invention or chimeric IFN-a2B molecule comprise the aminoacid sequence of the n amino acids that starts from SEQ ID (being selected from SEQ ID Nos:32 and 36, wherein n=24 ± 5) such as IFN-a2B-Fc.

In specific embodiments, IFN-b1 of the present invention or chimeric IFN-b1 molecule comprise the aminoacid sequence of the n amino acids that starts from SEQ ID (being selected from SEQ ID Nos:46,48,54 and 56, wherein n=22 ± 5) such as IFN-b 1-Fc.

In specific embodiments, IFN-g of the present invention or chimeric IFN-g molecule comprise the aminoacid sequence of the n amino acids that starts from SEQ ID (being selected from SEQ ID Nos:66,68,74 and 76, wherein n=21 ± 5) such as IFN-g-Fc.

In specific embodiments, IFNAR2 of the present invention or chimeric IFNAR2 molecule comprise the aminoacid sequence of the n amino acids that starts from SEQ ID (being selected from SEQ ID Nos:84,92,94 and 96, wherein n=27 ± 5) such as IFNAR2-Fc.

In specific embodiments, IL-10 of the present invention or chimeric IL-10 molecule comprise the aminoacid sequence of the n amino acids that starts from SEQ ID (being selected from SEQ ID Nos:104 and 108, wherein n=19 ± 5) such as IL-10-Fc.

In specific embodiments, IL-10Ra of the present invention or chimeric IL-10Ra molecule, such as IL-10Ra-Fc comprise the duty platform in SEQ ID (be selected from SEQ ID Nos:123,144,147 and 15O, wherein n=22 ± 5 or 32 ± 5) the aminoacid sequence of n amino acids.

The present invention further provides isolating albumen or its chimeric molecule or its nucleic acid molecule of encoding in diagnosis, prevention, treatment, the application during nutrition and/or research are used.Especially be noted that the method that the invention provides on tested animal treatment or preventing disease or alleviate disease symptoms, said method comprises isolating protein or its chimeric molecule that gives effective dose to described tested animal.

And the present invention has imagined to screen with albumen of the present invention or its chimeric molecule and may have different diagnosis, prevention, treatment, the small molecules of nutrition and/or research application effect.

The present invention has further imagined can use protein isolate or its chimeric molecule as immunogen, produces the antibody that is used for the treatment of and diagnoses.

The present invention has further imagined and can use protein isolate or its chimeric molecule and add the substratum that is used for culturing stem cells or related methods of treatment.

The present invention also provides the formula of medicine that contains albumen or its chimeric molecule in diagnosis, prevention, treatment, the application during nutrition and/or research are used.

The present invention also provides people's source protein or its chimeric molecule to be used as the purposes of the standard protein of immunoassay or its test kit.The present invention also provides the method for determining in biotechnological formulation people's cell expressing people's albumen or its chimeric molecule level.

Table 1

Sequence identifier

Sequence identifier	Sequence
		SEQ ID NO：1	Human IgG l Fc nucleotide sequence
SEQ ID NO：2	Human IgG l Fc aminoacid sequence
		SEQ ID NO：3	Human IgG l Fc nucleotide sequence (variant)
SEQ ID NO：4	Human IgG l Fc aminoacid sequence (variant)
		SEQ ID NO：5	Human IgG2 Fc nucleotide sequence
SEQ ID NO：6	Human IgG2 Fc aminoacid sequence
		SEQ ID NO：7	Human IgG 3 Fc nucleotide sequences
SEQ ID NO：8	Human IgG 3 Fc aminoacid sequences
		SEQ ID NO：9	Human IgG 4 Fc nucleotide sequences
SEQ ID NO：10	Human IgG 4 Fc aminoacid sequences
		SEQ ID NO：11	People IgA1 Fc nucleotide sequence
SEQ ID NO：12	People IgAl Fc aminoacid sequence
		SEQID NO：13	People IgA2 Fc nucleotide sequence
SEQ ID NO：14	People IgA2 Fc aminoacid sequence
		SEQ ID NO：15	People IgM Fc nucleotide sequence
SEQ ID NO：16	People IgM Fc aminoacid sequence
		SEQ ID NO：17	People IgE Fc nucleotide sequence
SEQ ID NO：18	People IgE Fc aminoacid sequence
		SEQ ID NO：19	People IgD Fc nucleotide sequence
SEQ ID NO：20	People IgD Fc aminoacid sequence
		SEQ ID NO：21	Human IgG l Fc forward primer (being used for pIRESbleo IP clone) (nucleotide sequence)
SEQ ID NO：22	Human IgG l Fc reverse primer (being used for pIRESbleo IP clone) (nucleotide sequence)
		SEQ ID NO：23	Human IgG l Fc forward primer (being used for pIRESbleo GSSNT clone) (nucleotide sequence)
SEQ ID NO：24	Human IgG l Fc reverse primer (being used for pIRESbleo GSSNT clone) (nucleotide sequence)
		SEQ ID NO：25	IFN-a2B forward primer (nucleotide sequence)
SEQ ID NO：26	IFN-a2B reverse primer (nucleotide sequence)
		SEQ ID NO：27	IFN-a2B nucleotide sequence (signal peptide)
SEQ ID NO：28	IFN-a2B aminoacid sequence (signal peptide)
		SEQ ID NO：29	IFN-a2B nucleotide sequence (mature peptide)
SEQ ID NO：30	IFN-a2B aminoacid sequence (mature peptide)
		SEQ ID NO：31	IFN-a2B nucleotide sequence (signal peptide+mature peptide)
SEQ ID NO：32	IFN-a2B aminoacid sequence (signal peptide+mature peptide)
		SEQ ID NO：33	IFN-a2B-Fc nucleotide sequence (mature peptide+GSSNT connexon+IgGl Fc)
SEQ ID NO：34	IFN-a2B-Fc aminoacid sequence (mature peptide+GSaNT connexon+IgGl Fc)
		SEQ ID NO：35	The IFN-a2B-F nucleotide sequence is used for complete structure (signal peptide+mature peptide+GSSNT connexon+IgGl Fc)
SEQ ID NO：36	(signal peptide+mature peptide+GsSNT connects the aminoacid sequence of IFN-a2B-Fc complete structure body

Sequence identifier	Sequence
			Meet son+IgGl Fc)
SEQ ID NO：37	IFN-b1 forward primer (nucleotide sequence)
		SEQ ID NO：38	IFN-b1 reverse primer (nucleotide sequence)
SEQ ID NO：39	IFN-b1 nucleotide sequence (signal peptide)
		SEQ ID NO：40	IFN-b1 aminoacid sequence (signal peptide)
SEQ ID NO：41	IFN-b1 nucleotide sequence (mature peptide)
		SEQ ID NO：42	IFN-b1 aminoacid sequence (mature peptide)
SEQ ID NO：43	IFN-b1 nucleotide sequence (mature peptide (variant))
		SEQ ID NO：44	IFN-b1 aminoacid sequence (mature peptide (variant))
SEQ ID NO：45	IFN-b1 nucleotide sequence (signal peptide+mature peptide)
		SEO ID NO：46	IFN-b1 aminoacid sequence (signal peptide+mature peptide)
SEQ ID NO：47	IFN-b1 nucleotide sequence (signal peptide+mature peptide (variant))
		SEQ ID NO：48	IFN-b1 signal amino acid sequence peptide+mature peptide (variant))
SEQ ID NO：49	IFN-b1-Fc nucleotide sequence (mature peptide+GSSNT connexon+IgGl Fc)
		SEQ ID NO：50	IFN-b1-Fc aminoacid sequence (mature peptide+GSSNT connexon+IgGl Fc)
SEQ ID NO：51	IFN-b1-Fc nucleotide sequence (mature peptide (variant)+GSSNT connexon+IgGl Fc)
		SEQ ID NO：52	IFN-b1-Fc aminoacid sequence (mature peptide (variant)+GSSNT connexon+IgGl FC)
SEQ ID NO：53	The nucleotide sequence of IFN-b1-Fc complete structure body (signal peptide+mature peptide+GsSNT connexon+IgGl Fc)
		SEQ ID NO：54	The aminoacid sequence of IFN-b1-Fc complete structure body (signal peptide+mature peptide+GSSNT connexon+IgGl Fc)
SEQ ID NO：55	The nucleotide sequence of IFN-b1-Fc complete structure body (signal peptide+mature peptide (variant)+GSSNT connexon+IgGl Fc)
		SEQ ID NO：56	The aminoacid sequence of IFN-b1-Fc complete structure body (signal peptide+mature peptide (variant)+GSSNT connexon+IgGl Fc)
SEQ ID NO：57	IFN-g forward primer (nucleotide sequence)
		SEQ ID NO：58	IFN-g reverse primer (nucleotide sequence)
SEQ ID NO：59	IFN-g nucleotide sequence (signal peptide)
		SEQ ID NO：60	IFN-g aminoacid sequence (signal peptide)
SEQ ID NO：61	IFN-g nucleotide sequence (mature peptide)
		SEQ ID NO：62	IFN-g aminoacid sequence (mature peptide)
SEQ ID NO：63	IFN-g nucleotide sequence (mature peptide (variant))
		SEO ID NO：64	IFN-g aminoacid sequence (mature peptide (variant))
SEQ ID NO：65	IFN-g nucleotide sequence (signal peptide+mature peptide)
		SEQ ID NO：66	IFN-g aminoacid sequence (signal peptide+mature peptide)
SEQ ID NO：67	IFN-g nucleotide sequence (signal peptide+mature peptide (variant))
		SEQ ID NO：68	IFN-g aminoacid sequence (signal peptide+mature peptide (variant))
SEQ ID NO：69	IFN-g-Fc nucleotide sequence (mature peptide+GSSNT connexon+IgGl Fc)
		SEQ ID NO：70	IFN-gFc aminoacid sequence (mature peptide+GSSNT connexon+IgGl Fc)
SEQ ID NO：71	IFN-g-Fc nucleotide sequence (mature peptide (variant)+GSSNT connexon+IgGl Fc)
		SEQ ID NO：72	IFN-g-Fc aminoacid sequence (mature peptide (variant)+GsSNT connexon+IgGl Fc)
SEQ ID NO：73	(signal peptide+mature peptide+GSSNT connects the nucleotide sequence of IFN-g-Fc complete structure body

Sequence identifier	Sequence
			Son+IgGl Fc)
SEQ ID NO：74	The aminoacid sequence of IFN-g-Fc complete structure body (signal peptide+mature peptide+GSSNT connexon+IgGl Fc)
		SEQ ID NO：75	The nucleotide sequence of IFN-g-Fc complete structure body (signal peptide+mature peptide (variant)+GSSNT connexon+IgGl Fc)
SEQ ID NO：76	The aminoacid sequence of IFN-g-Fc complete structure body (signal peptide+mature peptide (variant)+GSSNT connexon+IgGl Fc)
		SEQ ID NO：77	IFNAR2 forward primer (nucleotide sequence)
SEQ ID NO：78	IFNAR2 reverse primer (nucleotide sequence)
		SEQ ID NO：79	IFNAR2 nucleotide sequence (signal peptide)
SEQ ID NO：80	IFNAR2 aminoacid sequence (signal peptide)
		SEQ ID NO：81	IFNAR2 nucleotide sequence (mature peptide)
SEQ ID NO：82	IFNAR2 aminoacid sequence (mature peptide)
		SEQ ID NO：83	IFNAR2 nucleotide sequence (signal peptide+mature peptide)
SEQ ID NO：84	IFNAR2 aminoacid sequence (signal peptide+mature peptide)
		SEO ID NO：85	IFNAR2-Fc nucleotide sequence (mature peptide+IP connexon+IgGl Fc)
SEQ ID NO：86	IFNAR2-Fc aminoacid sequence (mature peptide+IP connexon+IgGl Fc)
		SEQ ID NO：87	IFNAR2-Fc nucleotide sequence (mature peptide+IP connexon+IgGl Fc (variant))
SEQ ID NO：88	IFNAR2-Fc aminoacid sequence (mature peptide+IP connexon+IgGl Fc (variant)
		SEQ ID NO：89	IFNAR2-Fc nucleotide sequence (mature peptide+GsSNT connexon+IgGl Fc)
SEQ ID NO：90	IFNAR2-Fc aminoacid sequence (mature peptide+GSSNT connexon+IgGl Fc)
		SEQ ID NO：91	The nucleotide sequence of IFNAR2-Fc complete structure body (signal peptide+mature peptide+IP connexon+IgGl Fc)
SEQ ID NO：92	The aminoacid sequence of IFNAR2-Fc complete structure body (signal peptide+mature peptide+IP connexon+IgGl Fc)
		SE0 ID NO：93	The nucleotide sequence of IFNAR2-Fc complete structure body (signal peptide+mature peptide+IP connexon+IgGl Fc (variant))
SEQ ID NO：94	The aminoacid sequence of IFNAR2-Fc complete structure body (signal peptide+mature peptide+IP connexon+IgGl Fc (variant))
		SEQ ID NO：95	The nucleotide sequence of IFNAR2-Fc complete structure body (signal peptide+mature peptide+GSSNT connexon+IgGl Fc)
SEQ ID NO：96	The aminoacid sequence of IFNAR2-Fc complete structure body (signal peptide+mature peptide+GSSNT connexon+IgGl Fc)
		SEQ ID NO：97	IL-10 forward primer (nucleotide sequence)
SEQ ID NO：98	IL-10 reverse primer (nucleotide sequence)
		SEQ ID NO：99	IL-10 nucleotide sequence (signal peptide)
SEQ ID NO：100	IL-10 aminoacid sequence (signal peptide)
		SEQ ID NO：101	IL-10 nucleotide sequence (mature peptide)
SEQ ID NO：102	IL-10 aminoacid sequence (mature peptide)
		SEQ ID NO：103	IL-10 nucleotide sequence (signal peptide+mature peptide)
SEQ ID NO：104	IL-10 aminoacid sequence (signal peptide+mature peptide)
		SEQ ID N0：105	IL-10-Fc nucleotide sequence (mature peptide+GSSNT connexon IgGl Fc)

Sequence identifier	Sequence
		SEQ ID NO：106	IL-10-Fc aminoacid sequence (mature peptide+GSsNT connexon IgGl Fc)
SEQ ID NO：107	The nucleotide sequence of IL-10-Fc complete structure body (signal peptide+mature peptide+GSaNT connexon IgGl Fc)
		SEQ ID NO：108	The aminoacid sequence of IL-10-Fc complete structure body (signal peptide+mature peptide+GSSNT connexon IgGl Fc)
SEQ ID NO：109	IL-10Ra forward primer (nucleotide sequence)
		SEQ ID NO：110	IL-10Ra reverse primer (nucleotide sequence)
SEQ ID NO：111	The nucleotide sequence of IL-10Ra (signal peptide)
		SEQ ID NO：112	The aminoacid sequence of IL-10Ra (signal peptide)
SEO ID NO：113	The nucleotide sequence of IL-1ORa (signal peptide (variant))
		SEQ ID NO：114	The aminoacid sequence of IL-1ORa (signal peptide (variant))
SEQ ID NO：115	The nucleotide sequence of IL-10Ra (mature peptide)
		SEQ ID NO：116	The nucleotide sequence of IL-10Ra (mature peptide (variant 1))
SEQ ID NO：117	IL-10Ra aminoacid sequence (mature peptide (normal and variant 1))
		SEQ ID NO：118	The nucleotide sequence of IL-10Ra (mature peptide (variant 2))
SEQ ID NO：119	The nucleotide sequence of IL-10Ra (mature peptide (variant 3))
		SEQ ID NO：120	The aminoacid sequence of IL-l0Ra (mature peptide (variant 2 and 3))
SEQ ID NO：121	The nucleotide sequence of IL-10Ra (signal peptide+mature peptide (normal or variant 2))
		SEQ ID NO：122	The nucleotide sequence of IL-10Ra (signal peptide+mature peptide (variant 1 or 3))
SEQ ID NO：123	The aminoacid sequence of IL-10Ra (signal peptide+mature peptide)
		SEQ ID NO：124	The nucleotide sequence of IL-1ORa-Fc (mature peptide+GIP connexon+IgGl Fc)
SEQ ID NO：125	The nucleotide sequence of IL-10Ra-Fc (mature peptide (variant 1)+GIP connexon+IgGl Fc)
		SEQ ID NO：126	The aminoacid sequence of IL-10Ra-Fc (mature peptide (normal and variant 1)+GIP connexon+IgGl Fc)
SEQ ID NO：127	The nucleotide sequence of IL-1ORa-Fc (mature peptide (variant 2)+GIP connexon+IgGl Fc)
		SEQ ID NO：128	The nucleotide sequence of IL-10Ra-Fc (mature peptide (variant 3)+GIP connexon+IgGl Fc)
SEQ ID NO：129	The aminoacid sequence of IL-10Ra-Fc (mature peptide (variant 2 and 3)+GIP connexon+IgGl Fc)
		SEQ ID NO：130	The nucleotide sequence of IL-10Ra-Fc (mature peptide+GIP connexon+IgGl Fc (variant))
SEQ ID NO：131	The nucleotide sequence of IL-10Ra-Fc (mature peptide (variant 1)+GIP connexon+IgGl Fc (variant))
		SEQ ID NO：132	The aminoacid sequence of IL-10Ra-Fc (mature peptide (normal and variant 1)+GIP connexon+IgGl Fc (variant)
SEQ ID NO：133	The nucleotide sequence of IL-10Ra-Fc (mature peptide (variant 2)+GIP connexon+IgGl Fc (variant))
		SEQ ID NO：134	IL-10Ra-Fc nucleotide sequence (mature peptide (variant 3)+GIP connexon+IgGl

Sequence identifier	Sequence
			Fc (variant))
SEQ ID NO：135	The aminoacid sequence of IL-10Ra-Fc (mature peptide (variant 2 and 3)+GIP connexon+IgGl Fc (variant))
		SEQ ID NO：136	The nucleotide sequence of IL-10Ra-Fc (mature peptide+GsSNT connexon+IgGl Fc)
SEQ ID NO：137	The nucleotide sequence of IL-10Ra-Fc (mature peptide (variant 1)+GSSNT connexon+IgGl Fc)
		SEQ ID NO：138	The aminoacid sequence of IL-10Ra-Fc (mature peptide (normal and variant 1)+GssNT connexon+IgGl Fc)
SEQ ID NO：l39	The nucleotide sequence of IL-10Ra-Fc (mature peptide (variant 2)+GSSNT connexon+IgGl Fc)
		SEQ ID NO：140	The nucleotide sequence of IL-10Ra-Fc (mature peptide (variant 3)+GSSNT connexon+IgGl Fc)
SEO ID NO：141	The aminoacid sequence of IL-10Ra-Fc (mature peptide (variant 2 and 3)+GSSNT connexon+IgGl Fc)
		SEQ ID NO：142	The nucleotide sequence of IL-10Ra-Fc complete structure body (signal peptide+mature peptide (normal or variant 2)+GIP connexon+IgGl Fc)
SEQ ID NO：143	The nucleotide sequence of IL-10Ra-Fc complete structure body (signal peptide+mature peptide (variant s 1 or 3)+GIP connexon+IgGl Fc)
		SEQ ID NO：144	The aminoacid sequence of IL-10Ra-Fc complete structure body (signal peptide+mature peptide+GIP connexon+IgGl Fc)
SEQ ID NO：145	The nucleotide sequence of IL-10Ra-Fc complete structure body (signal peptide+mature peptide (normal or variant 2)+GIP connexon+IgGl Fc (variant))
		SEQ ID NO：146	The nucleotide sequence of IL-10Ra-Fc complete structure body (signal peptide+mature peptide (variant 1 or 3)+GIP connexon+IgGl Fc (variant))
SEQ ID NO：147	The aminoacid sequence of IL-10Ra-Fc complete structure body (signal peptide+mature peptide+GIP connexon+IgGl Fc (variant))
		SEQ ID NO：148	The nucleotide sequence of IL-10Ra-Fc complete structure body (signal peptide+mature peptide (normal or variant 2)+GSSNT connexon+IgGl Fc)
SEQ ID NO：149	The nucleotide sequence of IL-10Ra-Fc complete structure body (signal peptide+mature peptide (variant s1 or 3)+GSSNT connexon+IgGl Fc)
		SEQ ID NO：150	The aminoacid sequence of IL-10Ra-Fc complete structure body (signal peptide+mature peptide+GSSNT connexon+IgGl Fc)
SEQ ID NO：151	The genomic nucleotide sequence of IFNa-2B
		SEQ ID NO：152	The genomic nucleotide sequence of IFN-b1
SEQ ID NO：153	The genomic nucleotide sequence of IFN-g
		SEQ ID NO：154	The genomic nucleotide sequence of IL-10

The tabulation of the abbreviation that this paper uses always is provided in table 4 and the table 5.

Table 4: be called for short and another name

Be called for short	Describe
		AAA	Amino acid analysis
AFC	Affinity chromatography
		bFGF	Basic Fibroblast Growth Factor, FGF2
BSA	Bovine serum albumin
		cDLC	Composite fuel part chromatography
CRD	Carbohydrate recognition domain
		CSF	G CFS
DCS	The donor calf serum
		DeoxGlc	2-deoxidation glucose
DLC	The false affinity chromatography of fuel part
		DSC	Differential scanning calorimetry
ECD	Extracellular domain
		EGF	Urogastron
ELISA	Enzyme Linked Immunoadsorbent Assay
		EPO	Erythropoietin
EST	The sequence label of expressing
		Fc	FC or constant region for immunoglobulin
FCS	Foetal calf serum
		FGF2	Basic Fibroblast Growth Factor, bFGF
FTIS	Fourier transform infrared spectroscopy
		Fuc	Trehalose
G-CSF	Granulocyte colony-stimulating factor
		Gal	Semi-lactosi
GalNAc，galactosamine	The 2-deoxidation, 2 galns
		GFC	Gel permeation chromatography
GlcA	Glucuronic acid
		GlcNAc，glucosamine	The 2-deoxidation, 2 glucosamines
Glc	Glucose
		GM-CSF	Granulocyte-macrophage colony stimutaing factor
HBS	The Hepes buffering salt
		hES	Human embryo stem cell
HIC	Hydrophobic interaction chromatograph

Be called for short	Describe
		HPAEC-PAD	The high pH anion-exchange chromatography that uses the pulse ampere electric current to detect
HPLC	High pressure liquid chromatography or high performance liquid chromatography
		HSA	Human serum albumin
HTS	High flux screening
		IdoA	Iduronic acid
IEC	Ion exchange chromatography
		IEF	Isoelectrofocusing
IFN	Interferon, rabbit
		IFN-a2B	Interferon alpha 2 b
IFNAR2	Interferon alpha/beta receptor β chain (IFNAR 2 for IFN-aR2, IFNAR2, IFNAR-2, and Ifnar-2, IFN-aRb); Interferon, rabbit (α β ω) acceptor 2; Interferon, rabbit (α and β) acceptor 2; Interferon alpha/beta receptor-2; IFNA; IFN-α-REC; IFN-R; I type Interferon Receptors
		IFN-b1	Interferon beta 1 (IFNB1); Inoblast (IFNB1); Interferon-; IFN-β 1; IFN-β; IFN-β 1a; IFB; IFNB; IFF; The fibroblast interferon (Fi-IFN, F-IFN); I type Interferon, rabbit pH2 stablizes Interferon, rabbit, the R1-GI factor
IFN-g	Interferon, rabbit Y (IFNG); IFN Y; The antigen induction Interferon, rabbit; Type II interferon IIF; IFG; IFI; The Type-2 Interferon, rabbit; The T Interferon, rabbit; The antigen induction Interferon, rabbit; The unstable Interferon, rabbit of pH2-; Azathioprine; Interferon, rabbit Y-lb (recombinant protein).
		Ig	Immunoglobulin (Ig)
IL	Interleukin
		IL-10	Interleukin-11 0 (IL10); ILlOA; The cell-derived T cell growth factor of B (B-TCGF); Cytokine synthesis inhibitory factor(CSIF) (CSIF); T cell growth inhibitory factor (TGIF)
IL-10Ra	Interleukin-11 0 acceptor a (IL10Ra); ILl0RA
		lacNAc	N-acetyl lactosamine
lacdiNAc	N.N’-diacetyllactosediamine
		LC	The fluid chromatography
MALODI-TOF	Matrix-auxiliary laser desorption ionization-flight time
		Man	Seminose

Be called for short	Describe
		MCC	Metal chelate chromatography
MS	Mass spectrum
		NacSial, NeuAc or NeuNAc	The neural preface L (N-acetyl neuraminicacid) of N-acetyl
NGlySial, NeuGc or NeuGly	N-hydroxyacetylneuraminic acid (N-glycolylneuraminic acid)
		PBS	Phosphate buffer soln
PCS	The photon correlation spectroscopy method
		PDGF-AA	Platelet-derived somatomedin A homodimer
PNGase	Tire-N4-(l-asparagine acid amides enzyme of N-ethanoyl-β-D-glvcosaminyl)
		RMLP	Receptor-mediated part chromatography
RPC	Reversed phase chromatography
		SDS PAGE	Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SEC	Exclusion chromatography
		Sia	Silicoaluminate
TCA	Trichoroacetic acid(TCA)
		TFF	Tangential flow filtration
TGF	Transforming growth factor
		TNF	Tumour necrosis factor
TNFR	Tumor Necrosis Factor Receptors
		Xyl	Wood sugar

Table 5: amino acid is called for short

Amino acid	The trigram code	The single-letter code
			L-Ala	Ala	A
Arginine	Arg	R
			L-asparagine	Asn	N
Aspartic acid	Asp	D
			Halfcystine	Cys	C
L-glutamic acid	Clu	E
			Glutamine	Gin	Q
Glycine	Gly	G
			Histidine	His	H
Isoleucine	lie	I
			Leucine	Leu	L
Methionin	Lys	K
			Methionine(Met)	Met	M
Phenylalanine	Phe	F
			Proline(Pro)	Pro	P
Serine	Ser	S
			Threonine	Thr	T
Tryptophane	Trp	W
			Tyrosine	Tyr	Y
Xie Ansuan	Val	V

Table 6: stem cell tabulation

Cell type
	Common stem cell type
Embryonic stem cell
	The plasmocyte 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 cell
Epithelium
	The human keratinocyte stem cell
The instantaneous amplifying cells of human keratinocyte
	People's melanophore stem cell
People's melanophore
	Skin
Human foreskin fibroblast
	Pancreas
People's ureter cell
	Human pancreas's island
Human pancreas's beta cell
	Kidney
The sophisticated kidney stem cell of people
	The human embryo kidney (HEK) epithelial stem cell
People's kidney epithelial cell
	Liver
People's liver elliptocyte
	People's liver cell
The single ductal epithelial cell of people
	People embryo endodermal stem cells
People's adult human liver stem cell (its existence is had dispute)
	Breast
The human breast epithelial stem cell

Cell type
	Lung
The stem cell of bone marrow derived
	People's lung fibroblast
Human bronchial epithelial cell
	The non-race of people II type pneumonocyte
Muscle
	People's skeletal muscle stem cell (satellite cell)
Heart
	People myocardial cell
Bone marrow interstital stem cell
	Simple squamous cell
Descending Aorta epithelial cell
	The arc epithelial cell of Aorta
The Aorta smooth muscle cell
	Eyes
The Limbal stem cell
	Corneal epithelial cell
The CD34+ hemopoietic stem cell
	Mescenchymal stem cell
Osteoblasts (precursor is a mescenchymal stem cell)
	Peripheral blood mononuclear progenitor cell (hemopoietic stem cell)
Between matter (precursor is above-mentioned cell type)
	Mesenchymal cell
Spleen
	People's spleen precursor stem cell
Human spleen cell
	Immunocyte
People CD4+T-cell
	People CD8+T-cell
NK cells of human beings
	The person monocytic cell
Human macrophage
	Human dendritic cell
People B-cell
	Nose
Goble t cell (the mucus secretory cell of nose)
	Pseudostrimatic ciliate stanchion cell (being positioned at below, nose regio olfactoria)

Cell type
	Pseudostratified ciliate epithelial cell (this cell is compared to ductus nasopharyngeus)
Tracheae
	Lamination epidermic cell (this cell comparison and formation tracheae)
Ciliate mast cell (this cell comparison and formation tracheae)
	Goblet cell (this cell comparison and formation tracheae)
Basal cell (this cell comparison and formation tracheae)
	Oesophagus
The cricopharyngeus cell
	Reproduction
Female vesica originally
	Male spermatogonium

Description of drawings

Fig. 1 is the diagram that coding proteinic cDNA of the present invention inserts clone's process of pIRESbleo3 or pIRESbleo3-Fc carrier.

Fig. 2 represents the cellulotoxic effect of the people IFN-a2b (square) of IFN-a2b of the present invention (circle) and E.coli cell expressing for GM-CSF inductive TF-1 cell proliferation.

Fig. 3 represents the cellulotoxic effect of IFN-b1 of the present invention (circle) for GM-CsF inductive TF-1 cell proliferation.

Fig. 4 is illustrated under the TNF-a existence condition, and the people IFN-g (square) of IFN-g of the present invention (circle) and E.coli cell expressing is for the retarding effect of people HT-29 cell proliferation.

Fig. 5 represents the cytotoxicity neutralizing effect of IFNAR2-Fc of the present invention for the GM-CSF inductive TF-1 cell proliferation of IFN-a2b mediation.

Fig. 6 is illustrated under the condition of IL-4 existence, and the people IL-10 (square) that IL-10 of the present invention (circle) and E.coli express is for the effect of MC/9 cell proliferation.

Fig. 7 is illustrated under the condition of IL-4 existence, and the soluble human IL-10R molecule (square) of IL-10R-Fc of the present invention (circle) and E.coli cell expressing is for the neutralizing effect of the MC/9 cell proliferation of IL-10 mediation.

Fig. 8 represent between people IL-10 (rhombus) the immune response characteristic of IL-10 of the present invention (square) and E.coll cell expressing in vitro tests relatively.Error line is represented the standard error of mean value.

Summary 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 possible change and so on. Also be interpreted as that the term purpose only is to describe specific embodiment and does not specially limit as used herein.

Should be noted that what this specification used, indefinite article and the definite article of singulative comprise plural number, unless context indicates in addition. Therefore, for example,, about " a kind of protein ", " a kind of cell factor " or " a kind of chimeric molecule " or " a kind of acceptor ", comprised that single parameter has also comprised two or more described parameters.

Term " compound ", " active factors ", " chemokines ", " pharmacology activating agent ", " medicament ", " active matter " and " medicine " use in this exchange, relate to a kind of compound and relate to especially a kind of protein or its chimeric molecule of inducing desired biochemistry and/or pharmacological effect. This term also comprises the acceptable and pharmacological active component of the pharmacy of these active factorses, at this, is particularly related to and includes, but are not limited to salt, ester class, amide-type, pro-drug, active metabolite, analog etc. While using term " compound ", " active factors ", " chemokines ", " pharmacology activating agent ", " medicament ", " activity " and " medicine ", be interpreted as that it comprises that active factors itself reaches pharmacy acceptable, pharmacology active salt, ester class, amide-type, pro-drug, active metabolite, analog etc.

The composition that comprises two or more active active materials about " compound ", " active factors ", " chemokines ", " pharmacology activating agent ", " medicament ", " active matter " and " medicine ", for example two or more cell factors. " composition " also comprises for example two parts composition of many parts, and wherein before formula, the factor is provided respectively and given or preparation or mixing respectively.

For example, many parts cartridge bag (multi-part pharmaceutical pack) can have to be selected from and comprises dimer 4-helical bundle, for example IFN-a2B, IFN-b1, IFN-g, IL-10 and their acceptors separately, IFN-a2B for example, IL-1ORa and chimeric molecule thereof, for example IFN-a2B-Fc, IFN-b1-Fc, IFN-g-Fc, IFNAR2-Fc, IL-10-Fc, 2 or greater protein matter or its chimeric molecule of IL-10Ra-Fc, preserve dividually.

As used herein the term of reagent " effective dose " and " treatment effective dose " expression protein or its chimeric molecule individually or in the composition of other reagent is arranged so that desired treatment or the sufficient amount of physiological effect or result to be provided. Undesirable effect, for example side effect, proof is followed desirable result for the treatment of sometimes; Therefore, the benefit that doctor's balance is possible and possible risk are to determine what is suitable " effective dose ". According to experimenter's species, age and comprehensive situation, mode of administration etc., definite amount essential between experimenter and experimenter can change. Therefore, can not specify one accurate " effective dose ". Yet, to suitable " effective dose " of any individual case, can use unique routine test to determine by this area those skilled in the art.

Use " pharmacy is acceptable " carrier, excipient or diluent represent that the pharmacy carrier comprises abiotic or non-undesirable substance, that is, material and selected active factors administration together do not cause any side effect or substantial side effect to the experimenter. Carrier can comprise auxiliary material and other additives, such as diluent, detergent, colouring agent, wetting agent or emulsifying agent, pH buffer, anticorrisive agent etc.

Similarly, " pharmacy is acceptable " salt, ester class, amide-type, pro-drug or the derivative at this composition that provides refers to abiotic or non-bad salt, ester class, amide-type, pro-drug or derivative.

Term " treatment " and " therapy " relate to the severity of the symptom that is treated disease and/or alleviating of frequency as used herein, the elimination of symptom and/or potential cause, the improvement of the prevention that the symptom of disease and/or their potential cause occurs and the infringement of accompanying diseases or remedy or take a turn for the better.

" treatment " experimenter can be included in the treatment to the clinical symptoms individuality of the prevention of disease in the individuality of susceptible or other bad physiology results and the symptom by improving disease.

" experimenter " relates to animal as used herein, in specific specific embodiments, and mammal, and in further specific embodiments, the people that can be benefited from pharmaceutical preparation of the present invention and method. In this not restriction of kind to the animal that can be benefited from the pharmaceutical preparation of present description and method. No matter being people or non-human animal, the experimenter can be called as individuality, patient, animal, host or acceptor. Compounds and methods for of the present invention is applied to physianthropy, veterinary science and general, that raise and train or wild animal breeding.

The above points out, in specific specific embodiments, animal is people or other primates for example orangutan, gorilla, ape, livestock animals, laboratory test animal, pairing animal or the wild animal that is captured and bird.

The laboratory test animal comprises mouse, mouse, rabbit, cavy and hamster for example. Rabbit and rodent, for example mouse and mouse, provide pilot system or animal model easily. Livestock animals comprises sheep, ox, pig, goat, horse and donkey. Nonmammalian for example bird, fish and amphibian comprises Xenopus, procaryon and non-lactation eucaryote.

Term " cell factor " is used for its most general meaning and comprises any range protein by emiocytosis, and it regulates immune system, regulates the functional activity of individual cells and/or tissue, and/or induces a series of physiological reactions. Term " cell factor " is construed as the cell factor that relates to " complete " and comprises one or more amino acid whose increases as used herein, disappearance or alternative, and basically keep its fragment of the BA of the intact cell factor, derivative or homologue or chimeric molecule.

" cytokine receptor " is the albuminous cell factor acceptor cell membrane association or soluble, with the cytokine signaling system or regulate relevant. Term " cytokine receptor " is construed as the cytokine receptor that relates to " complete " and comprises one or more amino acid whose increases as used herein, lack or substitute, and basically keep its fragment, derivative or homologue or the chimeric molecule of the BA of intact cell factor acceptor.

Term " protein " is used for its most general meaning and comprises cell factor and cytokine receptor. As used herein, term " protein " should be understood to relate to the protein of " complete " and comprises one or more amino acid whose increases, lack or substitute, and basically keep its fragment, derivative or homologue or the chimeric molecule of the BA of whole protein.

The present invention has imagined the protein or its chimeric molecule that separate, and it has measurable physical and chemical parameter (P_x) feature, wherein this character representation, be associated with one or more distinctive pharmacology characteristic (T_y) or form one or more distinctive pharmacology characteristic (T_y) basis. The protein or its chimeric molecule that separate are that protein isolate or chimeric molecule are a kind of albumen that contains dimer 4-helical bundle or its acceptor, comprising IFN-a2B, and IFN-a2B-Fc, IFN-b1, IFN-b1-Fc, IFN-g, IFN-g-Fc, IFNAR2, IFNAR2-Fc, IL-10, IL-10-Fc, IL-10Ra, IL-10Ra-Fc. IFN-a2B described here, IFN-a2B-Fc, IFN-b1, IFN-b1-Fc, IFN-g, IFN-g-Fc, IFNAR2, IFNAR2-Fc, IL-10, IL-10-Fc, IL-10Ra, IL-10Ra-Fc comprise whole polypeptide and its fragment that relates to. .

More particularly, the invention provides a kind of protein or its chimeric molecule of separation, it has the biochemical character that comprises a series of measurable physical and chemical parameters, { [P_x] ₁， [P _x] ₂、··[P _x] _n,, P wherein_xRepresent that measurable physical and chemical parameter and " n " are 〉=1 integer, wherein [P_x] ₁To [P_x] _nEach different measurable physical and chemical parameter naturally, the wherein numeric representation of any one or a plurality of measurable biochemical characters, be associated with a distinctive pharmacology characteristic T_yOr a series of distinctive pharmacology characteristic ([T_y] ₁、[T _y] ₂、....[T _y] _m, or form a distinctive pharmacology characteristic T_yOr a series of distinctive pharmacology characteristic ([T_y] ₁、[T _y] ₂、....[T _y] _mBasis, T wherein_yRepresent that a distinctive pharmacological characteristic and " m " are 〉=1 integer, and [T_y] ₁To [T_y] _mEach is a different pharmacology characteristic naturally.

Term " measurable physical and chemical parameter " (P as used herein_x) relate to the feature of protein or its chimeric molecule of one or more measurable separation. Representational " special measurable physical and chemical parameter " includes, but are not limited to: apparent molecular weight (P₁), isoelectric point (pI) (P₂), isoform number (P₃), the relative intensity (P of different isoform numbers₄), carbohydrate percetage by weight (P₅), N-connects the actual measurement molecular weight (P after the oligosaccharides deglycosylation₆), the actual measurement molecular weight (P after the oligosaccharides deglycosylation that is connected with O-that connects of N-₇), 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), N-connects the neutral percentage (P of oligosaccharides₁₃), N-connects the acid percentage (P of oligosaccharides₁₄), O-connects the neutral percentage (P of oligosaccharides₁₅), O-connects the acid percentage (P of oligosaccharides₁₆), N-connects the ratio (P of oligosaccharides₁₇), O-connects the ratio (P of oligosaccharides₁₈), N-connects the structure (P of oligosaccharide ingredient₁₉), O-connects the structure (P of oligosaccharide ingredient₂₀), N-connects position and the formation (P of oligosaccharides₂₁), O-connects position and the formation (P of oligosaccharides₂₂), altogether (P is modified in translation₂₃), posttranslational modification (P₂₄), acidylate (P₂₅), acetylation (P₂₆), amidatioon (P₂₇), deacylated tRNA amine (P₂₈), biotinylation (P₂₉), carbamylation (P₃₀), carboxylation (P₃₁), decarboxylation (P₃₂), disulfide bond forms (P₃₃), aliphatic acid acidylate (P₃₄), myristoylation (P₃₅), palmitoylation (P₃₆), octadecane acidylate (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), (P methylates₄₅), N or C end sealing (P₄₇), N or C end removes (P₄₈), nitrated (P₄₉), methionine oxidation (P₅₀), phosphorylation (P₅₁), the protease enzyme cuts (P₅₂), prenylation (P₅₃), farnesylation (P₅₄), Mang ox baseization (P₅₅), phosphopyridoxal pyridoxal phosphate addition (P₅₆), saliva acidifying (P₅₇), asialoglycoprotein (P₅₈), sulfation (P₅₉), ubiquitin (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 provides in table 2.

Term " distinctive (distinctive) pharmacology characteristic " is interpreted as by those skilled in the art any pharmacological or clinical relevant characteristic that comprises protein of the present invention or chimeric molecule. Representational " pharmacology characteristic " not only is defined as invention and comprises: result for the treatment of (T₁), dose therapeutically effective (TCID₅₀)(T ₂), bioavilability (T₃), from being administered into the time (T that keeps treatment level₄), absorption rate (T₅), discharge rate (T₆), special activity (T₇), heat endurance (T₈), freeze-drying stability (T₉), serum/plasma stability (T₁₀), serum half-life (T₁₁), the solubility (T in blood flow₁₂), immune response feature (T₁₃), immunogenicity (T₁₄), neutralizing antibody suppresses (T_14A), side effect (T₁₅), acceptor/part affinity (T₁₆), acceptor/part activation (T₁₇), tissue or cell category specificity (T₁₈), the penetration capacity of biomembrane or barrier (for example intestines, lung, blood-brain barrier, skin etc.) (T₁₉), generate the ability (T of blood vessel_19A), tissue absorbs (T₂₀), the degraded stability (T₂₁), freeze-thaw stability (T₂₂), protease stability (T₂₃), ubiquitin stability (T₂₄), administration reduces (T₂₅), mode of administration (T₂₆), with the compatibility (T of other pharmacy auxiliary materials or carrier₂₇), the residual (T in organism or environment₂₈), the stability (T in the preservation process₂₉), (T such as toxicity in organism or environment₃₀)。

In addition, protein of the present invention or chimeric molecule can have different biological effect (T in different cell categories₃₁), include but not limited to people's primary cell, lymphocyte for example, red blood cell, the retina cell, liver cell, neuron, horn cell, endothelial cell, the endoderm cell, ectoderm cell, mesoblastema, epithelial cell, nephrocyte, liver cell, osteocyte, bone marrow cell, LNC, dermal cell, fibroblast, the T cell, the B cell, thick liquid cell, NK, macrophage, bite neutrophil leucocyte, the grain cell of Langerhan, BMDC, bite sour granulocyte, bite the alkali granulocyte, mammary cell, little leaf cell, prostatic cell, pneumonocyte, the oesophagus cell, pancreatic cell, Beta cell (insulin secretion cell), angioblast, the myocyte, elliptocyte (liver cell), mesenchymal cell, brain microvessel endothelial cells in vitro, astroglia, spongiocyte, multiple stem cell comprises adult and embryonic stem cell, multiple CFU-GM, permanent, conversion or the cancer cell system with other people. biological effect in cell comprises multiplication effect (T₃₂), the differentiation (T₃₃), apoptosis (T₃₄), the growth (T of cell size₃₅), cell factor adhesion (T₃₆), cell adhesion (T₃₇), cell diffusion (T₃₈), cell mobility (T₃₉), migration and invade (T₄₀), chemotaxis (T₄₁), cytophagy (T₄₂), signal transduction (T₄₃), raise albumen to acceptor/part (T₄₄), the activation (T of JAK/STAT approach₄₅), the activation (T46) of Ras-erk approach, the activation (T of AKT approach₄₇), the activation (T of PKC approach₄₈), the activation (T of PKA approach₄₉), src activates (T₅₀), fas activates (T₅₁), TNFR activates (T₅₂), NFkB activates (T₅₃), p38MAPK activates (T₅₄), c-fos activates (T₅₅), the secretion (T₅₆), the acceptor (T that caves in₅₇), acceptor reciprocation (T₅₈), rise or the downward (T of surface markers₅₉), before FACs/change (T of other scattering feature₆₀), the change (T of subgroup ratio₆₁), differential gene expression (T₆₂), meronecrosis (T₆₃), cell agglutination (T₆₄), cellular rejection (T₆₅), with the combination (T of heparin sulfate₆₆), with the combination (T of glycosylation structure₆₇), with the combination (T of chondroitin sulfate₆₈), with combination (for example collagen, the fibronectin) (T of extracellular matrix₆₉), with combination (for example the support) (T of artificial material₇₀), with the combination (T of carrier₇₁), with the combination (T of confactor₇₂), independent or in containing the mixture of other protein to the effect (T of stem cells hyperplasia, differentiation and/or self₇₃) etc. The summary of these characteristics provides in table 3.

Term " distinctive " is relevant with the pharmacology characteristic of protein of the present invention or chimeric molecule as used herein, relates to the pharmacology characteristic of one or more protein or its chimeric molecule, and it is distinctive for special biochemical character. In specific specific embodiments, the protein that separates or one or more pharmacology characteristics of its chimeric molecule are different from, or particularity with respect to, the identical protein that produces in protokaryon or the eukaryotic such as low or even high inhuman eukaryotic or the form of chimeric molecule. In specific embodiment, by the pharmacology characteristic of the isolated protein of being tested or its chimeric molecule substantially similar in appearance to or function equivalence in the protein of generation naturally.

Term " protokaryon " relates to any prokaryotic as used herein, and it comprises any bacterial cell (comprising the actinomyces cell) or archeobacteria cell. The meaning of term " non-human eucaryote " is self evident as used herein. Yet for clear, this term comprises any non-human eucaryote especially, and it comprises: yeast is saccharomyces or pichia for example; Other fungies; Insect, comprise Drosophila and insect cell culture; Fish, comprise that chub mackerel belongs to; Amphibian, comprise Xenopus; Plant and plant cell culture.

Relating to " stem cell " comprises embryo or adult stem cell and is included in the stem cell of listing in table 6. Protein of the present invention or chimeric molecule can be used separately or with the protein in cocktail, be used, to induce one or more stem cells hyperplasias, differentiation or self.

it is determined that the primary structure of protein or its chimeric molecule can be used as amino acid sequence. it is determined that secondary structure can be used as quantity and/or the relative position of one or more secondary protein structures, alpha-helix for example, parallel beta sheet, antiparallel beta sheet or corner. tertiary structure is described the folding of polypeptide chain, and different secondary structure elements is assembled into special comparison. spiral is secondary building unit with folding, and domain is the tertiary structure unit. in Multidomain protein, tertiary structure comprises domain comparison each other. accordingly, tertiary structure can be to the existence of one or more protein domains, disappearance, and quantity and/or relative position are measured. representational domain is not only comprising of the present invention's restriction: single-screw, the helix turn helix domain, four-helix bundle, DNA is in conjunction with territory, the triple helical bundle, Greece's key helical bundle, spiral-spiral packaging structure territory, β-sandwich, β-tubbiness, up and down antiparallel beta sheet, Greece's key topological structure territory, jam volume topological structure territory, β-screw, β-clover, β-spiral, Rossman is folding, the α/β water chestnut, the α/β bucket, the alpha+beta topology, rich disulfide bond is folding, serine protease suppresses domain, the actinocongestin domain, EGF spline structure territory, complement c-modular construction territory, wheat phytotoxin domain, cobra (Cobra) neurotoxin domain, greenery cobra choline esterase inhibitor domain, the Kringle domain, mucoprotein sample district, spherical district, spacer region. quaternary structure is described the comparison of the different polypeptide chains with protein structure, and each chain has unique one-level, secondary and tertiary structure element. comprise for example with-or assorted-oligomer multimerization (for example dimer forms or tripolymer forms). in a specific embodiments, molecule of the present invention is selected from IFN-a2B, IFN-a2B-Fc, IFN-b1, IFN-b1-Fc, IFN-g, IFN-g-Fc, IFNAR2, IFNAR2-Fc, IL-10, IL-10-Fc, IL-10Ra, IL-10Ra-Fc, these molecules exist with homology or heterodimer, tripolymer or oligomer form.

for the primary structure that relates to, the invention provides protein or its chimeric molecule of separation, or its fragment, comprise SEQ ID NO:27 by what be selected from sequence table, 29, 31, 33, 35, 39.41, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 13, 115, 116, 118, 119, 21, 122 24, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149, or the nucleotide sequence that has about at least 60% homogeneity with above-named arbitrary sequence, or can encode with above-mentioned arbitrary sequence or their complementary type are hybridized under low stringency condition nucleotide sequence.

The present invention provides a kind of polypeptide of separation on the other hand, its spliced nucleotide sequence SEQ ID NO:151 by their mRNA separately that process by cell, 152,153,154 codings.

the present invention also provides a kind of separation on the other hand, the nucleotide sequence molecule of coded protein or its chimeric molecule or its functional part, described nucleic acid molecule comprise and are selected from sequence table and comprise SEQ ID NO:27, 29, 31, 33, 35, 39.41, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 113, 115, 116, 118, 119, 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, the nucleotides of selecting in 149 has 60% sequence similarity at least, or after best comparison and/or can with SEQ ID No:27, 29, 31, 33, 35, 39.41, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 113, 115, 116, 118, 119, 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149 or their nucleotide sequence of one or more hybridize under low stringency condition of complementary type.

in a specific embodiments, the present invention points to a kind of nucleic acid molecule of separation, and described molecule comprises a kind of protein of coding or its chimeric molecule, or the nucleotide sequence of its fragment, and it has basically as SEQ ID NO:28, 30, 32, 34, 36, 40, 42, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 112, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, amino acid sequence or a plurality of shown in one of 15O, or best comparison is rear and SEQ ID NO:28, 3O, 32, 34, 36, 40, 42, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 112, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, one or more amino acid sequences with about at least 60% similitude of 150.

on the other hand, the invention provides a kind of nucleic acid molecule of separation, encode protein molecule, or its fragment, comprise and be selected from SEQ ID NO:29, 31, 41, 43, 45, 47, 61, 63, 65, 67, 81, 83, 101, 103, 115, 116, 118, 119, 121, 122, in nucleotide sequence, its directly or through the nucleotide sequence of one or more codings protein connexon known in the art with basic as SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17 or 19 one or more shown in the constant region (Fc) of encoding human immunoglobulin (Ig) or the nucleotide sequence of framework region connect. in specific specific embodiments, the nucleotide sequence of encoding proteins connexon comprises and is selected from IP, GSSNT, the nucleotide sequence of TRA or VDGIQWIP.

On the other hand, the invention provides a kind of protein molecule or its fragment of separation, comprise and be selected from SEQ ID NO:30,32,42,44,46,48,62,64,66,68,82,84,102,104,117,120,123 amino acid sequence, its directly or through one or more protein connexons known in the art with substantially as SEQ ID NO:2,4,6,8,10,12,14,16,18 or 20 one or more as shown in constant region (Fc) or the framework region of human immunoglobulin(HIg) be connected.

In a specific embodiment, IFN-a2B of the present invention or chimeric IFN-a2B molecule, for example IFN-a2B-Fc comprises the amino acid sequence that starts from the acid of SEQ ID n bit amino, comprising SEQ ID Nos:32 and 36, n=24 ± 5 wherein.

In a specific embodiment, IFN-b1 of the present invention or chimeric IFN-b1 molecule, for example IFN-b1-Fc comprises the amino acid sequence that starts from the acid of SEQ ID n bit amino, comprising SEQ ID Nos:46,48,54 and 56, wherein n=22 ± 5.

In a specific embodiment, IFN-g of the present invention or chimeric IFN-g molecule, for example IFN-g-Fc comprises the amino acid sequence that starts from the acid of SEQ ID n bit amino, comprising SEQ ID Nos:66,68,74 and 76, wherein n=21 ± 5.

In a specific embodiment, IFNAR2 of the present invention or chimeric IFNAR2 molecule, for example IFNAR2-Fc comprises the amino acid sequence that starts from the acid of SEQ ID n bit amino, comprising SEQ ID Nos:84,92,94 and 96, wherein n=27 ± 5.

In a specific embodiment, IL-10 of the present invention or chimeric IL-10 molecule, for example IL-10-Fc comprises the amino acid sequence that starts from the acid of SEQ ID n bit amino, comprising SEQ ID Nos:104 and 108, n=19 ± 5 wherein.

In a specific embodiment, IL-10Ra of the present invention or chimeric IL-10Ra molecule, for example IL-10Ra-Fc comprises the amino acid sequence that starts from the acid of SEQ ID n bit amino, comprising SEQ ID Nos:123,144,147 and 150, wherein n=22+5 or 32 ± 5.

the present invention provides a kind of protein or its chimeric molecule or its fragment of separation on the other hand, comprise the SEQ ID NO:28 that is selected from sequence table, 30, 32, 34, 36, 40, 42, 44, 46, 48, 50, 52, 54, 56, 6O, 62, 64, 66, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 112, 114, 117, 120, 123.126, 129, 132, 135, 138, 141, 144, 147, 150 amino acid sequence, or with one or more amino acid sequences with about at least 65% similitude of above-mentioned sequence.

in specific embodiments, protein similar percentage or nucleotides homogeneity level comprise about at least 61%, or about at least 62%, or about at least 63%, or about at least 64 %, or about at least 65%, or about at least 66%, or about at least 67%, or about at least 68%, or about at least 69%, or about at least 70%, or about at least 71%, or about at least 72%, or about at least 73%, or about at least 74%, or about at least 75%, or about at least 76%, or about at least 77%, or about at least 78%, or about at least 79%, or about at least 80%, or about at least 81%, or about at least 82%, or about at least 83%, or about at least 84%, or about at least 85%, or about at least 86%, or about at least 87%, or about at least 88%, or about at least 89%, or about at least 90%, or about at least 91%, or about at least 92%, or about at least 93%, or about at least 94%, or about at least 95%, or about at least 96%, or about at least 97%, or about at least 98%, or about at least 99% similitude or homogeneity.

" derivative " of polypeptide of the present invention also comprises section or the part of total length parent polypeptide, and its part that keeps parent's polypeptide is transcribed activity and comprised variant. " biological active fragment " like this comprises depletion mutant and little peptide, for example, have at least 10, in specific embodiments, have at least 20 and in further specific embodiments at least 30 continuous amino acid, described continuous amino acid be show active necessary. This peptide can obtain or with conventional liquid phase or solid phase synthesis technique, synthesize by the recombinate application of nucleic acid technique of standard. For example, object of reference can synthesize or the synthetic preparation of solid phase with described solution, for example, be included in by Nicholson and edit by the 9th chapter in the publication that is called " synthetic Vaccines " of Blackwell Scientific Publlcations publication by Atherton and Shephard called after " Peptide synthesis ". Optionally, peptide can pass through with for example end0Lys-C, endoArg-C, endoGlu-C and staphylococcus V8 protease digestion amino acid sequence generation of the present invention of protease. Digestion fragment can be purified, for example, and high performance liquid chromatography (HPLC) technology. Any such fragment, irrelevant with the method that produces, can be understood to include in term used herein " derivative ".

Therefore, term " variant " relates to, be shown as sequence and the identical nucleotide sequence of reference nucleotide sequence basically or hereinafter definition under stringent condition with the polynucleotides of reference sequences hybridization. Term " nucleotide sequence ", " polynucleotides " and " nucleic acid molecule " can exchange and use and comprise that having one or more nucleotides is added or lacks at this, or the polynucleotides that replace with different nucleotides. In this respect, well known in the art, some changes comprise and can suddenly change to the reference nucleotide sequence, add, and disappearance and alternative, the polynucleotides that change thus keep biological function or the activity with reference to the polypeptide of polynucleotides or coding. Term " variant " also comprises spontaneous allelic variant.

Nucleic acid molecule of the present invention can be carrier or other nucleic acid construct forms.

In a specific embodiments, carrier is DNA and comprises selected marker arbitrarily.

The example of selected marker comprises to be given the compound gene of antibiotic resistance for example, gives the gene of the ability of growing in selective matrix, and coding produces can survey for example gene of the protein of fluorescence of signal. Multiple such gene is known and is available, comprises, for example for example neomycin resistance gene (neo) and hygromycin gene (hyg) of antibiotics resistance gene. Selected marker also comprise give some gene of cultivating energy for growth in matrix for example tk gene (thymidine kinase) or hprt gene (hypoxanthine phosphoribosyltransferase) its give the ability of growing (hypoxanthine, ammonia petrin and thymidine) in the HAT culture medium; With bacterium gpt gene (black purine/xanthine phosphoribosyl transferase), it allows growth (mycophenolic acid, adenine and xanthine) in the MAX culture medium. Other plasmids that are used for selected markers of mammalian cell and carry the multiple choices mark are at sambrook equimolecular clone-laboratory manual, the cold spring port, and New York, USA, have description in 1990.

Selected marker can rely on himself promoter to express and marker gene can obtain (for example being used in the protokaryon marker gene the purpose mammalian cell) from the organism very different from the purpose organism. Yet to replace original promoter be useful with the structure of transcribing of known function in recipient cell. A large amount of transcription initiation regions is useful to such purpose, for example, and metallothionein promoter, thymidine kinase promoter, beta-actin promoter, immunoglobulin promoter, SV40 promoter and human cytomegalovirus promoter. Widely used example is the pSV2-neo plasmid, the ability (antibiotic that a kind of neomycin is relevant) that it has the bacterium neomycin phosphotransferase gene under the control of SV40 early promoter and is endowed the anti-G418 of mammalian cell. Other a large amount of mutation can be used for strengthening the expression of selected marker at zooblast, for example the interpolation of the interpolation of poly (A) sequence and synthetic translation initiation sequence. Composition type and inducible promoter can use.

Hereditary construct of the present invention can also comprise 3 ' non-translated sequence. 3 ' non-translated sequence relates to the part of gene, comprises to contain polyadenylation signal and any other and can affect the DNA fragmentation of the conditioning signal of mRNA processing or gene expression. Polyadenylation signal has to be affected the polyadenylic acid chain and adds the feature of mRNA precursor 3 ' end to. The polyadenylic acid signal is identified by the existence of the homology with 5 ' AATAAA-3 ' normal form usually, although it is much to make a variation.

Correspondingly, comprise the hereditary construct of nucleic acid molecule of the present invention, effectively with promoter, be connected, can be cloned in suitable carrier to be delivered in the cell or tissue of regulating mistake, dysfunction or disappearance, to repair and/or to provide suitable adjusting. The carrier that contains suitable hereditary construct can be delivered in the purpose eukaryotic by the known many distinct methods of the technical staff of biology field.

Term " similitude " is included in accurate homogeneity between nucleotides or amino acid levels sequence relatively as used herein. Have nonidentity at nucleotide level, " similitude " comprises the difference between sequence, and it causes amino acid whose difference, amino acid whose difference still with mutual structure, function, biochemistry and/or conformation level are relevant. Have nonidentity at amino acid levels, " similitude " comprises and mutual structure, and function, biochemistry and/or conformation level be relevant amino acid still. In specific specific embodiments, the comparison of nucleotides and sequence is carried out rather than similitude in the homogeneity level.

The term that is used for describing the sequence relation of two or more polynucleotides or polypeptide comprises " canonical sequence ", " comparison block ", " sequence similarity ", " sequence homogeneity ", " sequence similarity percentage ", " sequence homogeneity percentage ", " substantially similar " and " substantially same ". " canonical sequence " for having at least 12, but be often 15 to 18 and often be at least 25 or above, for example 30 monomeric units, comprise nucleotides and amino acid residue, on length. Because two polynucleotides can all comprise (1) similar sequence (part of for example only having complete polynucleotide sequence) between two polynucleotides, (2) different sequence between two polynucleotides, article two, relatively generally by the sequence of two polynucleotides, relatively the carrying out of sequence between (or many) polynucleotide, go to identify and the similitude of comparative sequences regional area by " comparison block ". " comparison block " relates to notional fragment of general 12 continuous residues, and itself and canonical sequence contrast. For the comparison of the bests of two sequences, comparison block can comprise compares about 20% or still less interpolation or disappearance (for example gaps) with canonical sequence (wherein contain and add or disappearance). In order to compare comparison block, the best of sequence comparison can be by the computerization of algorithm or by checking and comparing (for example finally obtaining the highest percentage homology between whole comparison block) by the best of multiple selected any generation of method and realize. Contrast can also be obtained by the BLAST family of program, such as by discussing in detail of disclosed (Nucl Acids Rea 25:389, the 1997) sequence analyses such as Alschul, finding (In:Current Protocols in Molecular Biology, John Wiley Sons Inc. 1994-1998) in the Unit 19.3 of Ausubel etc.

term " sequence similarity " and " sequence homogeneity " relate to sequence on comparison block as used herein, nucleotides than nucleotides basis on or amino acid than amino acid basis on same or scope function or structural similarity. therefore, for example, the calculating of " percentage of sequence homogeneity " is to compare by two best sequences of comparing in comparison block, mensuration is present in two sequences and has identical nucleotide base (A for example, T, c, G, I) or identical amino acid residue (Ala for example, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) the numerical value in site, to produce the numerical value in pairing site, with the numerical value in pairing site divided by the sum in site in comparison block (for example, the size of frame), and result be multiply by 100 to produce sequence homogeneity percentage. for the purposes of the present invention, " sequence homogeneity " will be understood to that expression is by DNASIS computer program (Version 2.5 for windows, available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA) " the pairing percentage " that calculates with the standard error used in the appended comparison handbook of software. similar explanation application and sequence similarity.

comprise and comprise from least 0 at least about 15%v/v formamide with from 1M at least at least about 2M salt in this low stringency that relates to being used for hybridization, and at least approximately 1M is used for the washing condition at least about 2M salt. general, low stringency is from about 25-30 ℃ to about 42 ℃, for example 25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41 and 42 ℃. temperature can change and higher temperature is used for replacing formamide and/or optional stringency condition being provided. optional stringency condition can be used in the place of needs, moderate stringency for example, it comprises and comprises from 16%v/v at least to about at least 30 %v/v formamides, for example 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, for example 0.5, 0.6, O.7, 0.8 or O.9 M is used for the washing condition, or high stringency, it comprises and comprises from about at least 31%v/v to about at least 50%v/v formamide, for example 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50% and from least approximately O.01M to salt at least approximately O.15M, for example 0.01, 0.02, 0.0 3, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, O.14 be used for the washing condition with 0.15M. general, washing is at T_m=69.3+0.41 (G+C) % carries out (Marmur and Doty, J Mol Biol 5:109,1962). Yet, the T of double-stranded DNA_mThe 1 ℃ of right quantity of base mismatch of often successively decreasing increases by 1% (Bonner and Laskey, Eur J Biochem 46:83,1974. Formamide is optional under these hybridization conditions. Accordingly, strict level is determined as follows in specific embodiment: low stringency is 6 * SSC buffer solution, and O.1%w/v SDS is under 25-42 ℃; Middle stringency is 2 * SSC buffer solution, and 0.1%w/V SDS is under 20 ℃ to 65 ℃ of temperature ranges; High stringency is 0.1 * SSC buffer solution, and O.1%w/v SDS is at least 65 ℃ of temperature.

as used herein, term " is translated and is modified or posttranslational modification altogether " to relate to when peptide chain is translated or covalent bond occurs after translation and modifies. translation modification or posttranslational modification include but not limited to acidylate (comprising acetylation) altogether, amidatioon or deacylated tRNA amine, biotinylation, carbamylation (or carbamylation), carboxylation or decarboxylation, two sulphur alkali form, the aliphatic acid acidylate (comprises myristoylation, palmitoylation and octadecane acidylate), formylated, saccharification, glycosylation, hydroxylation, the selenocysteine combination, lipid, the addition of class resin acid, methylate, N-or C-endcapped, N-or C-end remove, nitrated, the methionine oxidation, phosphorylation, proteolytic cleavage, prenylation (comprises farnesylation, Mang ox baseization), the phosphopyridoxal pyridoxal phosphate addition, saliva acidifying or asialoglycoprotein, sulfation, ubiquitin (or ubiquitin) or Ubiquitin Like Proteins addition.

Acylation comprises that the hydrolysis of the initial methionine of N-end and acetyl group are attached to new N end amino acid. Acetyl group Co-A is the acetyl group donor of acylation.

Amidatioon is the covalent bond of the c-terminus of peptide and amide groups and normally essential for the stability of biologically active and albumen. Going amidatioon is that the hydrolysis of amide groups removes. The amidatioon of going that comprises the acid amides of amino acid residue is rare distortion, and it is by organism complete to recombinate 3D structure and change electric charge ratio/pI.

The biotinylation effect be a technology thus biotinyl be attached on molecule; carry out by holocarboxylase synthetase catalysis or external in the biosynthetic process of enzyme; trend towards by the visible special substrate with the hatching of biotin labeled probe and avidin, perhaps trend towards being connected to any antibiosis protein chain mycin of the many kinds of substance that is subjected to the biochemical analysis check.

It is for example amino that carbamylation (or carbamylation) is transferred to the acceptor part with carbamyl from the molecule (for example carbamyl phosphate) that contains carbamyl.

The carboxylation of glutaminic acid residue be the vitamin K dependent response its cause the formation (Gla residue) of Y carboxyglutamic acid. The Gla residue is present in the some protein of blood coagulation cascade, and its biological function for protein is essential. Carboxylation also can betide the asparatate residue.

Disulfide bond is the covalent bond of the disulphide of formation when the sulfydryl of two cysteines is oxidized. Many mammalian proteins matter comprise disulfide bond, and its generation for tertiary protein structure and keeping, and such BA is conclusive.

Protein in bacterium is synthetic to be comprised the formylated of N-end methionine and goes formylated. This formylated/go is formylated to be circulated in and does not occur in thin 2 born of the same parents' of eucaryon cytoplasm and be the exclusive feature of bacterial cell. Except the part of hydroxylating as the amidatioon process that occurs in glycine residue, hydroxylating can also occur in (Kivirikko et al.FASEB Journal 3:1609-1617,1989) on proline and lysine under proline and lysyl hydroxylase catalysis.

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

Glycosylation is that sugar unit is attached to the polypeptide main chain and will further describes hereinafter.

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

Selenoprotein is the protein that contains the selenium of rare element, by add unique amino acid, selenocysteine in translation process. The tRNA that is used for selenocysteine substitute serine and then the enzyme seleno to produce selenocysteine-tRNA. Terminator codon in the antisense codon of selenocystine-tRNA and mRNA (UGA) influences each other and substitutes the serine codon. An element in the 3 ' non-translational region (UTR) of selenoprotein mRNAs has determined that UGA pronounces terminator codon or selenocysteine codon.

Lipid is a covalently bound general name that comprises lipid on protein, and it comprises aliphatic acid acylation and prenylation.

The covalently bound thing that the aliphatic acid acylation comprises aliphatic acid is 14 myristic acids (myristoylation), 16 carbon palmitic acids (palmitoylation) and 18 carbon stearic acid (octadecane acidylate) for example. Aliphatic acid front-golgi's field every in be connected to protein and can regulate targeting (the Blenis and Resh Curr Opin Cell Bio15 (6): 984-9.1993) of protein to film. Therefore the aliphatic acid acylation is important (Bernstein Methods Mol Biol 237:195-204,2O04) in the functional activation of protein.

Prenylation comprises prenyl, i.e. the combination of 15 carbon farnesyls or 20 carbon Mang oxen-Mang ox base and receptor protein. Isoprenoid compounds, comprise farnesyl chloroquine or Mang ox benzylacetone chloroquine, obtains in the Biosynthesis of cholesterol approach. The isoprenoid base is attached to (wherein A is any aliphatic amino acid except alanine) on the cysteine residues that agrees with in sequence CAAX by thioether bond, be positioned the c-terminus of protein. Prenylation changes protein and is combined aminosal (G albumen) with the method modification with the ability of lipid membrane associating and the GTP-that all are known, and conduction is conclusive to signal to make prenylation. (Rando Biochim Biophys Acta 1300 (1): 5-l6,1996; Gelb et al.Curropin Chem Biol 2 (1) j:40-8,1998).

The class resin acid is vitamin sample antioxidant, as the free radical of scavenger. It is by the lipoic acid protein ligase, the class resin acid to be attached on lysine in conjunction with acid amides that lipoic acid lysine forms.

It is that a kind of common modification can be regulated the active of protein or produce new amino acid kind that albumen methylates. Protein methyltransferase is transferred to oxygen, nitrogen or the sulphur atom of the nucleophilic albumen with methyl from SAMe. The effect that methylates is divided into two kinds of general classification. The first, the relative level of transmethylase and methyl esterase can be controlled methylation on special carboxyl, and it regulates the activity of protein in turn. This methylating is reversible. Second group of protein methylation reaction comprises the irreversible modification of sulphur in protein or nitrogen-atoms. This reaction produces the new amino acid of the biochemical character with change, and it changes the activity (Clarke Curr Opin Cell Biol 5:977 983,1993) of protein.

Albumen is nitrated is important posttranslational modification, and it carries out in the conduction of nitrous oxide signal. The nitrated adjusting catalytic activity of protein, cell signal and cytoskeleton group structure.

Phosphorylation relates to the phosphate addition to protein kinase. Serine, threonine and tyrosine residue are the amino acid that is phosphorylated. Phosphorylation is a kind of important mechanism, and it regulates the biologically active of protein.

Most of protein is also modified by proteolytic cleavage. It can only comprise removing of initial methionine. Other protein are synthetic with the inactive precursor form, by restricted or specific proteolysis, activate. For secrete or with the burst of the synthetic 12-36 of having of membrane-bound albumen (front albumen) main hydrophobic amino acid, its after cut while passing through the ER film.

Phosphopyridoxal pyridoxal phosphate is the coenzyme derivative of vitamin B6 and the transamination of participating in amino acid side chain, decarboxylation, racemization, and a lot of the modification. All phosphopyridoxal pyridoxal phosphates-desirability enzyme works by the formation of Schiff alkali between amino acid and coenzyme. The enzyme that great majority dependence phosphopyridoxal pyridoxal phosphate base is combined with lysine residue is that the oneself activates.

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

Sulfation is in the tyrosine residue generation and by being present in the enzyme tyrosine protein sulfurtransferase catalysis of Gorky outside wire side. Determined by HepG2 emiocytosis 1 to 20 with interior albumen and by fibroblasts to secrete 1 to 3 with at least one tyrosine sulfate residue of interior albumen. It is influential to the biologically active of albumen that sulfation is found. Interested especially is CCR5, main HIV co-receptor, discovery is adhered to for the best of MIP-1 alpha/CCL3, MIP-1 beta/CCL4 and RANTES/CCL5 by the sulfation of one or more tyrosine residues in the N of tyrosine sulfation and CCR5-end extracellular domain and best HIV is total to-and the acceptor function is necessary (Moore J Biol Chem 278 (27): 24243-24246,20O3). Sulfation can also occur on carbohydrate. In addition, the sulfation of the carbohydrate of glycoprotein part can be by for example activity generation of GalNAc (β 1-4) GlcNAc (β 1-2) Man α 4 sulfurtransferases of sugared sulfurtransferase.

Posttranslational modification can comprise the protein-protein bonding. Ubiquitin is a kind of 76 aminoacid proteins, in mammalian cell its both can self in conjunction with also can being covalently attached to other protein. Peptide bond between the C end by ubiquitin and the amino of the lysine residue in other protein adheres to. The target that adheres to of the chain of ubiquitin molecule and target protein is tending towards surely by the proteasome proteolysis and for the stable state level of regulating protein, the protein relevant with the cell cycle for example, a kind of important mechanism (WWilkinson Annu Rev Nutr 15:161-89,1995). On the contrary, single ubiquitination can play important effect in the direct adjusting of protein function. Ubiquitin Like Proteins can also be covalently attached on protein to affect their functional metabolism, comprises NEDD-8, SUMO-1 and Apg12.

Glycosylation is sugared residue adhering on the polypeptide main chain. The sugar residue, monose for example, disaccharides and oligosaccharides include but 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 the polypeptide main chain at least seven kinds of modes, that is,

(1) be attached to common sequence Asn-X-Ser by the N-glycosidic bond; Asn-X-Thr; Or the R-of the asparagicacid residue in Asn-X-Cys base (N-glycosylation).

(2) be attached to serine by the O glycosidic bond, threonine, hydroxy-proline, the R-base (O-glycosylation) of tyrosine or oxylysine.

(3) the R-base C-by tyrosine connects mannose;

(4) the glycophosphatidyl inositol grappling is used for some protein fixing to cell membrane;

(5) be connected to the GlcNAc of R-base of serine or threonine as signal monose. This connection is generally reversible and adheres to (Yin-o-Yang) with inorganic phosphate;

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

(7) be connected to the R-base of cysteine by the S-glycosides key.

The glycosylation structure can comprise one or more CA determinants following in table 7.

Table 7: CA determinant list

The antigen title	The o antigen polysaccharide o structure
		Blood group H (O), 1 type	Fuc(cα1-2)Gal(β1-3)G1cNAc-R
Blood group H (O), 2 types	Fuc(α1-2)Gal(β1-4)G1cNAc-R
		Blood group A, 1 type	GalNAc(α1-3)[Fuc(cα1-2)]Gal(β1-3)G1cNAc-R
Blood group A, 2 types	GalNAc(α1-3)[Fuc(α(1-2)]Gal(β1-4)G1cNAc-R
		Blood group B, 1 type	Gal(α1-3)[Fuc(α1-2)]Gal(β1-3)G1cNAc-R
Blood group B, 2 types	Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)G1cNAc-R
		Blood group i	[Gal(β1-4)GlcNAc(β1-3)]Gal(β1-R
Blood group I	Gal(β1-4)G1cNAc(β1-3)[Gal(β1-4)GlcNAc(β 1-6)]Gal(β1-4)GlcNAc(β1-3)Gal(β1-R
		Lewisa(Le a)	Gal(β1-3)[Fuc(α1-4)]G1cNAc-R
Saliva acidic group Lewis a (sLea)	NeuAc(α2-3)Gal(β1-3)[Fuc(α1-4)]GlcNAc-R
		Lewis b (Le b)	Fuc(α1-2)Gal(β1-3)[Fuc(α1-4)]GlcNAc-R
Lewis x (Le x)	Gal(β1-4)[Fuc(α1-3)]GlcNAc-R
		Saliva acidic group Lewi s x (sLex)	NeuAc(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc-R
Lewi s y  (Le y)	Fuc(α1-2)Gal(β1-4)[Fuc(α1-3)]GlcNAc-R
		Forssman	GalNAc(α1-3)GalNAc(D1-3)Gal-R
Thomsen-Friedenreich (TF or T)	Gal(β1-3)GalNAc(α1-0)-Ser/Thr
		Saliva acidic group-TF (sTF) or sialic acid-T (sT)	Gal(β1-3)[NeuAc(α2-6)]GalNAc(α1-0)-Ser/Thr
Tn	GalNAc(α1-0-Ser/Thr
		Saliva acidic group Tn (sTn)	NeuAc(α2-6)GalNAc(α1-0)-Ser/Thr

Carbohydrate can also comprise some feeler structures, comprises list, and is two, and three and side structure all round. Glycosylation can be passed through the N linked glycosylation, the O linked glycosylation, and C joins mannose structures, and the existence of glycophosphatidyl inositol grappling, disappearance or pattern; Carbohydrate mass percent: Ser/Thr-GalNAc ratio; Single, two, three and the ratio of tetrose structure or by agglutinin or antibody in conjunction with mensuration.

The sialylation of protein can be measured by protein and the immunoreactivity of the antibody of anti-a kind of specific sialic acid structure. For example, the special antibody of Lewis x reacts with the CEACAMl that is expressed by granulocyte but with the restructuring people CEACAMl of 293 cellular expressions, does not react (Lucka et al Glycobiology 15 (1): 87-100,2005). Optionally, the mixture that the existence of sialic acid structure in protein can be processed by glycosidase is through suitable measuring process mass spectrum (MS) for example, and high performance liquid chromatography (HPLC) or saccharic amount finger-print (GMF) detect.

The apparent molecular weight of proteins, including protein complexes of all components (cofactors And non-covalent domain), and all of translational modification or post-translational modification (group covalently attached to the peptide Removal of the peptide or a monovalent group). Apparent molecular weight of usually translated by post-translational modification or repair Decorative effect. The apparent molecular weight of proteins by SDS-PAGE (sodium dodecyl sulfate poly Acrylamide gel electrophoresis) to determine, in the way of its analogs, 2D-PAGE (D PP Polyacrylamide gel electrophoresis) is also a two-dimensional. However, the apparent molecular weight of proteins by mass Spectrum (MS) - can be changed by generating an auxiliary matrix of electron-ion laser desorption ionization - Time of flight (MALDI-TOF) MS or generating a plurality of peaks with a charge EFI more sensitive Fog ionization (ESI) MS determined more precise. Protein or chimeric molecule of apparent molecular weight Can range from 1 to 1000kDa. Accordingly, the present invention is an isolated protein or chimeric Molecules 1,2,3,4,5,6,7,8,9,10,10,12,13,14,15, 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, 91,92,93,94,95,96,97,98,99,1 O0, 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, 141,142,143,144,145,145, 147,148,149,150,151,152, 153,154,155,156,157,158,159,16 O, 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, 201,202,203,204,205,206,2 O7, 208,209,210,210,212, 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, 261,262,263,264,265,266,267,268,269,27 O, 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, 297,298,299,300,3 O1, 302,303,304,305,306,307,308, 309,310,310,312,313,314,315,316,317,318,319,320, 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, 405,4 O6, 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, 585,586,587,588,589,590,591,590, 593,594,595,596, 597,598,599,600,601,602,603,604,6 O5, 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, 693,694,695,696,697,698,699,700,701,702,703,7 O4, 705,706,707,708,709,710,710,712,713,714,715,716, 717,7 l8, 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, 777,778,779,78 O, 781,782,783,784,785,786,787,788, 789,79 O, 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, 921,922,923,924,925,926,927,928,929,930,93 l, 932, 933,934,935,936,937,938,939,940,941,942,943,944, 945,946,947,948,949,95 O, 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, 981,982,983,984,985,986,987,988,989,990,991,992 993,994,995,996,997,998,999,1000 kDa apparent molecular weight. Egg White matter of the molecular weight or molecular weight may be determined by any convenient method, such as electrophoresis, MS, gradient centrifugation. ...

the isoelectric point of protein (or pI) is the pH of albumen while not carrying net charge. this attribute can be measured by isoelectric focusing (IEF), and it is also the one dimension of 2D-PAGE. the pI that experimental definite pI value is subjected to the impact of scope of common translation modification or posttranslational modification and therefore experiment can be up to 5 units with the difference between the pI of theory. accordingly, the protein of separation of the present invention or chimeric molecule can have 0, 1.0, 1.1, 1.2, 1.3, 1., 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.

The different kinds of molecules form of a kind of given albumen of term " isoform " expression as used herein, and be included in protein different on following level (1) primary structure (for example due to displacement RNA montage, or polymorphism); (2) secondary structure (for example due to different common translation modification or posttranslational modification); And/or (3) three grades or quaternary structure (for example because different subunits interacts, with-or different-oligomer multimerization). Special, term " isoform " comprises sugared type, and it comprises and has continuous primary structure but in secondary or tertiary structure, or translation is modified or posttranslational modification altogether, different glycosylation form for example, different protein or its chimeric molecule on level.

the chemical stability of protein can be measured with " half-life " form of protein in special solvent or environment. representational, have the half-life that the protein that is less than the 50kDa molecular weight had about 5 to 20 minutes. protein of the present invention or chimeric molecule focus on to have 1, 2, 3, 4, 5, 6, 7, 8, 9, 1O, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 3O, 31, 32, 33, 34, 35, 36, 37, 38, 39, 4O, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 6O, 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, 9O, 91, 92, 93, 94, 95, 96, 97, 98, the half-life of 99 or 100 hours. another kind of chemical stability be measured as easily especially protein or its resistance molecule resistance to the protease digestion effect, for example trypsase or pancreas milk reducing protease digesting effect.

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

The dissolubility of protein can be by Tot Prot and/or the wherein measurement of the ratio of protein dissolving that is dissolved in given solvent. Further, protein or its chimeric molecule be in heterogeneity polarity for example, pH, the ratio of dissolving in the solvent of temperature etc. and or level measurable biochemical characteristic of protein or its chimeric molecule can also be provided.

Any " measurable physical and chemical parameter " can be determined with known for a person skilled in the art any method, measure, and quantizes or limits. What the following describes is be used for to determine, measures, and quantizes or limit the methodological scope of measurable physical and chemical parameter of the protein of one or more separation or its chimeric molecule. Yet it should be understood to the present invention and never only is defined as described special method, or is defined as that to use these methods are measurable physical and chemical parameters.

Glycoprotein can be described to have two interactions to produce molecule element monoamino-acid sequence and carbohydrate or a sugared side chain as a whole. The carbohydrate part of molecule exists with the monose on the hydroxyl side chain that is attached to Asn amino side chain or Ser/Thr residue by N-or O-key or oligosaccharides side chain form respectively. Monose is that it is considered to a sugar, has (CH about the term of the minimum unit of carbohydrate₂O) _nBasic chemical formula and great majority usually form 5 or six-membered cyclic structure (being respectively pentose and hexose). Oligosaccharides is the compound with monose molding structure of Various Complex, and it can be linear or branch, but does not usually have the long-chain (it is a kind of form of polysaccharide) of series connection repetitive. Branch's level that oligosaccharides contains and the branch of end replace impact significantly and make the feature of as a whole glycoprotein, and play important effect on the biological function of molecule. Oligosaccharides is prepare and be attached on amino acid backbone in endoplasmic reticulum (ER) at cell and golgiosome. Different organisms and the kind of cell have the ratio of different glycosyl transferases and endoglycosidase and exoglycosidase and therefore produce different oligosaccharide structures. One of defense mechanism that health is main is to find and destroy abnormal isoform, and same to have correct glycosylated biological therapy agent be not only important for the discovery that is neutralized antibody for minimizing that also heightens the effect of a treatment.

Polysaccharide chains is usually with branch's formal representation, even and when it was linearity, such chain was subject to multiple modification usually. Therefore, the complete sequence of oligosaccharides be difficult to by single method complete and therefore need physics and chemical method repeatedly combination and the final details that obtains the structure of studying.

The mensuration of the glycosylation pattern of protein can be undertaken by using a lot of diverse ways, for example uses SDS-PAGE. This technology depends on glycosylated protein matter is divided a word with a hyphen at the end of a line with different diffusion zones usually in SDS-PAGE the fact. Differentiation between different isoforms be by with a series of reagent, processing protein. For example, consider with the change of the position of being connected glycosylated the distinguishing that N connects with the significant minimizing of bandwidth after the digestion of peptide-N4-(N-acetyl-β-GLUCOSAMINE base) asparagine acid amides enzyme (PNGase).

The glycosylated component that connects in order to measure N, N-sugar chain use from flavobacterium meningosepticum the clone's and at the PNGase of expression in escherichia coli, excise from protein. The N-sugar chain of excision can be from as Packer et al Glycoconj J 5 (8): 737-47,1998 described Alltech Carbograph SPE carbon posts (Deerfield, Illinois, USA) recovery. Then, sample can carry out the monose analysis with the Dionex system with GP50 pump ED50 pulsed amperometric meter or electric conductivity detector and multiple pH anion-exchange column, sialic acid analysis or sulfate analysis.

At first O-connects glycosylated degree can be by measuring from purpose protein excision O-sugar chain through β-elimination. The O-sugar chain of excision can as describedly in Packer et al. (1998, as above) reclaim from Alltech Carbograph SPE carbon post (Deerfield, Illinois, USA). Then, sample can carry out the monose analysis with the Dionex system with GP50 pump ED50 pulsed amperometric meter or electric conductivity detector and multiple pH anion-exchange column, sialic acid analysis or sulfate analysis.

The monose subunit of oligosaccharides has variable sensitivity to acid also therefore can be in slight trifluoroacetic acid (TFA) condition, moderate TFA condition, and discharge from purpose albumen under strength hydrochloric acid (HCl) condition. Then, mixture of monosaccharides is separated by the high pH anion-exchange chromatography (HPAEC) that uses multiple column filling medium, and with pulsed amperometric electrochemical assay (PAD), detects.

High pH anion-exchange chromatography and pulsed amperometric detection method (HPAEC-PAD) have been widely used for measuring monosaccharide component. Fluorescence one labeling method has been introduced into and much with the kit form, has used. The obvious advantage of fluorescence method is that sensitiveness strengthens (about 50 times). When a potential deficiency was coupling reaction, in hydrolysate and in the external standard mixture, different monose can show different fluorophors different selective. Yet, the enhancing of sensitiveness and differentiate the ability which monose exists from the sub-fraction of the total amount of the glycoprotein of reality, and, with the potentiality of the stronger sensitiveness of laser induced fluorescence, make the method very attractive. Electric conductivity detector can be used for measuring sulfate and phosphate component in addition. According to using rules, peak area can calculate the total amount of every kind of monose of existence. These data can represent that N-is connected glycosylated level with O-, the degree of saliva acidifying, and amino acid composition in compound, glycosylation percentage by weight, acidoglycoprotein percentage by weight.

A small amount of monose composition analysis of protein carries out with PVDF (PSQ) film after being preferably in the electric marking, or less amount is analyzed with dot blot. PVDF is that carbohydrate is analyzed desirable matrix, because in case through peracid or enzyme hydrolysis, monose and oligosaccharides all are not joined on film.

The mensuration of the oligosaccharide content of purpose molecule is undertaken by many technology. At first sugar excised by (for example with hydroxide β, eliminating) method of (for example with PNGase, the digesting) of enzyme or chemistry on amino acid backbone. Sugar can be by reducing stable or with fluorescence labeling, making and be easy to detect. Then, the free oligosaccharides that generates is separated, can pass through high pH anion-exchange chromatography and pulsed amperometric electrochemical assay (HPAEC-PAD), it can be used in known standard with the ratio of mensuration various structures and the level of saliva acidifying, or by fluorescence assisted carbohydrate electrophoresis (FACE), a kind of method that is similar to protein s DS-PAGE separation. In this process, oligosaccharides fluorophor mark, it has affected electrophoretic mobility. Their separated and banding patterns that obtain in the polyacrylamide gel of high percentage provide the feature of the oligosaccharide content of purpose molecule. By using standard sample, obtaining some information of the actual structure that exists or band can be cut and with mass spectrum, analyze, the structure of each of can measuring them.

Fluorescence assisted carbohydrate electrophoresis (FACE) is a kind of polyacrylamide gel electrophoresis system, is designed for and separates the single oligosaccharides that discharges from glycoconjugate. Oligosaccharides by the chemistry or enzyme method cut from sample protein matter, kept by this way reducing end. Then, oligosaccharides is digested is monose or keep complete, and by fluorescence labeling (charged or uncharged). High percentage polyacrylamide gel and multiple buffer system are used for migration oligosaccharides/monose, and its size/component with respect to them is to move with the almost identical mode of protein. Carbohydrate can be determined by fluoroscopic examination by the visual and sugared relative amount of optical densitometric method. This process is consistent with MALDI-TOF MS, so the method can be used for illustrating actual structure.

Quartz crystal microbalance and surface plasma resonance (being respectively QCM and SPR) are the two kinds of methods of biological information that obtain by the biochemical characteristic of molecule. The protein-protein Indirect Interaction is measured in the change of the physics feature of the fabricated chip that both cause by interaction. Single quartz crystal slice is used and the processing such as the interactional acceptor/antibody of target ligand in QCM. This chip vibrates by microbalance and the frequency of chip is recorded. Target protein is allowed through chip and with the interaction of molecules of combination, causes the frequency shift of thin slice. The change of the interactional condition by part and chip, can measure the binding characteristic of purpose molecule.

Apparent molecular weight is also a kind of biochemical character, and it can be used for measure protein that the present invention and those produce with mode selectively or the similitude between chimeric molecule.

As used herein, term " molecular weight " is defined as the summation of the atomic weight of composed atom in molecule, sometimes also relates to " 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 by one or more analytical technologies molecular weight of measuring of SDS page or ultracentrifugation for example, and depends on the relation between molecule and detection system. The apparent molecular weight of protein or its chimeric molecule can be measured with any of series of experiments method. Analytical method that be used for to measure the molecular weight of protein comprises, be not limited to, exclusion chromatography (SEC), gel electrophoresis, Reyleith scanttering light scattering, analytical ultracentrifugation and, in a way, time-of-flight mass spectrometry (TOFMS).

Gel electrophoresis be protein some biochemical characters (particularly apparent molecular weight and DI) assay method and about with molecular separation, being the dielectrophoresis of isoform, thereby the information of protein product posttranslational modification is provided. Particularly, electrophoresis is to force charged molecule (for example protein or DNA) to be divided a word with a hyphen at the end of a line by the method for gel-type vehicle (most of common polyacrylamide or agarose), the use of the current potential by passing colloid. The most general electrophoresis form that is used for protein is isoelectric focusing, non-sex change, and sds polyacrylamide gel electrophoresis. Protein is placed in the polyacrylamide gel with the pH gradient of passing therebetween in isoelectric focusing. The albumen position in gel of dividing a word with a hyphen at the end of a line, having at this place's albumen is zero net charge, thereby provides the isoelectric point of albumen.

Saccharic amount fingerprint (GMF) is a kind of method, and through the method, the oligosaccharides feature of one of protein or its isoform is identified by electrophoresis and special mass-spectrometric technique subsequently. The 1D SDS-PAGE that measures or the 2D gel electrophoresis that is used for special isoform characteristic are purified sample protein matter by being used for total protein. Protein band/spot excises and decolours to remove pollutant from gel. Carbohydrate discharges by chemistry or enzyme process and uses nanoflow LC system desalination/separation and subsequently the oligosaccharides that is present in sample identified. LC stream can directly be expelled to electronic spraying mass spectrograph (be used for quality measurement and discriminating amount subsequently be present in sample), and it provides feature or the fingerprint of each isoform, can be in conjunction with for example Dionex analysis of quantitative technique, to measure total component of the molecule of being tested.

Primary structure can be by measuring the biochemical character assessment of protein of the present invention or chimeric molecule.

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

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

In order to measure the sequence of amino acid backbone, the terminal chemistry order-checking of N-, the order-checking of polyphone mass spectrum or both combinations can be used.The terminal chemistry order-checking of N-utilizes Edman chemistry (Edman P. " Sequence determination " Mol Biol Biochem Biophys 8:211-55,1970), wherein put down in writing peptide bond between protein N-end amino acid and 2 s' the amino acid than peptide bond every other in the sequence a little less than.By using the moderate acidic conditions,-terminal amino acid is cut, derive to having fluorophor (FTIC) and be determined at retention time in the reversed-phase HPLC post, and with the standard model comparison to determine being which kind of amino acid.This method can be measured the primary structure of molecule reality but not be quantitative.Choose wantonly, nanoflow liquid chromatography polyphone mass spectrum can be used (Lc-MS/MS).In this method, use special endo-protease with the molecular weight of proteolysis as peptide and mensuration peptide.Then with energetic encounter gas for example nitrogen or argon fracture peptide bond and measure the quality of the peptide that obtains.The change of the quality by calculating peptide can be measured the sequence (every seed amino acid has unique quality) of each peptide.By using different proteolytic enzyme, peptide can overlap with the order of measuring them and measure proteinic complete sequence thus then.

Clearly, the combination of enzymic digestion, chemically derived, liquid chromatography (LC)/MS and polyphone MS provides a kind of very effective instrument, is used for the AA sequential analysis.For example, the detailed structure of recombinant soluble CD4 acceptor is characterized by the combination of method, determine to surpass the primary structure of this 36AA glycoprotein of 95% and had been found that N-and all characteristics of C-end, the attachment position of saccharan, the correct configuration of saccharan structure and disulfide bridge bond (carr et al.J BiolChem 264 (35): 21286-21295,1989).

Mass spectrum (MS) is a kind of method of measuring molecular mass by the deduction of the behavior of molecule in the charged environment under the vacuum.MS is very useful in stability study and quality control.This method at first requires to carry out (trypsinase with proteolytic ferment, V8 proteolytic enzyme, Quimotrase, subtilisin and shuttle mattress proteolytic enzyme) 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, by the separating digesting sample of reverse-phase chromatography (RPC).In conjunction with LC-MS, peptide mapping can be used to survey genetic stability with tryptic digestion, and the homology of product batch, and fermentation are purified, proteinic stability in formulation preparation and the storage process.

Before the mass analysis, some modes are used for HPLC is connected to mass spectrograph: 1) direct liquid infusion; 2) supercutical fluid; 3) conveyer belt system; 4) thermal spraying.The HPLC/MS that is used for Caprioli ' s operation connects the sample probe joint that use fused quartz capillary column is transported to the post elutriant mass spectrometric chamber.When sample solution appears at the kapillary joint, bombard the probe joint continuously with the Xe atom of high energy, cause the sample solution sputter.Quality is by instrumental analysis (Caprioli et al.Biochem Biophys Res Commun146:291-299,1987) then.

MS/MS is connected with LC/MS and has expanded the MS potential and use.MS/MS allows part, deacylated tRNA amine site and the isomerized direct discriminating (Carr et alAnal Chem 63:2802-2824,1991) of the above peptide complete sequence of 25AAs.RPC or capillary electrophoresis (CE) combine with MS and can carry out proteinic high sensitive analysis (Figeys and Aebersold, Electrophoresis 19:885-892,1998; Nguyen et al.J ChromatogrA 705:21-45,1995).LC/MS allows the LC method of isolated peptides before entering MS, for example the constant current FAB that is connected with micropore HPLC (Caprioli et al.1987, as above)." connection " of back allows the order-checking from the one peptide of complex mixture: selected peptide by the first time Ms rupture, then through the ionic cloud of collision cell: CID (collision induced dissociation).Collision produces segmental characteristic set, can infer sequence thus, and not need to know other information, for example the cDNA sequence.In independent MS experiment, the not classification mixture of peptide (for example, from enzymic digestion) inject and the quality of leading ion with estimate by the cDNA sequence those compare.The sequence of recombination human interleukin-2 is verified (Fukuhara et al.J Biol Chem260:10487-10494,1985) by fast atom bombardment(FAB) (FAB)-MS analysis and the proteolysis of CNBr.

Electro-spray ionization Ms (ESI-MS) uses proteolytic aerosol to insert in the pin under high-voltage, produces a series of electric charge peaks with same molecular of multiple electric charge.Because each produces the estimation that produces molecular weight from the peak of different charge species, these estimations can be in conjunction with the total accuracy with the estimation of increase molecular weight.Matrix assisted laser desorption ionization MS (MALDI-MS) uses the high density chromophoric group.The energy evaporation section matrix that the higher-strength laser pulse can absorb and absorb by matrix and almost completely bring protein example into gas phase.Analyze the ionic MS flight time that obtains then.The ionization of moderate can strengthen the ability that this method provides quaternary structure information.MALDI-MS can be 15 with interior operation like a cork.It does not need the fragmentation molecule and when SDS-PAGE gel densitometric scan, the result explains easily, is used for more than the mass range 100kDa.

Can determine aminoacid sequence by the DNA that measures coded protein or its chimeric molecule.But, actual once in a while protein sequence may be different.Usually, dna sequencing reaction and the PCR reacting phase that duplicates (DNA sex change, duplicate) DAN are seemingly.By the dna clone technology, can clone this gene and definite kernel acid sequence.

Can use one or more the following systems analysis protein or the aminoacid sequence of chimeric molecule.

Description to the complete sequence of protein or its chimeric molecule requires to describe this product usually.Amino acid sequencing comprises: carry out tryptic digestion on gel, with RPC-HPLC the peptide that digests is carried out fraction, by the peptide peak that MALDI-TOF MS screening has the most symmetrical absorption feature, use edman degradation first peptide (N-end).The primary sequence data that Ai Deman chemically derives are to determine proteinic classical way on molecular level.MALDI-TOF MS can be used for the N-terminal sequence analysis.But, all enzymic digestion that is used for HPLC and peptide sequencings are all recommended to differentiate to reduce consuming time and to reduce cost through MS or MS/MS protein earlier.Behind the isolating protein, separating the aminoacid sequence that can obtain inside from sDS-PAGE with HPLC through the original position tryptic digestion or in the digestion of the Lysyl endopeptidase on the matrix.

Recommendation moves the order-checking of the inside of standard peptide and analyzed sample together and makes instrument maintains in peak performance.Surpass 80% the eukaryotic protein sealing amino-terminal end that has been in the news, hinder the direct amino acid order-checking.When running into the eukaryotic protein of sealing, the existence of internal standard can guarantee that instrumentation is normal.

Can adopt edman degradation method to the directly order-checking of N end by chemical process, the described method N-terminal amino acid of deriving discharges amino acid, exposes next amino acid whose N-terminal.The Ai Deman order-checking comprises: 1). by micropore HPLC, carry out the N-terminal sequential analysis repeatedly by the Ai Deman chemical cycle, each Ai Deman chemical cycle can be identified an amino acid.2). in the gel or the conjugated protein digestion of PVDF back is carried out inner sequential analysis of protein with the polypeptide that the HPLC separating digesting generates by the edman degradation chemical method.

Use micropore HPLC and kapillary HPLC and use the RPC-HPLC method to analyze and the purified peptide mixture.Use sample and PVDF sample in the different pillar purifying gels.After separating, the HPLC segmentation can adopt the auxiliary mastrix-assisted laser desorption ionization time of flight mass spectrum of matrix (MALDI-TOFMS) analysis to carry out the N-terminal analysis.Choice criteria is: the 1) apparent purity of HPLC fraction.2) quality of peptide and the length of estimation thus.The peptide quality information is specified useful to conclusive evidence Ai Deman order-checking amino acid, and useful to the possible detection of translation or posttranslational modification.

(In-gel) digestion on gel is suitable for the purifying in the highly sensitive HPLC system.Inner sequential analysis of protein at first carries out enzymic digestion by sds page (SDS-PAGE).Albumen in the SDS-PAGE microgel can only be digested in gel by trypsinase reliably.With RPC-HPLC purified peptide fragment, to analyze with MALDI-TOF MS then, screening is fit to the peptide of Ai Deman sequencing analysis.Albumen can only be analyzed with the internal sequence analytical method in the gel, but can obtain peptide quality very accurately, and this can provide extra amino acid is specified and the database search Useful Information.

Conjugated protein N-terminal and the inner Ai Deman sequencing analysis of being suitable for of PVDF.PVDF is conjugated protein by suitable enzymic digestion (trypsinase, endoproteinase Lys-C, endoproteinase Glu-C, clostripain, endoproteinase, Asp-N, thermophilic mattress proteolytic enzyme) and non-ionic detergent, for example hydrogenation Triton X-100.In PVDF was conjugated protein, the stain remover that the peptide that is used for digesting discharges from film can disturb the auxiliary mastrix-assisted laser desorption ionization time of flight mass spectroscopy of matrix.Before adding enzyme, with dithiothreitol (DTT) (DTT) reduction Gelucystine (Cys), and generate the Carboxylamide Gelucystine that methylates with the iodo-acid amide alkylation, can in the N-terminal sequencing analysis, be identified.

Form for the amino acid of determining protein or its chimeric molecule, in gas phase, use the catalytic strength hydrochloric acid of phenol (HCl) acidic conditions hydrolyzation sample.In case hydrolysis is finished, the amino acid with a kind of fluorophor compound deriving discharges makes bonded divide anti-phase characteristic special on the subband.Anti-phase high speed liquid chromatography (RP-HPLC) separates derivative amino, and detects with fluorimetric detector.Use outside and internal standard, come each amino acid whose quantity in the calculation sample by the peak area that observes.This information can be used for identifying sample and to the protein quantification in the sample.For example, theoretical deviation with result reality can be used for determining at first the possibility at a desamidation position.Analysis combines with monose, and it can determine glycosylation weight percent composition and acid glycoprotein weight percentage.The limitation of this method is that it can provide because of environmental pollution and the amino acid whose accidental skeleton actual sequence information that produces inherent variation of destroying.For example this method can not detect the point mutation of sequence.

It is the method that another kind can be confirmed protein or its chimeric molecule that the peptide quality fingerprinting is analyzed (PMF).Its process comprises initial with electrophoresis (1 dimension or 2 dimensions) sample separation, cuts from gel a little/is with and digest with special endo-protease (typically using pig trypsinase).Elution goes out peptide and the quality of the peptide determining with mass spectroscopy to exist from the gel fragment.Subsequently the peptide quality of generation and the proteinic Theoretical Mass crumb data storehouse of all announcements are compared (or theoretical peptide quality of the implementation sequence that makes up).It is this unique fact that this technology depends on proteinic " fingerprint " (being its peptide mass combination).Can identify reliably that (surpassing 90% accuracy) little sequence to 4 peptides and 30% covers.Can identify modification in the PMF stage of analyzing, for example fat part and desamidation.Further analyze by tandem mass spectrometry (Ms-MS) with the peak that the protein of identifying does not conform to, the energy that the MS-MS technology has used collision gas to collide generation interrupts the weak bond of PTM.Subsequently the molecule of new release and original peptide are carried out quality and analyze the peptide fragment of identifying that posttranslational modification and it are adhered to again.

Special component according to size, electric charge, hydrophobicity, function or target biological molecules is divided into different patterns with HPLC.Substantially, adopt two or more chromatographys to come a kind of protein of purifying.When selecting chromatogram mode, the most important thing is to consider the characteristic of protein and sample solvent

Can estimate their secondary structure by the characteristic that characterizes protein of the present invention or its chimeric molecule.

The secondary structure that can use one or more following systems to come analysing protein or its chimeric molecule.

In order to study proteinic secondary structure, should adopt and more the most frequently used several spectroscope methods.Can use the radiation continuous wave, determine electromagnetic energy according to the size and the shape of ripple.Different spectroscope methods is used different electromagnetic energy.

Wavelength is the length (two continuous wave maximum value between distance) of the single ripple of radiation.When radiant increased, wavelength shortened.Pass between frequency and the wave number is:

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

Molecule is to absorption involving vibrations and the rotational transition and the transition of electron of electromagnetic radiation.The most popular method that is used to identify the mensuration molecular vibrational energy of secondary structure is infrared (IR) and Raman spectrum.But their method is different with the mensuration molecular absorption.

The scattered radiation energy is less than the stokes line incident radiation.The scattered radiation energy is greater than the incident radiation of anti-Stokes spectral line.Excite the base electronic state vibrational energy of increase or energy that reduces and molecule relevant at interval.Therefore, stokes and anti-Stokes spectral line wave number are that the direct detection of molecular vibrational energy is measured.

Only observe Stokes shift (Stokes shift) at Raman spectrum.The wave number of stokes line is less than (or wavelength greater than) exciting light.Can use high-energy excitaton source (for example laser) to strengthen Raman scattering efficient.Excitaton source should be unicolor, because we are interested in exciting with the capacity volume variance (wave number) of stokes line.

In order to make vibration have infrared active (IR active), the moment of dipole of molecule must change.Therefore, the symmetry of carbonic anhydride stretch be do not have infrared-active because moment of dipole does not change.It is that variation has taken place moment of dipole that the asymmetry stretching, extension has infrared-active reason.Change in order to make vibration have Raman active (Raman active), the polarizability of molecule that vibration must take place.The symmetry of carbonic anhydride stretches Raman active, because change has taken place the polarizability of molecule.Therefore, Raman spectrum has replenished infrared spectra (Herzbergetal.Infrared andRaman Spectra of Polyatomic Molecules, Van Nostrand Reinhold, New York, NY, 1945).For example, in default of the moment of dipole motion, infrared can not the detection with the nuclear diatomic molecule, but Raman spectrum can detect, and makes the polarizability of molecule that change take place because key stretches and shrinks, and change has also taken place in the interaction between this exoelectron and the nuclear.

For the symmetric polyatomic molecule of the height that has invert center (for example benzene), might in infrared spectra, there be bands of a spectrum active and in Raman spectrum, do not have the bands of a spectrum activity, vice versa.In the low or chiral molecular, might in infrared and Raman spectrum, activity be arranged all in symmetry.

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

Infraredly characterize molecular vibration by the photoabsorption that detects corresponding to molecule particular energy of the vibrational excitation of (or higher) state from v=0 to v=1.The selective rule that the ability of some decision infrared spectra detection molecules is arranged, infrared rays 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, for example α spiral, βZhe Die, βZhuan Jiao and erratic composition.The most useful infrared band in the protein analysis is acid amides I (1620-17OOcm ^-1), acid amides II (1520-1580cm ^-1) and acid amides III (122O-135Ocm ^-1).Acid amides I is the strongest proteinic absorption band.It comprises the stretching vibration of C=O (70-85%) and C-N group (10-20%).Definite band position is by bone framework image and the decision of hydrogen bond pattern.Acid amides II is than acid amides I complexity.Acid amides II is by N-H bending (40-60%) in the plane, C-N (18-40%) and C-C (10%) stretching vibration decision.Acid amides III band use little (Krimm andBandekar, Adv Protein Chem 38:181-364,1986).Most of FTIR acid amides I band B pleated sheet structures are usually located at 1629cm ^-1About, minimum 1615cm ^-1, maximum 1637cm ^-1, less important part may be near 1696cm be little (minimum 1685cm ^-1) showing the peak, the α spiral is mainly at 1652c ^-11680cm ^-1Near be βZhuan Jiao.

The principle of Raman scattering is different with infrared absorption.The inelastical scattering light wavelength and the intensity of Raman spectrum detection molecules.Raman scattering, the molecular vibration energy changes lambda1-wavelength, produces Raman diffused light.

For Raman active is arranged, to vibrate and be inelastical scattering, its key is that polarizability changes in vibration.In symmetry stretched, the electronics bonding strength was different between minimum and maximum kernel spacing.Therefore, polarizability changes in the vibration, and this vibration modes scattering Raman scattering light, and this vibration has Raman active.In asymmetric stretching, extension, the easier polarization of the electronics in the key of stretching, extension, the more difficult polarization of the electronics in the key of compression.Polarizability does not change generally, and asymmetric stretching, extension is (the Herzberg et al. 1945, as above) that does not have Raman active.

The circular dichroism performance is used to detect any unsymmetric structure, for example protein.The optically-active chromophoric group absorbs the dextrorotation and the left-handed rotation of different amounts, and this different absorption causes positive and negative absorption spectrum (usually, deducting the right avertence vibrational spectrum from left-hand polarization spectrum).Usually, extreme ultraviolet or acid amides zone (190-250nm) are mainly contributed by peptide bond, the information of amido linkage carbonyl environment is provided, therefore the information of secondary protein structure is provided, the α spiral usually 208, the 222nm place shows two negative peaks (Holzwarth et al J Am Chem Soc 178:350,1965), βZhe Die shows a negative peak at 196nm, and random coil shows a negative peak at 218nm.The peak of near ultraviolet region (250-350nm) is by the environment contribution of fragrant chromophoric group (Phe, Tyr, Trp).Disulfide linkage increases near the minimum CD band the 250nm.

Circular dichroism is followed the folding three-dimensional structure of side-chain structure height tightly usually.Most protein sex change Free up Memory steric hindrance, denaturation degrees increases, and circular dichroism spectrum weakens.For example, the side chain circular dichroism spectrum of hGH is very responsive to the partially denaturing that the adding denaturing agent causes.Some reversible molecular chemistries change, and for example disulfide bond reduction or alkalimetric titration will change the side chain circular dichroism spectrum.For hGH, the variation of removing chromophore fully or influencing circle two response of specific chromophore can cause these spectral differences, but sex change or conformational change do not cause spectral difference (Aloj et al.J BiolChem 247:1146-1151,1971).

Ultra-violet absorption spectrum is one of effective means that detects protein characteristic.It can provide the information of protein concn and chromophoric group direct environment.Protein function group, for example amino, pure (or phenol) hydroxyl, carbonyl, carboxyl or sulfhedryl can be converted into strong chromophoric group.As seen be used to monitor two types chromophore with near-ultraviolet spectrum: metalloprotein (greater than 400nm) and contain the protein (260-280nm) of Phe, Trp, Tyr residue.The change of ultraviolet or fluorescent signal can be negative or male, depends on protein sequence and solution properties.

The fluoroscopic examination molecule is radiated the energy of launching behind the excited state.The die aromatischen Aminosaeuren of numerous protein is excited in 250 to 300nm, and emission 300 is to the interior fluorescence of 400nm scope.The albumen mass-energy that only has Phe, Trp, Tyr residue is detected, and intensity is Trp in proper order " Tyr " Phe.Fluorescence spectrum can reflect the microenvironment information that influenced by protein folding.For example, the Trp that imbeds is in hydrophobic environment usually, arrive emitting fluorescence in the 330nm scope 325, but residue that exposes or free amino acid arrives 355nm place emitting fluorescence 350.A kind of common agents of surveying the protein stretching, extension is Bis-ANS.The fluorescence of Bis-ANs is pH dependent form.Though a little less than its signal in water, it can increase signal (James and Bottomley Arch BiochemBiophy 356:296-300,1998) by the hydrophobic site that is attached to the exposure of stretching in the protein.

Effective cancellation of Tyr and Trp causes protein to stretch the remarkable increase of back signal in the unfolded protein.A kind of simple solute also can cause this variation.In order to make the detection sensitivity maximization, can use signaling rate.For example, in research rFXIII stretches, use the ratio (Kurochkin et al.J M0l Biol 248:414-430,1995) of 350nm and 330nm fluorescence intensity.Can and absorb the method research conformational change that excitation energy is changed between the acceptor by the fluorescence donor, because efficiency of conversion relies on the distance (Honroe et al.Biochem J 258:199-204,1989) between these two kinds of chromophores.Detect antitrypsin conformation (K wonand Yu, Biophim Biophys Acta 1335:265-272 with fluorescence, 1997), determine Tm (the Farrugia et al.Int J Biol Macromol 20:43-51 of HAS, 1997), and detect MerP and stretch interact (Aronsson et al.FEBS Lett.411:359-364,1997).

Under neutral pH, the fluorescence emission spectral intensity is Trp in proper order〉Tyr.Under acid pH, because conformational change has destroyed the ability conversion, the fluorescence of Tyr is better than Trp.Fluorescence experiments has also been proved conclusively protein that guanidine causes and has been stretched intermediate in changing.

Three grades of the biochemical form of protein of the present invention or chimeric molecule and quaternary structure are to finding out that its function also is important.

Can adopt one or more following systems to measure three grades and quaternary structure of protein or their chimeric molecule.

NMR and X ray diffractive crystal analysis are the most common technique of research protein 3D structure.The method of detecting tertiary protein structure of other summary comprises the CD of near ultraviolet region, UV spectrum secondary derive (Ackland et al.J Chromatogr 540:187-198,1991) and fluorescence.

NMR is one of main method of studying in the structure biology molecular structure and molecular interaction.Except the research protein structure, NMR can also be used for studying the present invention's protein or the carbohydrate structure in the chimeric molecule.The chemist uses the structure of simple one-dimensional NMR spectral technology research chemical substance usually.Can use two dimensional technique to measure the more structure of complicated molecule.Time domain NMR is used to detect molecular dynamics in the solution.Solid state NMR is used to measure the solid-state molecular structure.NMR can be used to study the structure and the dynamics of protein, low-molecular weight compound that nucleic acid is relevant with multiple biology, pharmacology and medical science.But not every nuclear all has the suitable characteristic that can be read by NMR, and for example not every nuclear all has spin, and spin is that NMR is needed.Spin causes that nuclear produces the NMR signal, brings into play the function in little magnetic field.

Can use the crystalline texture of one or more following systems measurement protein or their chimeric molecule.

The X ray diffractive crystal analysis is a kind of experimental technique, and X ray can be by crystalline diffraction, and (in the dust scope ,-10-8cm) X ray is by the suitable electronic cloud institute diffraction of the atom of size to have suitable wavelength.According to the molecule of cycle combination in the crystal or the diffraction pattern of atom X-ray diffraction, electron density can be by reconstruct.Can or mend the diffraction experiment from diffraction data and obtain additive phase information, finish reconstruct.Progressively set up experiment electron density model subsequently, according to data-optimized, the result obtains molecular structure very accurately.

The X ray diffractive crystal analysis has developed into the structure of studying the material of all states with any ray, for example ion, electronics, epithermal neutrons and proton, and wavelength is similar with the distance between atom to be measured or molecular structure.

Light scattering spectrum is based on so simple principle, and macroparticle is more than the light of small-particle scattering.In the 310-400nm zone, based on the macroparticle scattered light is the baseline of an inclination, for example aggregate Schmid et al.Protein structure, a practicalapproach, Creighton Ed., IRI Press, Oxford, England, 1989 in the solution).

Light scattering spectrum can be used to evaluating protein matter molecular weight, and it is a kind of simple tool of monitoring quaternary structure of protein or protein aggregate.The protein aggregation degree can detect with simple turbidity and characterize.The final pharmaceutical solutions that produces carries out the transparency inspection, presents cloud and lacteous because great majority are assembled albumen.Quasi-elastic light scattering spectrum (QELSS), sometimes being called as photon correlation spectroscopy (PCS) or dynamic light scattering (DLS), is the non-intrusion type exploration technology of a kind of macromole (protein, polysaccharide, synthetic polymer, micelle, colloidal solid and aggregate) 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 spread coefficient that QELSS obtains can be estimated size.In polydisperse system, it estimates the width of molecular weight distribution.For accurate mensuration, use the 200-500mW laser energy, extensively adopt conventional Ar+/Kr+ gas laser (Phillies Anal Chem62:1049A-1057A, 1990).Detected protein aggregate (Liet al.Biochemistry 34:5162-5772,1995) with human relaxin.

The stability of protein or its chimeric molecule also is an important determinative of function.The analytical procedure of this characteristic comprises DSC, TGA and lyophilize cryomicroscope, analyzes freezing one and melts resistance and protease resistant.

Can be more stable after protein of the present invention or the chimeric molecule freeze-drying (lyophilize).Freeze-drying is used to increase the stability and/or the shelf-life of product, because product stores with powder type, rather than liquid form.Its process comprises the freezing sample of beginning, removes liquid by dehydration subsequently under vacuum.Net result is " cake " of a kind of exsiccant protein and auxiliary material (other material composition of use).The consistence of the cake that finally obtains is crucial to successfully rebuilding.Freeze-drying process can cause protein to change, and especially by crosslinked aggregated forms, also has desamidation and other modification.These can be lost, reduce active or induce immune response at aggregate, thereby reduce effect.In order to detect freeze-drying stability, use stablizer (for example N.F,USP MANNITOL, trehalose, tween 80, human serum albumin etc.) that protein is pressed the prescription freeze-drying.If activity of proteins can be measured by suitable bioassay method, detect the proteinic amount of recovering with ELISA after the freeze-drying.Can detect protein aggregate with HPLC or western blot analysis.

Before freeze-drying, should determine the Tg or the Te (definition of T g or Te) of composition, the maximum permissible temperature of product in the drying first is set.Equally, the degree of crystallinity of composition or amorphous degree information help more rational design freeze-drying circulation.Can use differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) or freeze-drying cryomicroscope to obtain these thermal parameters product informations

Differential scanning calorimetry (DSC) is a kind of physics heat analysis method, and the thermal characteristic of detection, sign and analysis of material is also determined thermal capacitance, thawing enthalpy and corresponding transition point.DSC scans a temperature range with the linear ratio.According to " power back-off zero balance " principle, the discrete thermal source in the instrument is respectively sample and provides heat with reference to dish.In the physical transformation process, energy absorption or send the imbalance that causes the amount of energy that offers sample container.Rely on the different calorifics behavior of sample, energy will removed or diffusion from sample, and temperature contrast will be detected to passing to computer telecommunication number.As a result, the automatic adjustment of well heater makes the temperature of sample container and equates with reference to holder.Electric energy that compensation needs and calorimetric effect equate.

Can estimate organic purity with DSC according to the temperature of shape and DSC thawing heat absorption.Under the same conditions, compare power back-off DSC with hot-fluid DSC very high resolving power is provided.Power back-off DSC produces determinacy and accuracy is better melted regional area, does not resemble time resolution factor hot-fluid DSC at a narrow temperature fuzzy at interval because melt regional area.Power back-off DSC itself just can produce the better local zone of melting, and therefore can carry out better purity check.By the help of StepScan DSC, use traditional and method time-proven, power back-off DSC can provide direct thermal capacitance to detect, do not need deconvolution or extract sinusoidal wave amplitude.

Thermogravimetric analysis (TGA) test sample mass loss and rate of weight loss are as the function of temperature or time.

In DSC, the freeze-drying low-temperature device can reach a wide temperature range fast.At present, as preplanning and experimental tool planning, simulation freeze-drying circulation provides the platform of best small-scale research protein component thermal parameters in the freeze-drying low-temperature device.The freeze-drying microscope can be predicted the influence of component and process factors in the freezing and drying.Low temperature test only needs the 2-3mL sample, makes this technology become the instrument of a valuable research medicine rare, that be difficult to obtain.It is refrigerating effect, ratio, the dryness factor in the research freeze-drying circulation, a kind of good instrument of thawing rate.The experiment of freeze-drying cryomicroscope can help annealing research.Because the widespread use of freeze drying technology, and to the heavy demand of extremely expensive medicine (for example protein and gene therapy medicament) stabilization, wish that pharmaceutical industry is in the near future with the micro-monitoring in the implementation procedure.

In the following system of energy use one or more are measured the freezing thawing resistance of protein or its chimeric molecule.

In the translation or posttranslational modification, for example glycosylation can protected protein matter stands to freeze repeatedly/melt circulation.In order to detect it, can comparison protein or the carrier free resemblance that produces of chimeric molecule of the present invention and intestinal bacteria.Protein or its chimeric molecule are diluted in the suitable medium (cell growth medium for example, PBS or similar other material), use several different methods freezing subsequently, for example freezing at once in liquid nitrogen, be positioned over-70 ℃ slow freezing or in dry ice quick freezing.Though at room temperature melt fast or slowly melt samples at 4 ℃.Subsequently that some samples are freezing once more, this process repeats some circulations.Detect the protein mass that exists with ELISA, it is active that experienced staff selects suitable biological detecting method to measure.Activity/remaining proteinic value is compared with original material, determine many freezing/resistance after the thaw cycle.

Protein or chimeric molecule of the present invention can change calorifics stability in solution.External can be by following mensuration calorifics stability of the present invention.

Protein or chimeric molecule of the present invention can be mixed in the damping fluid, for example contain carrier proteins (for example human serum albumin) phosphate buffered saline buffer, and are incubated the specific time (for example 37 ℃, 7 days) under specified temp.Can measure the remaining quantity of handling back protein or its chimeric molecule with ELISA, and think comparison with the material that is stored in-70 ℃.The experienced personnel of association area carry out suitable biological detection and measure the remaining protein or the biological activity of its chimeric molecule.

Can use the protease resistant of one or more following systems measurement protein or its chimeric molecule.

In order to compare protease resistant, the solution that contains the solution of protein or chimeric molecule of the present invention and the resemblance that contains escherichia coli expression can be hatched (proteolytic enzyme of for example unpurified serum protein enzyme, purifying, recombinant protein enzyme) the different time period with the proteolytic enzyme of selecting.Adopt suitable ELISA (the identification epi-position of for example catching and detect antibody is cut apart by proteolytic enzyme cutting site) to measure remaining protein content, the suitable biological detecting method that adopts experienced personnel to select is measured the activity of remaining protein or its chimeric molecule.

Can use the bioavailability of one or more following systems measurement protein or its chimeric molecule.

Bioavailability be after the administration a kind of medicine or other material by the available degree of destination organization.Bioavailability depends on the transformation period of medicine or the ability of its arrival destination organization.

The mixture that contains protein or chimeric molecule of the present invention adopts subcutaneous or intramuscular injection.Egg is measured the level of protein in the blood or its chimeric molecule subsequently with ELISA or radiocounting.Perhaps, the protein active with in the suitable biological detecting method detection blood sample of experienced personnel's selection for example, stimulates the propagation of specific targeted cell population.Because sample comes from blood plasma or serum, it is influential to detected activity therefore to have some other molecule.This can control by the neutralizing antibody that uses testing protein.Therefore, any remaining biological activity all is that other serum composition causes.

Can use the stability or the transformation period of one or more following systems measurement protein or its chimeric molecule.

Protein or chimeric molecule of the present invention may have the transformation period of variation in serum and blood plasma.External can be by the following mensuration transformation period of the present invention.The mixture that contains protein or chimeric molecule of the present invention can be mixed in the human serum and hatch specified time (for example 37 ℃, 4 hours, 12 hours etc.) under specified temp.Can measure the remaining protein in back or the amount of its chimeric molecule handled with ELISA.The suitable biological detecting method that adopts the experienced personnel of association area to select is measured the biological activity of remaining protein or its chimeric molecule.The serum of selecting can come from different human blood group (for example A, B, AB, O etc.).

Also can measure the transformation period of protein or its chimeric molecule in vivo.The mixture that contains protein or chimeric molecule of the present invention, can be with radioactive tracer or other method mark, can be in the species of experimental selection by intravenously, subcutaneous, back eye socket, tail vein, intramuscular or peritoneal injection, for example mouse, rat, pig, primate, people.Injection back different time points obtains blood sample, analyzes the protein or its chimeric molecule (precipitating radiocounting with ELISA or with TCA) that exist.With the protein that contains intestinal bacteria or CHO production or the mixture contrast as a comparison of its chimeric molecule.

In order to determine the transformation period of protein or chimeric molecule of the present invention, in vivo, can be to male Wag/Rij rat or other suitable a kind of protein of animal intravenous injection or its chimeric molecule.

Before giving material, the 200 μ l EDTA blood of taking a sample are as negative control.Different time points after injection adopts the constructed 20O μ l EDTA blood of taking a sample from animal.The last time behind the blood sampling, kill animals.Sample is at room temperature centrifugal 15 minutes in back 30 minutes of collection.Measure the concentration of protein in each plasma sample or chimeric molecule of the present invention with special ELISA.

Protein or chimeric molecule of the present invention can pass hemato encephalic barrier.

Can adopt following assay method vitro detection protein or chimeric molecule of the present invention whether in conjunction with the human brain epithelial cell.

The protein of energy detection of radioactive labels or chimeric molecule of the present invention are in conjunction with the epithelial ability of human brain kapillary.Adopt methods known in the art with isolating protein or chimeric molecule of the present invention manually in conjunction with last radio-labeling, realize certain specific activity, for example chloramine-T method mark 125I or 3H.

Epithelial former being commissioned to train of human brain supported and can be grown in flat 96 orifice plates, cover with back 5 days fixing gently with acetone.Cell is dissolved, transfers on the glass fibre membrane.Can use liquid scintillation counter detection of radioactive labels albumen or chimeric molecule of the present invention.

Can adopt following assay method to detect protein or chimeric molecule of the present invention in vivo in conjunction with the human brain epithelial cell.

Use FF (not through fixing), in cryostat, cut into slices, be positioned on the sheet glass and detect the combination of human specific protein or chimeric molecule of the present invention and human brain capillary vessel with the human brain tissue section of acetone fixed.The combination of 3H-protein or chimeric molecule of the present invention in the use quantitative autoradiography detection brain section.

Can in the primates system, detect the tissue distribution and the blood clearance of human specific protein or chimeric molecule of the present invention in vivo.

In two male stump-tailed macaques or other suitable primate, protein or chimeric molecule of the present invention are used to the albumen of definite 14C-mark or the tissue distribution and the blood of chimeric molecule of the present invention is removed, and the control protein of protein or chimeric molecule of the present invention and 3H mark is had the animal of intravenous catheter simultaneously.In experimentation, collect blood sample and measure the removing of protein from circulation.In injection back 24 hours, kill animals was also selected organ, collected representational tissue and measured isotopic distribution and removing naturally.In addition, according to people's such as Triguero (J of Neurochemistry 54:1882-1888199O) method the sample from the brain different zones is carried out kapillary and reduce experiment.This method has been removed the vascular system (Triguero etc. quote above) that surpasses 9O% from brain tissue homogenate.

Radiolabeled protein or the time-dependent of chimeric molecule of the present invention from the capillary portion to the substantial part time-dependent migration of distribution and protein or chimeric molecule of the present invention again are consistent by hemato encephalic barrier.

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

Can use methods known in the art to measure the latent effect of the vasculogenesis of protein or chimeric molecule of the present invention.For example, can in angiogenesis model, sprout the vasculogenesis degree of measuring by capillary blood vessel.In this detects, press people such as Shepherd (ArteriosclerThromb vase Biol 24:898-904,2004) method is separated rat fat microvessel fragment (RFMFs), collects the epididymal adipose tissues pad from the animal of putting to death, the former enzymic digestion of chopping blended rubber.From fat and adipocyte, isolating RFMFs and separate cell by centrifugation method, and be suspended among the 0.1%BSA PBS.Continuous filtration RFMF suspension removes the fragment of tissue in the fragment, unicellular and red corpuscle.With 15, the 000RFMF/ml RFMFs that in cold, pH neutral rat tail type i collagen, suspends, and be layered in the hole in 48 orifice plates (for example O.25ml/ hole) and cultivate.After the collagen polymerization, add the DMEM that contains 10%FBS of equal volume in each glue.After forming gel, the blood vessel extended characteristic of vasculogenesis is being cultivated progressively appearance in the 4th day.Follow form in default of anchor, unstriated muscle, be easy to distinguish these newly-generated blood vessels and parent blood vessel fragment.Can cultivate and measure newly-generated length of vessel on the the 5th and 6 day with protein or chimeric molecule processing RF MF 3-D culture of the present invention.

Can also measure the vasculogenesis latent effect of protein or chimeric molecule of the present invention with the interior vasculogenesis detection method of the body that people such as 6uedez (Am J Pathol 162:1431-1439,2003) describe.This detection method be included in subcutaneous transplantation semi-closure silicone right cylinder in the nude mice (the vascular reaction device, angioreactor).The pre-mixing or the extracellular matrix of pre-mixing protein or chimeric molecule of the present invention have not been filled in the vascular reaction device.Intravenous injection fluorescein isothiocyanate (FITC)-dextran is come the vascularization in the quantitative vascular reaction device before recovery, carries out fluorescence spectrophotometry subsequently and detects.The vascular reaction device is carried out the fluorescence spectrophotometry detection can show the cell of different developmental phases and the new vessel of intrusion.

Protein or chimeric molecule of the present invention can have the immunoreactivity feature that different available immunoassaies are measured, and comprise with one or more directly at the interaction of the antibody of molecule.The example of immunoassay comprises that the crosslinked immunosorption of enzyme detects (ELISA), Dot blot and immunochromatography and detects, for example flow test or strip test.

Can use the immunoassay process to measure the level of protein or its chimeric molecule, for example, a kind of ELISA test kit of commercial distribution.Because the specificity of the antibody that provides in the immunoassay kit, protein or chimeric molecule of the present invention can have different immunoreactivity features to the protein or its chimeric molecule of inhuman cell expressing.For example, the antibody of catching and/or detect of immunoassay can be specific directly at the human protein of inhuman cell expressing or the antibody of its chimeric molecule.

In addition, the incorrect folding meeting of the human protein of inhuman cell expressing or its chimeric molecule causes that unexposed epitope exposes in the human protein of correct folding people's cell expressing or its chimeric molecule.Non-correct folding can passing through for example, excessively produces foreign preteins and produces in inhuman cell cytosol, for example, and intestinal bacteria (Baneyx Current Opinion inBiotechnology, 70:411-421,1999).Further, the human protein of inhuman cell expressing or its chimeric molecule can have different protein or chimeric molecule posttranslational modification form of the present invention.For example, the human protein of inhuman cell expressing or its chimeric molecule can have sugared structure, phosphate radical, sulfate radical, fat or other residue of unusual quantity and/or type.This may cause the exposure of unexposed epitope in protein or chimeric molecule of the present invention.Opposite, the change of posttranslational modification pattern may cause lacking the epitope in protein or the chimeric molecule of the present invention, exposes in human protein that described epitope is expressed in inhuman cell or its chimeric molecule.

Any one or the above-mentioned factor that makes up may cause as following non-correct detection:

(a) human protein of natural generation in laboratory sample or people's tissue, or

(b) laboratory sample, people tissue or in human embryo stem cell (hES) substratum recombinant human protein's matter of people's cell expressing or its chimeric molecule

Use the human protein of people's cell expressing that suitable immunoassay measures or the immunoreactivity feature of its chimeric molecule that proteinic immunogenic sign in the human body can be provided, as mentioned below.

Most biological products cause the antibody response at them of certain level.Antibody response can cause possible serious side effects and/or loss effect in some cases.For example, some patients that accept the recombinant protein of inhuman cell expressing or the treatment of its chimeric molecule may produce neutralizing antibody, especially in long-term treatment is used, and therefore weaken proteinic effect and/or facilitate side effect.Therefore the human protein of people's cell expressing or chimeric molecule unlikely produce neutralizing antibody, compare with the human protein of inhuman cell expressing or its chimeric molecule to have strengthened curative effect.

Can use the immunogenicity of one or more following systems measurement protein or its chimeric molecule.

Most biological products cause the antibody response at them of certain level.Antibody response can cause possible serious side effects and/or loss effect in some cases.For example, some patients that accept recombinant epo treatment may produce neutralizing antibody, and and patient's self EPO cross reaction.In this example, simple erythroid aplasia can take place in them, and treatment produces resistance to EPO, causes the frequent dialysis of needs.

Immunogenicity is the ability that can cause immunne response in organism.Immunogenicity partly depends on the size of described material, and partly depends on the dissimilar degree of it and host's molecule.Because have new plysiochemical characteristic, protein or its chimeric molecule may have the immunogenicity of change.Therefore for example, the glycosylation structure of protein or its chimeric molecule may shield or weaken the identification of antibody to epitope, prevents or weakens antibodies on protein or its chimeric molecule.Perhaps, some antibody can be identified in non-existent glycopeptide epi-position in the non-glycosylated protein matter.

Can determine that patient's sample identification has the ability of the protein or its chimeric molecule of distinctive plysiochemical form by different method of immunity, as described here.Design suitable method of immunity and comprise the directly suitable detection of consideration, quantitative and sign antibody response.Some are listed in people's such as Mire-Sluis JImmunol Methods 289 (1-2): 1-16 to immunoassay designs and the suggestion optimized, in 2004, be incorporated herein by reference document.

Can use the protein of one or more following systems measurements use in therapeutic is transplanted or its chimeric molecule.

The present invention expands and uses protein or its chimeric molecule to operate stem cell.A main stem-cell therapy application is tissue, cartilage or bone regeneration.In one embodiment, the cell in biocompatible 3 dimension matrix might be introduced in the human body.Transplanting will comprise cell mixture, skeleton, somatomedin and necessary composition, biological example degradable polymer, proteoglycan etc.The behavior that protein or its chimeric molecule are regulated cell is mixed in plan in these matrix in building process.This transplanting can be used to bone formation, neural from growth of progenitor cells and other application.The cell-derived protein of people can reduce proteinic quantity of recessive allele and/or the kind under the stem cell cultivation conditions, and has therefore reduced the risk of inhuman pathogenic infection.

Different interactions can take place with the matrix that is used to form graft in protein of the present invention or chimeric molecule, and the cell of regulating mixes in the graft.Estimate to unite and use protein of the present invention or chimeric molecule and graft composition will cause one or more following pharmacology proterties, for example high proliferations, promote differentiation, keeping of the differentiation state of wishing, stronger differentiation pedigree specificity, increase the secretion of matrix components, form better three-dimensional structure, enhancing signal, better structure properties, reduce toxicity, reduce side effect, reduce inflammation, reducing immunocyte soaks into, reduce injection, the graft time length is longer, graft function is more permanent, the graft peripheral cell is better stimulated, better tissue regeneration, better organ dysfunction, better tissue remodeling.The effect that can use one or more following systems measurement protein or its chimeric molecule that different genes is expressed.

Can analyze the gene expression of cells difference that is exposed to protein or its chimeric molecule.

The mRNA that microarray technology can be measured gene nearly all in the organism genome simultaneously expresses.This method use gene " chip ' ', the oligonucleotide of corresponding different genes sequence is attached on the solid carrier in " chip ".The cDNA and the chip of the mark that the mRNA that utilization is extracted from interested cell or tissue obtains are hatched, the complementary sequence hybridization that allows cDNA and adhere to.Also need to use contrast, hybridizing subsequently and cleaning the back and compare both signals.Using special software to measure which gene raises or reduces or change of which expression.Each chip can be analyzed thousands of genes.For example use the Affymetrix technology, people's gene group U133 (HG-U133) set, comprise two GeneChip (registered trademark) array, contain about 45000 probes set, represented more than 39000 transcripton that from the Human genome that about 33,000 quilts well confirm, extracts.GeneChip (registered trademark) mouse genome 4302.O contains more than 39000 transcripton in individual array.

Such analysis has disclosed the variation of overall mRNA expression pattern, can find that therefore the unknown expression of gene of being regulated and control by the particular stimulation thing changes.Therefore this technology is suitable for analyzing the inductive genetic expression relevant with protein of the present invention or chimeric molecule.

Determine to be subjected to the known and new gene of particular stimulation thing regulation and control will help to confirm biological chemistry path important in specific protein of the present invention or chimeric molecule biological activity.These information will be useful to determining the novel therapeutic target spot.

This system can also be used to observe and compares protein of the present invention or chimeric molecule inductive changes in gene expression with the product that can buy.

Can use the binding ability effect of one or more following systems measurement protein or its chimeric molecule.

Can study the binding ability of protein of the present invention or chimeric molecule and different substances, described material comprises extracellular matrix, artificial material, heparin sulfate, carrier or cofactor.

Can use following assay method to determine that specified protein in protein or its chimeric molecule is in conjunction with the effect of extracellular matrix.

Pan coating extracellular matrix protein in suitable damping fluid includes but not limited to collagen, vitronectin, Fiberonectin, ln.Binding site can be with methods known in the art bag quilt, for example and BSA solution hatch.Clean surface for example, is used PBS solution, adds the solution that contains albumen to be detected (protein for example of the present invention or chimeric molecule) subsequently on the surface.Bag by after, clean surface and and the antibody incubation of identification of protein or its chimeric molecule.Detect bonded antibody subsequently, for example, use a kind of identification one anti-enzyme di-to resist.By to hatch and observe color change reactions visual with bonded antibody with suitable material.Not glycosylated protein is compared, and what glycosylated protein can be stronger adheres on the extracellular matrix protein.

Perhaps, (specifying) protein of the present invention or chimeric molecule with equal amount with ELISA concentration or biological assay activity unit, or the protein of the present invention of all inhuman cell expressings or chimeric molecule resemblance, hatch together with the aperture of matrix bag quilt, clean aperture subsequently, detect in conjunction with quantity with ELISA.Can be educated the reactive decline indirect measurement of back ELIsA in conjunction with quantity by 9 gusts on surface by sample and bag.

Can use one or more following systems measurement protein or its chimeric molecule ability in conjunction with artificial material.

In order to measure protein or its chimeric molecule ability in conjunction with artificial material, surface bag in suitable damping fluid is included but not limited to metal, support by artificial material.Clean surface for example, is used PBS solution, adds the solution that contains albumen to be detected (protein for example of the present invention or chimeric molecule) subsequently on the surface.Bag by after, clean surface and and the antibody incubation of identification of protein or its chimeric molecule.Detect bonded antibody subsequently, for example, use a kind of identification one anti-enzyme di-to resist.By to hatch and observe color change reactions visual with bonded antibody with suitable material.

Perhaps, (specifying) protein of the present invention or chimeric molecule with equal amount with ELISA concentration or biological assay activity unit, with protein of the present invention or chimeric molecule resemblance with inhuman cell expressing, hatch together with the aperture of artificial material bag quilt, clean aperture subsequently, detect in conjunction with quantity with ELISA.Can be hatched the reactive decline indirect measurement of back ELISA in conjunction with quantity by the surface by sample and bag.

Ability in conjunction with artificial surfaces may have biology effect, for example in support bag quilt.Perhaps, the support that is coated with a kind of protein of the present invention or chimeric molecule is used to repopulating cell.Monitor the growth and the differentiation of cell subsequently, and with do not wrap quilt or the bag compared by different supports.

Can use the ability of one or more following systems measurement protein or its chimeric molecule combined sulfur heparin.

Because the biochemical form of protein of the present invention or chimeric molecule estimates that they and heparin sulfate have different interactions.Estimate that these difference are tangible in experimental models such as cell proliferation, differentiation, migration.In TA, unite and use protein or its chimeric molecule and heparin sulfate in advance in respect of distinctive pharmacology proterties.May be that serum half-life prolongs, bioavailability increases, reduce that immunity is relevantly removed, stronger effect, reduce dosage and reduce side effect and relevant advantage.

Can use one or more following systems measurement protein or its chimeric molecule ability in conjunction with carrier or cofactor.

When protein or its chimeric molecule were present in the blood plasma, they will be in conjunction with other molecule.These molecules can be named as " carrier " or " cofactor ", and will influence the bioavailability or the serum half-life of these factors.

Hatch and can measure the interaction of protein of the present invention or chimeric molecule and their binding partner with molecular-exclusion chromatography analysis gained solution with the plasma proteins of purifying.If protein or its chimeric molecule are in conjunction with a kind of cofactor, the mixture that obtains will have bigger molecular weight, cause elution time to change.Can compare the biological activity of mixture, external or interior transformation period of body and bioavailability.

Can use one or more following systems measurement protein or its chimeric molecule effect to biological assay.

Can carry out the activity that multiple bioassay method detects protein of the present invention or chimeric molecule, comprise and measure cell proliferation, cytodifferentiation, apoptosis, cell size, cytokine/cytokine receptor adhesion, cell adhesion, cell dispersion, cell movement, migration and infiltration, chemotaxis, ligand receptor combination, receptor activation, signal conduction and isoform ratio vary.

Can use the effect of one or more following systems measurement protein or its chimeric molecule on cell proliferation.

Cell, in specific embodiment, the cell of exponential growth is hatched in the growth medium that contains protein of the present invention or chimeric molecule.This can carry out in narrow-necked bottle or 96 orifice plates.Cell growth for some time is used the method counting cells of directly (for example cell counting) or indirect (MTT, MTS, tritiated thymidine) subsequently.The contrast that only contains substratum is compared and is measured the increase or the minimizing of propagation.In parallel serial experiment, can use the protein of different concentration or its chimeric molecule to obtain the dose response feature.Can measure ED with it ₅₀And ED ₁₀₀(producing the needed dosage of response effect half maximum and maximum).

Can use differentiation of one or more following systems measurement protein or its chimeric molecule pair cell or cell to keep the effect of undifferentiated state.

Cell is hatched in the growth medium that has protein of the present invention or chimeric molecule.After suitable for some time, the differentiation indicator is carried out cell analysis.It can be the expression of cell surface special sign thing, the expression of endochylema mark, the variation of cell size, shape or kytoplasm feature.Mark may comprise protein, sugared structure (for example glycosaminoglycan, for example heparin sulfate, chondroitin sulfate etc.), fat (glycosphingolipid or lipid bilayer composition).Can use multiple technologies to detect these variations, described technology comprises microscope, protein blot, FACS dyeing or forward direction/lateral scattering feature.

Can use the effect of one or more following systems measurement protein or its chimeric molecule pair cell apoptosis.

Apoptosis is defined as programmed cell death, waits other cell death way different with necrosis.It is characterized by the cellular change of determining, for example activation of signal path (for example Fas, TNFR) causes a proteolytic enzyme subgroup that is called as caspase (caspases) to be activated.The activation that starts caspase causes the activation of deadly caspase, and its cutting various kinds of cell albumen causes nuclear fragmentation, the nuclear lamins fracture, and tenuigenin bubbles and also destroys cell.Apoptosis can be induced by protein ligands, for example FasL, TNFa, lymphotoxin or by signal induction, for example UV-light and cause the material of dna damage.

Cell is hatched in the growth medium that contains protein or its chimeric molecule or other suitable reagent that detects.For example, may need to block the reagent of transcribing (dactinomycin) or translation (cycloheximide).After hatching suitable for some time, measure cell quantity with suitable method.Cell quantity descends may mean apoptosis.Can obtain other apoptosis by cell dyeing and characterize, for example, annexin or observe distinctive trapezoidal dna form.Can detect the apoptosis mark subsequently by hatching the expression that prevents the apoptosis mark, further confirm apoptosis with Premeabilisation of cells caspase inhibitor (for example z-VAD FMK).

Protein of the present invention or chimeric molecule may provide the existence signal to prevent apoptosis by cells survival path (for example Bcl2 or Akt path).Can confirm the activation of these paths by cell Bcl2 expression increase or the protein blot that uses direct phosphoric acid specific antibody to detect Akt activation form (phosphorylation) increase at Akt.

For these detections, cell cultures is containing or is not containing under Survival Factor (for example IL-3 and certain immunocyte) condition.Cultivation is prolonging apoptosis under the cultivation at the part cell that lacks under the Survival Factor condition, and the cell of cultivating under sufficient amount Survival Factor condition will survive or breed.Can pass through protein blot, immunocytochemistry and facs analysis and measure the activation of the cell pathway of being responsible for these effects.

Can use one or more following systems measurement protein or its chimeric molecule to suppress the effect of apoptosis.

It is low and lack the activity that occurs necrocytosis under the glucose condition in oxygen level at external protection rat, mouse and people's cortex neurocyte to have detected protein of the present invention or chimeric molecule.For this reason, from rat embryo, extracted cell culture.In order to measure the effect of protein of the present invention or chimeric molecule, cell is containing 30mM glucose serum free medium and 95% wetness air/5%CO ₂(oxygen level is normal) or do not containing glucose serum free medium and 95% wetness nitrogen/5%CO ₂Under (hypoxemia and glucose lack) condition, lacking or existing under protein of the present invention or the chimeric molecule condition, the module in water jacketed mcubator is cultivated in the storehouse and was kept under 37 ℃ 48 hours.Cell culture is exposed to and is less than 24 hours under hypoxemia and the glucose shortage condition and returned to the oxygen level normal condition subsequently following 24 hours.With the fluorometric analysis cytotoxicity of Alamar indigo plant, this method is measured cell viability by metabolic activity.

In another method, the neuronal cell cultures thing is under the oxygen level normal condition, lacking or existing under the protein of the present invention or chimeric molecule condition of different concns, be exposed to 1mML-L-glutamic acid or alpha-amino group-3-hydroxyl-5-methyl oxazole-4-propionic acid (AMPA) 24 hours.With the fluorometric analysis cytotoxicity of Alamar indigo plant, this method is measured cell viability by metabolic activity.

Protein or its chimeric molecule can influence growth, apoptosis, growth or the differentiation of various kinds of cell.But in other detect parameters, these variations can be grown the cell size variation and the variation of kytoplasm complexity that cause by the cell within a cell device and be reflected.For example, suspension culture is induced the keratinocyte differentiation, shows as surface marker (for example beta 1 integrin, β 1 intetegrins) downward modulation, and it is big that cell becomes, and the kytoplasm complexity increases.Can use the effect of one or more following systems measurement protein or its chimeric molecule pair cell size or kytoplasm complexity.

FACS measures the amount of light that scatters after beam of laser incides on the cell.Usually use the argon laser of wavelength as 488nm is provided.Cell is big more, and it is many more that the forward direction light beam is blocked, and therefore preceding levels of scatter is relevant with the cell size.In order to measure the variation of cell size, the cell of handling with protein of the present invention or chimeric molecule is diluted in the sheath fluid (sheath fluid) and is injected into into streaming cell instrument (FACSVantage SE, Becton Dickinson).Untreated cell in contrast.Cell is by a branch of light, and the forward scatter quantity of light is relevant with the cell size.

Also can measure the variation (kytoplasm complexity) of cell within a cell device g and D with FACS.The cell within a cell device is to side-scattered light.Therefore, the variation of the light side-scattered quantitative measurement kytoplasm complexity that can cause with the aforesaid method cell, and can be by measuring the level of complexity and the cell granulations degree level of the light side-scattered level determination cell within a cell device that cell sends.

Can use FACS to measure the effect of protein or its chimeric molecule pair cell size or kytoplasm complexity, come the signal of the untreated cell emission of signal characteristic that comparative example sends as the cell of 20000 processing and equal amount.By comparing the signal of different treatment cell mass, can detect the relative variation of cell size and kytoplasm complexity.

Can use the effect of one or more following systems measurement protein or its chimeric molecule cell growth, apoptosis, growth or differentiation.

Can with dyestuff for example propidium iodide (propidium iodine) the mark DNA that handles cell measure the apoptosis of protein induce and cell growth or cell cycle and change, the excitation wavelength of propidium iodide is transmitted in the 620nm zone in the 488nm zone.It is condensing that apoptotic cells DNA takes place, and size is also different with granularity.These factors have provided forward size scattering signatures and fluorescent signal, and therefore will the apoptotic cells group just be taken place and normal cell distinguishes.DNA quantity in the cell has reflected that also cell is in which of cell cycle in stage.For example, G ₂The DNA quantity of phase cell is G ₀The twice of phase.This can pass through G ₂The fluorescent signal that the phase cell sends twice obtains reflection.Can use the signal of the untreated cell emission of fluorescent signal that the FACS comparative example sends as the cell of 20000 processing and equal amount, measure the effect of protein or its chimeric molecule.

Protein of the present invention or chimeric molecule can also change the expression of multiple proteins.Can use one or more following systems measurements protein of the present invention or chimeric molecule effect to protein expression.

For increase and the reduction of measuring protein expression in the whole cell, can fix and penetrating cell, be the crosslinked antibody incubation of fluorescein of target spot subsequently with proteins of interest matter epitope.In the argon laser system, can use a variety of fluorescent marks.Normal FITC, Alexa Fluor 488, Cyanine 2, Cyanine 3 these fluorescence molecules of using in this experiment.Can be by the epi-position of having only exposure on the labeled cell surface by the non-penetrating cell of antibody labeling, this method can also be used to measure surface marker and protein expression changes.The signal of the untreated cell emission of fluorescent signal that the FACS comparative example sends as the cell of 20000 processing and equal amount be can use, protein or its effect of closing molecule measured.

Can use one or more following systems measurement protein or its chimeric molecule to the adherent effect of ligand/receptor.

Compare with known biochemical forms before those, protein of the present invention or chimeric molecule may have lower or high-adhesiveness more.Interaction can be and protein acceptor, because sugared structure (is for example selected albumen, for example L-selects albumen and P-to select albumen) and extracellular matrix components (for example Fiberonectin, collagen, vitronectin and ln) or and non-protein component glycan molecule (heparin sulfate, other glycosaminoglycan) for example.

Protein or its chimeric molecule can also for example different interactions takes place in tissue culturing plastic, medicine equipment composition (for example support or other graft) or dental material with non-biological material.For medicine equipment this may change graft immigration rate, graft and specific cell type or and the body connection type between interaction

Can use any suitable protein adherence detection method.For example, in certain embodiments, solution and the lip-deep binding partners of immobilization of containing protein of the present invention or chimeric molecule are hatched.After hatching, detect the protein in the solution or the quantity of chimeric molecule with ELISA, the quantity variance between the remaining and parent material is the amount that is attached on the mating partner.For example, can use the aperture of 96 orifice plates of ECM albumen (for example Fiberonectin) the first layer bag quilt to measure interaction between protein or chimeric molecule and the extracellular matrix proteins.Hatch with BSA solution subsequently and stop non-specific combination.After cleaning, add a kind of protein of concentration known or its chimeric molecule solution and reach definite time.Subsequent removal solution is measured the protein remaining in the solution or the amount of its chimeric molecule.Measure the amount that is attached on the ECM albumen by using at the antibody incubation aperture of protein or its chimeric molecule, use subsequently a kind of suitable system (perhaps two of a kind of mark anti-or with vitamin H one avidin enzyme complex for example ELISA is employed) detect.

The method that mensuration is attached to the amount on other surface can comprise from inertia graft surface hydrolysis protein or its chimeric molecule, measures the amino acid in the solution subsequently.

Can use one or more following systems measurement protein or its adherent effect of chimeric molecule pair cell.

Cell adhesion is numerator mediated by integrin to small part to matrix (for example extracellular matrix components for example Fiberonectin, vitronectin, collagen, ln etc.).The integrin molecule comprises α and beta subunit, and α and beta subunit uniqueness increase binding specificity at specific ligand (for example a2b1 integrin incorporating collagen, a5b1 binding fiber conjugated protein etc.) in conjunction with physical efficiency.Integrin subunit has that big extracellular domain is used for binding partner and short cytoplasmic domain structural domain is used for interacting with cytoskeleton.When having part, the cytoplasmic domain structural domain is responsible for inducement signal transduction incident (signal transduction in outer).The extracellular signal event can be regulated and control the affinity of integrin to their part, causes the variation (inside and outside signal transduction) of integrin cytoplasmic tail subsequently.

Hatch and to change cell adhesion by certain methods with protein of the present invention or chimeric molecule.At first, it can change the expression of specific integrin subgroup qualitatively, causes the change of binding ability.Secondly, can change the expression amount of specific integrin, cause cell to change with combining of its target matrix.The 3rd, can under the situation of the surface expression that does not change specific integrin, change the avidity (inside and outside signal transduction) of integrin.All these variations can change the combination of cell to a pedigree part, or change the combination to certain specific part.

Can adhere to detection method with the cell that in 96 orifice plates, carries out usually-ECM and detect protein of the present invention or chimeric molecule., wrapped by the Kong Zhongwei binding site with BSA subsequently by aperture with the matrix bag.The hole of bag quilt and the cell of set amount are hatched, and the unconjugated cell of flush away is hatched the bonded cell under the condition that contains or do not contain protein or its chimeric molecule subsequently.By indirect method for example MTT/MTS measure the quantity of cell.Perhaps, (for example with radioactively labelled substance ⁵¹Cr) labeled cell, the radioactivity (being cell) of adding dose known amounts in each hole.Mensuration is calculated the ratio that loads quantity that accounts in conjunction with radioactivity.

Cell also adheres on other cell, and for example a group cell adhesion is to the another kind of cell of individual layer.In order to detect this situation, suspension cell is labeled radioactivity and joins on the monolayer cell.Cell is hatched under the condition that has or do not exist protein or its chimeric molecule subsequently.The unconjugated cell of flush away dissolves remaining cytomixis colony and detects radioactivity.

Can use the effect of one or more following systems measurement protein or its chimeric molecule pair cell expansion.

Protein of the present invention or the expansion of chimeric molecule pair cell have different effects.It is the committed step of the cell mobility and the behavior of infiltration that cell begins to spread.Can begin diffusion in many ways at cell in vitro.Suspension cell is layered on the adhesion and the part combination that will cause on the ECM composition by integrin.

This has started the signal transduction incident, causes the activation of the little GTPases of Cdc42, Rac and Rho family.These proteinic activation cause actin polymerization and sheet foot (lamellipodium) to extend, and cause cell to flatten gradually and their acceptor touches more integrin.Final cell is put down flat fully and is formed and sticks together spot (macrostructure that contains integrin and signal protein).Can also be by start cell expansion, the formation that also causes the proteinic activation of cdc42/Rac/Rho and stick together spot with the adherent cell of factors stimulated growth.

Can detect a large amount of cells by different time points after protein or the stimulation of its chimeric molecule comes the pair cell expansion to carry out quantitatively.Can use image analysis software to measure the zone of each cell, and cell can be expanded per-cent and cell degree of expansion and time and compare.The intensity of activation that the Cdc42/Rac/Rho path is stronger starts expansion faster, and is temporary in addition, can measure the qualitative and quantitative difference of their activated when protein of the present invention or chimeric molecule are arranged.This has reflected the variation of protein of the present invention or chimeric molecule inductive signal event successively.

Can use the effect of one or more following systems measurement protein or its chimeric molecule pair cell mobility, migration and infiltration.

Adhering to cell on the tissue culture ware is not that immobilized keeps being anchored on the site, but the stretching, extension that continues and shrink their cell paste.When observing with time lapse photography, can observe cell and in plate, stroll about, isolating individual cells or cell colony are not always the case.This motion both can be " random walk " (promptly not towards specific direction), also can be directed.Can strengthen two type of motion by adding somatomedin.Can use time lapse photography quantitative total distance and general direction of covering of cell in the preset time section.

In directional migration, cell will be by experiencing the source motion towards chemoattractant of chemical gradient and their migration machine of orientation.In many cases, chemoattractant is a somatomedin.Can carry out imaging with the time lapse photography pair cell to the migration in source then by chemoattractant source (for example by long transfer pipet) is provided, come the quantitative assay directional migration.

The another kind of system that measures directional migration is that Boyden chamber detects.In this detected, cell was placed in by cutting apart in aperture in the film and the last chamber that following chamber links to each other.All add growth medium in two chambers, but, cause having diffusion gradient between two chambers only adding chemoattractant in the chamber down.The grown factor of cell source attracts and moves by cutting apart the aperture in the film, enters into the lower surface of film.After a few hours, take film away and the cell of moving to the film bottom is counted mensuration.

The cellular infiltration process has been used many assemblies identical with migration.Can use cellular infiltration multi-layer cellular epimatrix to be used as the cellular infiltration model.For example, matrigel is the mixture (ECM composition, somatomedin etc.) of basement membrane components, is liquid state down at 4 ℃, but forms gels rapidly at 37 ℃.Can utilize it to wrap, add chemoattractant in lower floor by the upper surface of Boyden chamber.For the cell that passes through to the film lower surface, they must use enzyme (for example collagenase and matrix metalloproteinase (MMP)) to come matrix degradation glue and carry out directional migration towards chemoattractant.The required various procedures of cellular infiltration has been simulated in this detection.

Can use one or more following systems measurement protein or its chimeric molecule to chemotactic effect.

Can in Boyden chamber, measure the migration of cell external towards the chemoattractant direction.Protein of the present invention or chimeric molecule are placed to down the chamber, and suitable target cell group is placed into the chamber.Can detect migration, simulate the external process of the migration of immunocyte from blood to the inflammation part by a confluent monolayer cells.Coat the upper surface in the hole of Boyden chamber with a confluent monolayer cells that covers with, described cell is epithelium, endothelium or inoblast for example, by the hole in the hole, immunocyte adheres to needs on the monolayer cell and migration arrives detected albumen place by it with blocking immunity cell Direct Transfer for this.Only at the Boyden chamber lower surface in the hole of handling or exist cell to show the chemotaxis ability of protein or chimeric molecule in the substratum of hole down with protein or its chimeric molecule.In order to show that this effect is that protein or its chimeric molecule are specific, can allow albumen and neutralizing antibody hatch together in the chamber down.

In addition, prevent chemotactic ability in order to detect a kind of material (chemical substance, protein, sugar), in the following chamber of Boyden chamber, this material and the solution that contains known chemotaxis ability (can be the conditioned medium of specificity chemokine, the cell cell source or that secrete a series of chemokines) are hatched together.Add the permissive cell group in last chamber subsequently, detect by above-mentioned.

Can use one or more following systems measurement protein or its chimeric molecule to ligand receptor one bonded effect.

Protein of the present invention or chimeric molecule may have different parts one receptor binding capacity.Can be by multiple parametric measurement part one receptors bind, for example, dissociation constant (Kd), dissociation rate constant (k ^-), association rate constant (k ⁺).The difference of part one receptors bind may be relevant with activation with the different signal time limits, causes different biological result.

Can by scatchard plot method or other method for example the Biacore method measure and analyze part one receptors bind.

For the scatchard plot method, (for example with radio-labeling for example ¹²⁵I) come the protein of mark or its chimeric molecule, under the condition of the competitor of cold protein that has different quantities or its chimeric molecule and cell or its extract of expressing respective ligand or acceptor hatch together.Measure the protein of specificity bonded mark or the quantity of its chimeric molecule, and the calculations incorporated parameter.

For the Biacore method, match mutually with detection unit with protein or the corresponding reorganization part of its chimeric molecule or acceptor.The solution that contains selected protein or its chimeric molecule passes through to detect cell subsequently, and measures combination by the variation that detects unit character.Solution by will containing protein or its chimeric molecule by detect cell up to record a fixed reading (when the site of all permissions all sometimes occupied) measure association rate constant.The solution that does not contain protein or chimeric molecule is by cell, and protein disintegrates down from respective ligand or acceptor, has provided the rate constant of dissociating.

Can use one or more following systems measurement protein or its chimeric molecule effect to receptor activation.

Can contrast the interaction between protein or its chimeric molecule and corresponding part or the acceptor by the difference in the cell intrinsic protein inductive signal event.Can characterize the time limit that interacts accurately with combination/dissociation rate constant or dissociation constant with protein or its chimeric molecule.

Take the photograph the activated acceptor in cell is frequent.The cell surface that recirculates of receptor/ligand complex dissociation (for example, the low pH in the cell vesicles causes part to break away from), and acceptor subsequently.In addition, this mixture also may become the target spot of degraded.In this process, acceptor will effectively be reduced and can not produce further signal, but then can the repeat signal process when they recirculate.Different receptors bind or activation may cause acceptor to change the path that recirculates into from degraded, cause stronger biological response.

Can use one or more following systems measurement protein or its chimeric molecule effect to the signal conduction.

Part or receptors bind may be sent signal by multiple plasmosin on protein or its chimeric molecule, wherein may comprise reverse signal.When the part of film combining form by solubility or film combining form acceptor when coming conducted signal in conjunction with it, reverse signal takes place.Reverse signal also may occur in by after a kind of antibodies film binding partner.These signal events cause (comprising the reverse signal incident) variation of gene and protein expression.Therefore, different signal conduction or other signal conduction incident in the different paths can be induced or be suppressed to protein of the present invention or chimeric molecule, the for example activation of JAK/STAT path, Ras-erk path, AKT path, the activation of PKC, PKA, Src, Fas, TNFR, NFkB, p38MAPK, c-Fos, protein is raised acceptor, receptor phosphorylation is taken the photograph in the acceptor, and acceptor is crosstalked or secreted.

Part or the acceptor raised on protein or its chimeric molecule may be unique to protein of the present invention or chimeric molecule, because induced the different conformation of part or acceptor.A method measuring these differences is to come immunoprecipitation part or acceptor with the antibody on a kind of sepahrose of being linked to globule.After immunoprecipitation and the cleaning,, analyze the comparison point pattern with sample 2D gel power supply on the protein.Can downcut discrepant point and identify with mass spectrum.

Can use one or more following systems measurement protein or its chimeric molecule the effect that goes up the mediation downward modulation to surface marker.

Cell may have multiple response to protein of the present invention or chimeric molecule.There are a series of protein to be responsible for communication between cell and the extracellular matrix at cell surface.By regulation and control encytosis and exocytosis process, multiple proteins is transported to and transports out of cell surface.The typical protein of finding at cell surface comprises acceptor, conjugated protein, modulin and signaling molecule.The variation of protein expression and degradation rate also changes the level of cell surface proteins.Some protein also are stored in the cell inner jar, and distinctive signal can the transportation of induced protein between these containers and cytolemma.

Cell is hatched the suitable time in the substratum that contains protein of the present invention or chimeric molecule, compares with the cell in the substratum that is exposed to identical not conforming to and has protein of the present invention or chimeric molecule.Protein on the energy dissolved cell film is also by centrifugal and cellular segregation.Can measure the expression level of particular proteins with protein imprinted method.Can also use the crosslinked antibody labeling cell of fluorescein, visual or with the Laser Scanning Confocal Microscope system by fluorescence-activated cell sorting method (FACS) counting.This will detect any variation of protein expression and distribution on the cell.Can also utilize the variation of Laser Scanning Confocal Microscope and immunoprecipitation research protein dependent interaction by using a plurality of antibody.Similar, these experiments can expand in the interior animal model of body.Can extract cell from the animal privileged site of handling with protein of the present invention or chimeric molecule, detect with identical method.

Induce the cell of differentiation will express different marks by adding protein of the present invention or chimeric molecule in external or body, this mark is with these cells with for handling cellular regions separately.Some cells, for example, progenitor cell or stem cell can be divided into multiple isoform, distinguish by their surface marker.Protein of the present invention or chimeric molecule may stimulate progenitor cell to be divided into multiple isoform in specific ratio.

Protein of the present invention and chimeric molecule thereof might repel effectively by pair cell.

Protein or its chimeric molecule are cellular rejection detection methods easily in the effect of regulating cell and neure growth and sensing.

The interaction that destroys between subunit and other assembly of protein is a kind of method of the biological effect of arrestin matter or its chimeric molecule.Identify the compound that suppresses this biological effect by several different methods.

The high flux screening project is used little chemical entities storehouse (compound or peptide) to produce lead compound to be used for clinical development.Can use some detection methods to screen the ability of the biological relevant end of the final point of compounds affect in the storehouse.Detect each potential compound in the storehouse in the independent aperture in one-time detection, determine the effect of compound.The embodiment of some detections is provided below.

To this detection, cell is taped against in the micro plate (96 orifice plates, 384 orifice plates etc.).Cell will have protein or its chimeric molecule activated is read mechanism.This can comprise inducing (for example CAT, beta-galactosidase enzymes, fluorescin), detect apoptosis and detecting differentiation of the stimulation that detects the cell growth, detect specific passageways (for example based on FRET technology), examining report gene.Cell is exposed in protein of the present invention or the chimeric molecule existing or lack under the specific micromolecular condition subsequently.Can be before adding protein or its chimeric molecule, afterwards or among add medicine.After the suitable time period, use suitable method to indivedual holes reading (for example inducing of the fluorescence of FRET or fluorescin, the cell quantity among the MTT, betagalactosidase activity etc.).The control wells that does not add any medicine or cytokine as a comparison.Any molecule that can suppress acceptor/cytokine mixture will provide and contrast different readings.Also need further experiment to show the specificity of inhibition.In addition, medicine may influence detection method (false positive) by the acellular factor, non-receptor mechanism.

The part or the acceptor of protein or its chimeric molecule are immobilized in solid surface.Add protein or its chimeric molecule and compound to be detected subsequently.Can add protein or its chimeric molecule earlier, add compound subsequently; Add compound earlier, add protein or its chimeric molecule subsequently; Perhaps compound and protein or its chimeric molecule add together.Subsequently by suitable detection antibody test bonded protein or chimeric molecule.Can be with a kind of enzyme (for example be used for colorimetry detect alkaline phosphatase or horseradish peroxidase) or a kind of fluorescence labels marker detection antibody that is used for fluoroscopic examination.

In addition, can with a kind of suitable technique mark (biological example element, radio-labeling) protein or chimeric molecule and with a kind of suitable technique (for example, for biotin labeling, streptavidin and colorimetric detection system; For radio-labeling, dissolving mixture and counting) detect.Measure the inhibition of protein bound by the decline of comparing reading with control wells.

The soluble ligand of protein or its chimeric molecule or receptors bind are on globule.This association reaction both can be that a kind of adsorption process also can relate to their chemically crosslinkeds to plate.In a suitable aperture, globule and protein or chimeric molecule and a kind of candidate compound are hatched.Can add protein or chimeric molecule earlier, add compound subsequently; Add compound earlier, add protein or chimeric molecule subsequently; Perhaps compound and protein or chimeric molecule are added together.The detection antibody that adds a kind of protein of fluorescently-labeled identification or its chimeric molecule subsequently.Remove unconjugated antibody and globule is passed through FACS.If the interaction of compound arrestin matter or its chimeric molecule and its acceptor, the amount of fluorescence of detection will descend.

In order to screen protein and respective ligand/acceptor thereof and a kind of a plurality of interactions that suppress between the compound, need to use the ability of FACS instrumental analysis scattering signatures.The ball of larger diameter will have the scattering signatures different with less ball, and can be separated and be used for analyzing (" gating ").

The protein that some are different, wherein a kind of is protein of the present invention or chimeric molecule, all is linked on the ball with special diameter.In containing a kind of ball mixed solution of candidate compound, add above-mentioned proteinic ligand/receptor mixture subsequently.Subsequently with the one species specific fluorescently-labeled two anti-bonded ligand/receptor that detects.Antibody is identical detection fluorophor on the mark all.Measure the ability that compound stops its ligand/receptor of protein bound by operation sample on the FACS instrument and to the ball gating of each known dimensions subsequently.Analyze independent each subsequently respectively in conjunction with the result.The main benefit of this analytical procedure is to have reduced screening time accordingly with each compound of the parallel detection screening of some protein.

Protein or its chimeric molecule can also characterize by its crystal result.The biochemical form of protein or its chimeric molecule can provide 3 unique dimension crystalline structure.In addition, can also use protein of the present invention or chimeric molecule to produce the crystalline structure of protein one ligand/receptor mixture.Because the invention provides and similar basically protein of the natural existence form of people or its chimeric molecule, this mixture might be the more suitably representative of the body inner structure of naturally occurring protein one ligand/receptor mixture.In case the acquisition crystalline structure just can determine that protein or its chimeric molecule and potential suppress the interaction between this interactional compound.

In case determine just can begin to carry out design and rational medicine process by the potential compound by high flux screening or from protein one ligand/receptor compound crystal structure.

Carrying out adopting some steps in the stand-in design usually according to compound with design specified characteristic.At first, determine crucial and/or important compound privileged site in the decision desired characteristic.For peptide, can finish above-mentioned work by the amino-acid residue in the systematic change peptide, for example replace each residue successively.The normal L-Ala that uses scans this peptide motif of refining.These parts or residue have constituted the reactive site of compound, and described reactive site is called as its pharmacophoric group.

In case found pharmacophoric group, use the data in a series of sources to come its structure of modeling according to its physical property, described physical property is stereochemistry, Cheng Jian, size and/or electric charge for example, and the data in described series source are spectroscopy techniques, X ray diffracting data and nucleus magnetic resonance for example.Can use computational analysis, similarity to do figure (electric charge of pharmacophoric group and/or volume-based model, rather than interatomic Cheng Jian) and other technology at this modeling process.

In a variant of this method, make the 3 d structure model of part and binding partners thereof.This for part and/or binding partners in conjunction with the time change conformation and be particularly useful, in the simulation thing, can allow modeling consider this problem.Can use modeling to produce interactional inhibitor takes place with linear order or a kind of 3 dimension conformations.

The template molecule that selection subsequently can be transplanted up the chemical group of simulation pharmacophoric group.Can select template molecule easily and transplant the chemical group get on, so stand-in are synthesized easily, might be acceptable on the pharmacology, and not degrade in vivo, keeping the biological activity of lead compound.Perhaps, when stand-in are based on peptide, can increase its rigidity by the peptide cyclisation is obtained further stability.Can screen by this method the stand-in of finding subsequently and observe them and whether have target property, perhaps they show the target property of much degree.Can further optimize or modify subsequently, enter in the body one or more final stand-in or clinical detection.

The target of rational drug design is to produce interested biologically active polypeptides or micromolecular analog, they and these polypeptide or small molecules interaction (for example agonist, antagonist, inhibitor or toughener), so that formation medicine, for example, activity or more stable polypeptide form are more arranged, perhaps, for example strengthen or disturb the function of polypeptide in vivo.See, for example Hodgson (Bio/Technology9:19-21,1991).In a process, at first by x ray crystallography, by microcomputer modelling or three-dimensional structure most typical, combine to determine protein of interest matter by several different methods.Can also obtain useful information by structural modeling based on homologous protein about polypeptide structure.The example of a rational drug design is that the exploitation hiv protease suppresses (Erickson et al. Science 249:527-533,1990).In addition, can scan evaluating objects molecule (wells, MethodsEnzymol 202:2699-2705,1991) by L-Ala.In this technology, an amino-acid residue is replaced by Ala, and determines that it is to the active influence of peptide.Each amino-acid residue of analyzing peptide is in this way determined the important area of peptide.

Also may isolate the target specific antibody, select, and resolve its crystalline structure subsequently by functional analysis.In principle, this method produces a drug core, can carry out follow-up medicinal design based on it.Might omit bak protein crystallography fully by having antibody function, that pharmacological activity is arranged to produce antiidiotypic antibody to one.Therefore the binding site of antiidiotypic antibody is expected for the analogue of original acceptor as the mirror image of mirror image.Therefore antiidiotypic antibody can be used to identify and isolated peptides from chemistry or biogenic peptide storehouse.Use the peptide of selecting as drug core subsequently.

On the one hand, protein of the present invention or chimeric molecule are used as immunogenic and produce antibody.The biochemical form of protein of the present invention or chimeric molecule can increase the antibody at protein or chimeric molecule; Antibody at the glycopeptide of protein of the present invention or chimeric molecule; Or directly in another translation in protein or its chimeric molecule or the antibody of the peptide of posttranslational modification.

Protein of the present invention or its chimeric molecule may have the epi-position of not accessible under the normal circumstances (but might exist) in the body.For example, might in receptor domain, there be a zone that under normal circumstances contacts with the part of a heteropleural acceptor.Can be with the monoclonal antibody that these epi-positions produce and the endogenous recipient intersection is replied.This antibody can be blocked a receptor element and another interaction, and therefore anti-stop signal conduction.This can use as treatment when overexpression cytokine or acceptor.When can also and not needing part just to produce signal at the acceptor overexpression, do not use antibody as treatment.

Antibody can also be used for detecting in the treatment disease level (for example, serum level is determined the transformation period) of protein or its chimeric molecule.

In addition, antibody can be used for diagnostic assay and whether have protein of the present invention or chimeric molecule in specific sample.

" antibody " form of ownership that comprises the antibody of being mentioned of being mentioned includes but not limited to: complete anti-(for example containing complete Fc zone), comprise for example monoclonal antibody; Antigen binding antibody fragment comprises for example Fv, Fab, Fab ' and F (ab ') ₂Fragment; Humanized antibody; Human antibodies (for example, transgenic animal or by biting the antibody that mattress body display technology produces); With the immunoglobulin (Ig) derived peptide that produces by genetic engineering technique.With other refer in particular to different, term " antibody " and comprise complete anti-as described here and its Fab.

Unless otherwise noted, the present invention represents this antibody basically only in conjunction with its target antigen about the specificity of antibody, not irrelevant proteic appreciable combination.But, might will be designed or be chosen as by an antibody in conjunction with two or more related proteins.Related protein comprises different same protein or comes from the splice variant or the fragment of the homologous protein of different plant species.This antibody is considered to that also those protein are had specificity, and is included among the present invention.Term " basically " means the detectable combination that non-target antigen is not higher than the substrate level under this background, and is promptly non-specific.

Can prepare antibody of the present invention by the method for knowing.Referring to, for example, people (eds.), Plenum Press, New York (1980) such as MonoclonalAntibodies, Hybridomas:A New Dimension in Biological Analvses, Kcnnet; With ask month Antibodies:A Laboratory Manual, Harlow and Lane (eds.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY, (1988).

A method that produces antibody of the present invention (for example comprises protein of the present invention producing with animal or chimeric molecule or its immunogenicity part, a peptide that comprises the receptors bind structural domain, antibody comes in conjunction with polypeptide in protein or its chimeric molecule or immunogenicity part by the receptors bind structural domain) immunity a non-human animal, for example a mouse or a transgenic mice.Have several different methods to increase the antigenicity of specified protein or its chimeric molecule, for example give adjuvant or conjugated antigen, comprise antigen and another element at the response of required antibody, these methods are all known in the art and be to adopt.Typical immunization comprises initial immunity and a series of enhancing immunity subsequently.Can get antibody titers in blood and the serum analysis to animal.Can strengthen animal, up to the plateau that reaches titre.Can in the reconstitution cell culture, merge the preparation conjugant with protein.Equally, aggregating agent prepared therefrom for example alum be applicable to enhancing immunity response.

Polyclone and monoclonal antibody can be by this method preparations.The method that obtains two types of antibody is well known in the art.Polyclonal antibody uses less but relatively easy preparation, partly inject suitable animal with the protein of the present invention of effective quantity or chimeric molecule or its immunogenicity, from animal, collect serum and use any known immunoadsorbent technics to separate specific antibody at protein or its chimeric molecule.Less relatively by the antibody use that this method produces, because there is the potential unhomogeneity in product.

The reason of using monoclonal antibody significantly to be had a preference for is that they can mass production, and product has homogeneity.Can pass through ordinary method manufacture order clonal antibody.

The term of Shi Yonging " monoclonal antibody " is meant the antibody that obtains from a large amount of homogeneous antibodies herein, that is, and and the independent antibody of forming by identical colony except the sudden change of the possible natural generation of a small amount of existence.Monoclonal antibody is a high specific, directly at single antigen site.In addition, with the polyclonal antibody preparation difference that typically comprises at the different antibodies of different antigenic determinants (epi-position), each monoclonal antibody is directly at single antigenic determinant on the antigen.The feature that modifier " mono-clonal " is meant antibody obtains from abundant homologous antibody colony, rather than needing to be interpreted as by any specific method generation antibody.For example, can prepare by the hybridoma method that people Nature 256:495 (1975) such as Kohler at first announce, maybe can prepare (seeing that for example U.S. Patent number 4,816,567) by recombinant DNA method according to monoclonal antibody used in the present invention." monoclonal antibody " can also separate from bite mattress body antibody library, uses for example people Nature 352:624-628 such as Clackson, and 1991 and people J Mol Biol 222:581-597 such as Marks, 1991 described methods.

The present invention has imagined a kind of method that produces a kind of hybridoma cell line, comprises with protein of the present invention or chimeric molecule immunity a kind of non-human animal, for example mouse or transgenic mice; From the animal of immunity, gather in the crops splenocyte; The splenocyte and the myeloma cell line of results are merged and produce hybridoma; And identify producing can be in conjunction with the hybridoma cell line of the monoclonal antibody of a kind of protein or its chimeric molecule.

The monoclonal antibody that this hybridoma cell line and they produce is included among the present invention.By routine techniques purifying hybridoma cell line excretory monoclonal antibody.The monoclonal antibody that can further screen hybridoma or their generations identifies the monoclonal antibody that has required particular characteristics, for example suppresses the ability of its signal by cytokine receptor.

Can be used for the protein of immune animal or its chimeric molecule or its immunogenicity part in the initial period of producing antibody of the present invention should come from the people and express the source.

Can produce antigen-binding fragments of antibodies of the present invention by ordinary method.This segmental example includes but not limited to: Fab, Fab ', F (ab ') ₂With the Fv fragment, comprise strand Fv fragment (called after sFv or scFv).By antibody fragment and derivative that genetic engineering produces, for example stable Fv fragment (dsFv), the strand variable region structural domain (Abs) of curing according to the present invention, also imagined miniantibody and binary and has been used for using.

Can prepare thisly directly at the fragment and the derivative of the monoclonal antibody of protein or its chimeric molecule and screen desired characteristic by known technology, described technology comprises detection described herein.These detections provide the fragment of the antibody of the present invention of identifying energy conjugated protein or its chimeric molecule and the method for derivative, and can identify that those also keep the active fragment and the derivative of the signal of arrestin matter or its chimeric molecule.These technology must comprise the DNA of the polypeptide chain (or its part) among the interested mAb of separation coding and operate these DNA by recombinant DNA technology.Can another interested DNA goes up or other method (for example by mutagenesis or other routine techniques) increases, deletes or replace one or more amino-acid residues by DNA is fused to.

Can be from the DNA that separates the encoding antibody polypeptide the B cell of protein of the present invention or chimeric molecule mice immunized (for example heavy or light chain, only variable region or total length).Can use the conventional procedure DNA isolation.Phage display is the example of another known technology, can prepare the derivative of antibody by it.In one approach, in any suitable recombinant expression system, express polypeptide, and polypeptide expressed can assembled formation antibody molecule as an a kind of part of antibody interested.

Can heavy chain and variable region of light chain (Fv district) fragment be coupled together the formation single-chain antibody by amino acid bridge (small peptide linker), form a single polypeptide chain.Prepare this strand Fvs (scFvs) by the DNA that between the DNA of two variable region polypeptide of coding (VL and VH), merges the coded polypeptide linker.According to the length that connects son flexibly between two variable domains, the gained antibody fragment capable forms dimer or tripolymer people Protein such as Engineering 10:423 such as (, 1997) Kortt.The technology of the manufacture order chain antibody that development is come out comprises United States Patent (USP) 4,946,778; The described technology of people (Nature334:544,1989) such as people such as Bird (Science 242:423,1988), Huston (Proc Natl Scad Sci USA 85:5879,1988) and ward.The single-chain antibody that derives from antibody provided herein is included in the present invention.

In one embodiment, the invention provides can be in conjunction with antibody fragment or chimeric molecule, recon or the synthesized form of the antibody of the signal of protein of the present invention or chimeric molecule and arrestin matter or its chimeric molecule.

Known technology can be obtained the antibody of different subclass or isotype from interested antibody, that is, and and the subclass conversion.Therefore, for example can obtain IgG1 or IgG4 monoclonal antibody from the IgM monoclonal antibody, vice versa.This technology can produce the new antibodies that has the antigen binding characteristic of specifying antibody (female antibody), but also shows the biological characteristics that antibody isotype or subclass had different with female antibody.Can adopt recombinant DNA technology.In this process, can use the cloned DNA of coding specific antibodies polypeptide, the DNA of the constant region of the required isotype antibody of for example encoding.

Can use above-mentioned mono-clonal production process in animal, for example mouse comes the production monoclonal antibody.The conventional antibodies that obtains from these animals, for example mouse antibodies is considered to not be suitable for human body usually, because they can cause immunne response.Therefore, this antibody may need modification to be applicable to human body.The process of preparation chimeric molecule and/or humanized antibody is known in the art, will be described in further detail below.

Monoclonal antibody herein is particularly including " chimeric " antibody, identical or the homology of corresponding sequence of heavy chain and/or light chain variable structural domain and the antibody that comes from inhuman species (for example mouse) wherein, and the identical or homology of corresponding sequence of rest part in the chain and the antibody that comes from human body, the fragment of this antibody also is like this, so they show required biologic activity (United States Patent (USP) 4,816,567; With father Morrison people .Proc Natl Acad Sci USA81:6851-6855,1984).

" humanization " form of inhuman (for example mouse) antibody is a chimeric antibody, wherein contains a spot of sequence that comes from non-human immunoglobulin.For most part, humanized antibody is human normal immunoglobulin (recipient's antibody), wherein the complementary determining region of recipient's antibody (CDRs) is replaced by inhuman species (donor antibody) corresponding C DRs, and described inhuman species for example have mouse, rat, rabbit or the non-human primate of desired characteristic (for example specificity and affinity).In some instances, the framework region residue of people's immune globulin is replaced by corresponding inhuman residue.And humanized antibody can be included in undiscovered residue in receptor antibody or the donor antibody.Carry out the performance that these modify further refining antibody.In a word, humanized antibody will comprise all at least one basically, and the typical case is two a variable domains, and wherein all or all substantially complementary determining regions are corresponding to non-human immunoglobulin, and all or all substantially framework region residues are the human normal immunoglobulin sequences.Humanized antibody also will optionally contain the constant region for immunoglobulin (Fc) of at least a portion, be typically human normal immunoglobulin.Further details people Nature 332:323-329,1988 such as people Nature 321:522-525,1986:Reichmann such as has referring to Jones; Presta, Curr Op Struct Biol 2:593-596,1992; People Proc Natl Acad Sci USA 84:3439,1987 such as Liu; People Bio/Technology 7:934,1989 such as Larrick; With Winter and Harris, TIPS 14:139,1993.

In a further embodiment, the invention provides immunity detection reagent, can detect the level of the expressed human protein of people's cell in a kind of biological products, described biological products comprise and comprise the proteic biological products of natural generation people.

The biological products that can use immunity detection reagent to detect of the present invention include but not limited to laboratory sample, cell, tissue, blood, serum, blood plasma, urine, ight soil, saliva and phlegm.

Immunity detection reagent of the present invention comprises a kind of solid phase supported matrix, is not limited to but comprises microtest plate at the bottom of a kind of film, dipstick, pearl, gel, pipe or porous, flat, round bottom or the v shape, for example 96 orifice plates; Direct antibody preparation (capture antibody) at interested human protein; Bag is by pharmaceutical solutions (for example BSA or casein); Two anti-preparations (detection antibody), directly at interested human protein and with suitable detection molecules (for example alkaline phosphatase) conjugation; A kind of chromogenic substrate solution (for example nitro blue tetrazolium); A kind of interpolation substrate solution (for example 5-bromo-4-chloro-3-indoles phosphoric acid salt); A kind of substrate buffer solution mother liquor (for example 0.1M Tris-HCL (pH7.5) and 0.1M NaCl, 50mM MgCl ₂); The protein of the present invention of concentration known (standard) or the preparation of chimeric molecule; And working instructions.

Can select suitable detection molecules to be attached to from the tabulation that comprises reagent such as enzyme, dyestuff, fluorescence molecule, chemoluminescence agent, isotropic substance or Radioactive colloidal gold includes, but is not limited on SP or the streptococcal protein G equimolecular.

In a specific embodiments, catching and detect antibody is monoclonal antibody, and its preparation comprises with protein of the present invention or chimeric molecule immunity non-human animal, and for example mouse or transgenic mice as indicated abovely subsequently carry out standard method.The method production with reorganization of all right alternative of monoclonal antibody, as indicated above, and can comprise people or chimeric antibody part or structural domain.

In another embodiment, catching and detect antibody is polyclonal antibody, and its preparation comprises with protein of the present invention or chimeric molecule immunity non-human animal, and for example mouse or rabbit, sheep or horse as indicated abovely subsequently carry out standard method.

The assembly of detection kit provides by predetermined proportion, and the reagent concentration in the solution is kept in the variation that the relative populations of different reagent is suitable, allows detection sensitivity maximize basically.Especially, can provide reagent in the mode of dry powder, normally freeze dried, comprise auxiliary material, each reagent solution of dissolving relief all has suitable concentration for biological products to be measured.

Working instructions can be introduced the using method of immunity detection reagent of the present invention in detail.For example, the capture antibody solution that working instructions can be introduced with preparation wraps under suitable concentration by the method for solid phase supported matrix, and for example 4 ℃ are spent the night.Working instructions can be described in further detail with the preparation bag by the solution bag by the method for nonspecific proteins matter binding site; (for example 37 ℃ 1 hour or room temperature 2 hours) adds the sample that contains protein of the present invention or chimeric molecule of serial dilution and hatches under conditions suitable, use suitable damping fluid known in the art to carry out series subsequently and clean, for example contain 0.1MPBs (pH7.2) solution of 0.05% polysorbas20.In addition, specification sheets can provide, and hatches under conditions suitable behind the applying detection antibody preparation, and for example, 37 ℃ of 1 hour or room temperatures 2 hours are carried out a series of cleanings subsequently.The preparation of the detection substrate that utilization provides and substrate buffer solution detects the working solution of damping fluid, adds subsequently in each hole, remains under the appropriate condition, from room temperature 5 minutes to 37 ℃ 1 hour.Can be by adding 1N NaOH or 2N H ₂SO ₄Stop producing colour response.

In another alternate embodiment, working instructions can provide any combination of any or all said components to be added that can be simultaneously adds in the reservation ratio, the reagent concentration in the solution is kept in the suitable variation of relative populations of different reagent, allows mixture form the detectable signal that is produced and maximizes basically.

Can use dull and stereotyped reader of ELISA or spectrophotometer, under suitable optical density(OD) (OD), or as exciting light, use spectrophotometer, photofluorometer or flow cytometer, under suitable wavelengths, or use radioactive counter, under suitable power spectrum, or by photodensitometer, or, detect coloured product or fluorescence or chemoluminescence or radioactivity or other level by the signal of combination, the generation of conjugation joint detection reagent by carrying out visual comparison with chart or index.Serial dilution solution with the parallel examination criteria preparation of above-mentioned sample.Produce typical curve or chart, according to the level of protein or its chimeric molecule in typical curve or chart interpolation (interpolated) calculation sample.

The present invention also provides people's derived protein or its chimeric molecule, as the standard protein in the immunodetection.The present invention also further expands to the method for the level of a kind of people's albumen that is determined at people's cell expressing in the biological products or its chimeric molecule, comprise the suitable mensuration people's albumen or the assay method of chimeric molecule, this detection method comprises that (a) directly combines biological products and one or more at the antibody of people's albumen or its chimeric molecule, (b) measures the level of people's albumen in this antibody or each the antibodies biological products or chimeric molecule; (c) with normal man's albumen or a kind of chimeric molecule sample and between one or more antibody at people's albumen or chimeric molecule combine; (d) measure the level in conjunction with people's albumen or chimeric molecule in the biological products of this antibody or each antibody; (e) level of the level of people's albumen or chimeric molecule and this antibody or each antibodies normal man albumen or chimeric molecule sample in this antibody or each the antibodies biological products relatively:

Especially, normal man's albumen or chimeric molecule sample are a kind of preparations that comprises protein of the present invention or chimeric molecule.

Biological products include but not limited to laboratory sample, cell, tissue, blood, serum, blood plasma, urine, ight soil, saliva and phlegm.As indicated above or by methods known in the art, biological products can be in conjunction with one or more capture antibodies.For example, at first under appropriate condition, wrap by solid phase supported matrix (for example, 4 ℃ are spent the night) with the capture antibody solution for preparing; Sealed the binding site of nonspecific proteins matter subsequently by solution with the bag of preparation; Add subsequently and contain the series of diluted samples of protein of the present invention or chimeric molecule and under appropriate condition, to hatch (for example 37 ℃ 1 hour or room temperature 2 hours), use suitable damping fluid known in the art to carry out series subsequently and clean (0.1MPBS (pH7.2) solution that for example contains 0.05% polysorbas20).

Subsequently, biological products and one or more detection antibody that are combined with suitable as described here detection molecules combine.For example, under conditions suitable, hatch (for example, 37 ℃ 1 hour or room temperature 2 hours) behind the applying detection antibody preparation, carry out a series of cleanings subsequently.

Can be as indicated above or measure in conjunction with level by methods known in the art.For example, utilize to detect the working solution that the preparation of substrate and substrate buffer solution detects damping fluid, add subsequently in each hole, remain under the appropriate condition, from room temperature 5 minutes to 37 ℃ 1 hour.Can be by adding 1N NaOH or 2N H ₂SO ₄Stop producing colour response.

In a specific embodiments, the present invention's imagination as indicated above know clearly isolating protein or chimeric molecule.

In a specific embodiment, with physical and chemical parameter (P _x) and pharmacological characteristics (T _x) feature characterize IFN-a2b of the present invention, it comprises

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

Iso-electric point pI (P ₂) be 2-14, as 2,3,4,5,6,7,8,9,10,11,12,13,14, and in a specific embodiments, be 4.5-7; Have

About 2-100 isoform (isoform), as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,4O, 41,42,43,44,45,46,47,48,49,5051,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,9O, 91,92,93,94,95,96,97,98,99,100 isoforms, in a specific embodiments, isoform number (P ₃) be 2-22.

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

Contents of monosaccharides (P ₉) and sialic acid content (P ₁₀) be standard with galn (GalNAc), be respectively 1: the 0-3 trehalose, 1: 0-3 n acetylglucosamine n (GlcNAc), 1: the 0-6 semi-lactosi, 1: 0-3 seminose and 1: 0-5N-n acetylneuraminic acid n (NeuNAc), in specific embodiments, it is 1: the 0-1 trehalose, 1: 0-1 n acetylglucosamine n (GlcNAc), 1: 14 semi-lactosi, 1: 0-1 seminose and 1: 0-2N-n acetylneuraminic acid n (NeuNAc).

Sialic acid (P ₁₀) be expressed as the per-cent of the contents of monosaccharides among the IFN alpha 2b of the present invention, be 0 to 50%, as O, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,3O, 31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50%, in a specific embodiments, be 0-10%.

O-connects the neutral per-cent (P of oligosaccharides ₁₅) approximately be 60%-100%, for example: 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, P ₁₅Should be 80%-100%.

O-connects the acid per-cent (P of oligosaccharides ₁₆) approximately be 0-4O%, for example: 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,2O, 21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40%, in a specific embodiment, P ₁₆Should be 0-20%.

With the different biological activity of people IFN-a2b that inhuman cell system is expressed, in a specific embodiment, IFN-a2b of the present invention suppresses GM-CSF inductive TF-1 cell proliferation (T ₃₂) ability be the people IFN-a2b that expresses in the E.coli cell 250-600 doubly.

In a specific embodiment, with physical and chemical parameter (P _x) and pharmacological characteristics (T _y) feature characterize IFN-b1 of the present invention, it comprises

Apparent molecular weight (the P of 1-250kD ₁), as: 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,1O0,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250kDa, in a specific embodiment, be 15 to 40kDa.

PI (P ₂) scope be about 2 to 14, for example: 2,3,4,5,6,7,8,9,10,11,12,13,14.

About 2-100 isoform (P ₃), for example 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,2O, 21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,6O, 61,62,63,64,65,66,67,68,69,7O, 71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100 isoforms, in a specific embodiment, be the 1-50 isoform.

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

In a specific embodiment, with physical and chemical parameter (P _x) and pharmacological characteristics (T _y) feature characterize IFN-g of the present invention, it comprises

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

PI (P ₂) scope be about 2 to 14, for example: 2,3,4,5,6,7,8,9,10,11,12,13,14, in a specific embodiment, be 4-14.

About 2-100 isoform (P ₃), for example 2,3,4,5,6,7,8,9,1O, 11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100 isoforms, in a specific embodiment, be the 4-16 isoform.

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

N-connects the actual measurement molecular weight (P after the oligosaccharides de-glycosylation ₆) be approximately 10-25kDa, in a specific embodiment, be 12-20kDa.

Actual measurement molecular weight (P after the oligosaccharides de-glycosylation that is connected with O-that N-connects ₇) be approximately 10-25kDa, in a specific embodiment, be 12-20kDa.

PNGase handles the N-glycosylation (P that identify by PMF the back ₂₁) the site, comprise N-48 and N-120 (by signal sequence end numbering);

With the different biological activity of people IFN-g that the non-human cell system is expressed, in a specific embodiment, in the presence of TNF-a, IFN-g of the present invention suppresses HT-29 cell proliferation (T ₃₂) ability be the people IFN-g that expresses in the E.coli cell 11-17 doubly.

In a specific embodiment, with physical and chemical parameter (P _x) and pharmacological characteristics (T _y) feature characterize worker FNAR2-Fc of the present invention, it comprises

Apparent molecular weight (the P of 1-250kD ₁),

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

PI (P ₂) scope be about 2 to 14, for example: 2,3,4,5,6,7,8,9,10,11,12,13,14, in a specific embodiment, be 4-7.

About 2-100 isoform (P ₃), for example 2,3,4,5,6,7,8,9,10,11, l2,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,5O51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100 isoforms, in a specific embodiment, be the 10-25 isoform.

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

Actual measurement molecular weight (P after the oligosaccharides de-glycosylation that N-connects ₆) being approximately 40-l00kDa, a concrete scope is 45-95kDa.

The molecular weight that actual measurement molecular weight (P7) after the oligosaccharides de-glycosylation that is connected with O-that N-connects records is approximately 40-90kDa, in a specific embodiment, is 45-80kDa.

In a specific embodiment, with physical and chemical parameter (P _x) and pharmacological characteristics (T _y) feature characterize IL-10 of the present invention, it comprises

Apparent molecular weight (the P of 1-250kD ₁), as: 1,2,3,4,5,6,7,8,9,10,11, l2,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250kDa, in a specific embodiment, be 10 to 23kDa.

PI (P ₂) scope be about 2 to 14, for example: 2,3,4,5,6,7,8,9,10,11,12,13,14, in a specific embodiment, be 6-10.

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

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

N-connects the actual measurement molecular weight (P after the oligosaccharides de-glycosylation ₄) be approximately 8-23kDa, in a specific embodiment, be 10-23kDa.

Actual measurement molecular weight (P after the oligosaccharides de-glycosylation that is connected with O-that N-connects ₇) be approximately 8-23kDa, in a specific embodiment, be 10-23kDa.

Immunoreactivity characteristics (T ₁₃) different with the people IL-10 of inhuman cell system expression, in a specific embodiment, if the IL-10 protein concentration adopts the enzyme-linked immunosorbent assay ELISA test kit of the people IL-10 that contains the E.coli cell expressing to carry out assay among the present invention, the content that then records is on the low side;

Biological activity is different with the people IL-10 that inhuman cell system is expressed.IL-10 of the present invention under the condition that IL-4 exists, induce the ability of MC/9 cell proliferation be ugly coli cell expressing people IL-10 10-25 doubly.

In a specific embodiment, with physical and chemical parameter (P _x) and pharmacological characteristics (T _y) feature characterize IL-10Ra-Fc of the present invention, it comprises

Apparent molecular weight (the P of 1-250KD ₁), 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, be 50 to 100kDa.

PI (P ₂) scope be about 2-14, for example: 2,3,4,5,6,7,8,9,10,11,12,13,14, in a specific embodiment, be 4.5-9.5.

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

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

Actual measurement molecular weight (P after the oligosaccharides de-glycosylation that N-connects ₆) be approximately 35-95kDa, in a specific embodiment, be 40-85kDa.

Actual measurement molecular weight (P after the oligosaccharides de-glycosylation that is connected with O-that N-connects ₇) molecular weight that records is approximately 30-95kDa, in a specific embodiment, is 36-85kDa.

Monose (P ₉) and sialic acid (P ₁₀) content, when being standard with galn (GalNAc), 1: 0.1-4 trehalose, 1: 2-34 acetylglucosamine (GlcNAc), 1: the 0.5-8 semi-lactosi, 1: 1-13 seminose and 1: 0-3 N-n acetylneuraminic acid n (NeuNAc), when being standard with 3 times of seminoses, 3: 0.1-2 trehalose, 3: 0.01-3 N-acetylgalactosamine (GalNAc), 3: 1-30 n acetylglucosamine n (GlcNAc), 3: 0.1-4 semi-lactosi and 3: 0-3N-n acetylneuraminic acid n (NeuNAc).

Sialic acid content (P ₁₀) be expressed as the per-cent of contents of monosaccharides among IL-10R α-Fc, scope is 0-50%, for example: 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 0-10%.

Vitriol and phosphate content (P ₁₁), when being standardized as GalNac, the vitriol scope is 1: 0-3 in a specific embodiment, is 1: 0-1.5.When being standard with 3 times of seminoses, the vitriol scope is 3: 0-1 in a specific embodiment, is 3: 0-0.6.

Sulfuration (P ₅₉) be expressed as molecule contents of monosaccharides per-cent, be 0-50%, 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50, in a specific embodiment for example:, be 0-3%.

N-connects the neutral per-cent (P of oligosaccharides ₁₃) approximately be 40%-85%, for example: 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%, in a specific embodiment, be 55%-75%.

N-connects the acid per-cent (P of oligosaccharides ₁₄) approximately be 15-60%, for example: 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%, in a specific embodiment, be 25-45%.

PNGase handles the N-glycosylation (P that identify by PMF the back ₂₁) the site, comprise N-110, N-154, N-177 and N-323 (by signal sequence end numbering); The different biological activity of expressing with inhuman cell system of people IL-10Ra-Fc, in a specific embodiment, IL-10Ra-Fc of the present invention under the condition that IL-4 exists, in and IL-10 inductive MC/9 (T ₃₂) ability of cell proliferation be the soluble human IL-10Ra molecule of expressing in the E.coli cell 18-150 doubly.

In one embodiment, protein of the present invention or chimeric molecule are at N-crosslink part (P ₁₉) in contain at least one following structure.In these charts, " u " or the different header structure of " " expression be a or b, and/or crosslinked position is 2,3,4 and/or 6.

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)] GicNAc+ "+3xGal (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+ "+3xGal (1-) GlcNAc (1-)+Fuc (1-) "

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-) [Gal (b1-4) G1cNAc (b1-2) [Gal (b1-4) GlcNAc (b1-6)] Man (a1-)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ "+3xGal (b1-4) GlcNAc (b1-3) "

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

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-) [Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) G1cNAc (b1-6)] Man (a1-)] Man (b1-4) G1cNAc (b1-4) GlcNAc+ "+3 x Gal (b1-4) G1cNAc (b1-3)+Gal (b1-3) GlcNAc (b1-3) "

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

Glycan structures Gal (1-) GlcNAc (1-) [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 x 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)] G1cNAc+ "+4xGal (1-) GlcNAc (1-)+Fuc (1-) "

Glycan structures Gal (1-) GlcNAc (1-) [Gal (1-) GlcNAc (1-)] Man (a1-3) [Gal (1-) GlcNAc (1-) [Gal (1-) G1cNAc (1-)] Man (a1-6)] [GlcNAc (1-4)] Man (b1-4) GlcNAc (b1-4) [Fuc (1-6)] GlcNAc+ "+5xGal (1-) GlcNAc (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 "

Glycan structures NeuAc (a2-) Gal (b1-4) (GlcNAc (b1-3) Gal (b1-4)) jGlcNAc (b1-2) Man (a1-) [Gal (b1-4) fGlcNAc (b1-3) Gal (b1-4)) kGlcNAc (b1-2) Man (a1-)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ " Where j+k=14 ﹠j, k 〉=13 "

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

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

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

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

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

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

Glycan structures NeuAc (a2-) Gal (b1-4) (GlcNAc (b1-3) Gal (b1-4)) kGlcNAc (b1-2) Man (a1-3) [NeuAc (a2-) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4)) and jGlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ " Where j+k=14 ﹠amp; J, k 〉=1 "

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

Glycan structures NeuAc (a2-) Gal (b1-4) (GlcNAc (b1-3) Gal (b1-4)) jGlcNAc (b1-2) Man (a1-) [Gal (b1-4) fGlcNAc (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 ﹠amp; J, k 〉=1 "

Glycan structures NeuAc (a2-) Ga1 (b1-4) (GlcNAc (b1-3) Gal (b1-4)) kGlcNAc (b1-2) Man (a1-3) [NeuAc (a2-) Gal (b1-4) (GlcNAc (b1-3) Gal (b1-4)) jGlcNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ " Where j+k=14 ﹠ j, k 〉=1 "

Glycan structures GlcSAc (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

Weary sugared structure GlcNAc (b1-2) Man (a1-3) [GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc of bridle

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

Weary sugared structure GlcNAc (b1-2) Man (a1-6) Man (b1-4) GlcNAc (b1-4) [Fuc (the a1-6)] GlcNAc of bridle

Han a1---3 Han a1---6 Han b1---4 GlcNRc b1---, 4 GlcNRc glycan structures Man (a1-3) Man (a1-6) Man (b1-4) GlcNAc (b1-4) GlcNAc

NeuHc a2---u Gal b1---4 GlcNHcb1---2 Hdn a1---3 Hdn b1---, 4 GlcNHc glycan structures NeuAc (a2-) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) Man (b1-4) GlcNAC

Luo Liang sugar structure HSO3 (4) GalNAc (b1-4) GlcNAc (b1-2) Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

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

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

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

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

Glycan structures GlcNAc (b1-2) [GlcNAc (b1-4)] gan (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) G1cNAc

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

Luo Chen sugar structure Gal (b1-4) GlcNAc (b1-2) Man (a1-6) [Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

Weary sugared structure Gal (b1-4) GlcNAa (b1-2) Man (a1-3) [Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (the a1-6)] GlcNAc of bridle

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) G1cNAc (b1-4) GlcNAc

Glycan structures Gal (b1-4) GlcSAc (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) GlcSAc (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) G1cNAc (b1-2) Man (a1-3) [G1cNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc

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

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

Glycan structures NeuAc (a2-6) GalNAc (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) G1cNAc (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

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

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) G1cNAc (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) GlcNhc (b1-4) [Fuc (a1-6)] GlcSAc

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) GlclqAc (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) G1cNAc (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) GlclqAc (b1-4) [Fuc (a1-6)] GlcNAc

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

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

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

Glycan structures NeuAc (a2-6) Gal (b1-4) GlcNAc (b1-2) Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] [G1cNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc

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) GleNAc (b1-2) Man (a1-6)] [GlcNAc (b1-4)] Man (b1-4) GlcNAc (b1-4) GlcNAc

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

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

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

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

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

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

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

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

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

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

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

Glycan structures Ga-1 (b1-4) GlcNAc (b1-2) Man (a1-3) [Man (a1-3) [Man (a1-6)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc

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

Glycan structures Gal (bl-4) GlcNAc (b1-2) [Gal (b1-4) G1cNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ "+Fuc (a1-3) "

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

Weary sugared structure 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 of heap of stone

Weary sugared structure 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 of bridle

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

+3x NeuRc(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 x NeuAc (a2-) "

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

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

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

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

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [Gal (b1-4) G1cNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ "+Fuc (a1-2) "

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

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

Glycan structures NeuAc (a2-6) Gal (b1-4) G1cNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

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

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

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

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

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

Glycan structures NeuAc (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

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

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

Glycan structures Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) G1cNAc (b1-4)] Man (a1-3) [Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc+ "+2xHSO3 (6)+2xNeuAc (a2-3)+NeuAc (a2-6) "

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

Glycan structures Ga1l (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+ "+Gal (b1-2) GlcNAc (b1-3)+3xNeuAc "

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

Glycan structures 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) "

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)+2xNeuAc (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

Weary sugared structure 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) GlcNAe (b1-4) GlcNAc of bridle

Weary sugared structure 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+ "+3xNeuAc (a2-) " of bridle

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) GlcSAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc

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

Another name for Sichuan Province lacks sugared structure NeuAc (a2-) Gal (b1-4) GlcNAc (b1-2) [NeuAc (a2-) Gal (b1-4) GlcNAc (b1-4)] Man (a1-3) [NeuAc (a2-) Gal (b1-4) GlcNAc (b1-2) [NeuAc (a2-) Gal (b1-4) GlcNAc (b1-6)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

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

Easily weary sugared structure Gal (b1-4) GlcNAc (1-) [Gal (b1-4) GlcSAc (1-)] Man (a1-3) [Gal (b1-4) GlcNAc (1-) [Gal (b1-4) GlcNAc (1-)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ "+2xFuc "

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

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

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

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

Weary sugared structure 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 (the a1-6)] GlcNAc of scolding

Sieve lacks sugared structure Gal (a1-3) Gal (b1-4) GlcNAc (b1-4) [NeuAc (a2-) Gal (b1-4) GlcNAc (b1-2)] Man (a1-3) [Gal (a1-3) Gal (b1-4) GlcNAc (b1-2) Man (a1-6)] Man (b1-4) GlcNAc (b1-4) [Fuc (a1-6)] GlcNAc

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

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

Glycan structures Gal (b1-4) GlcNAc (b1-) Gal (b1-4) GlcNAc (b1-6) [Gal (b1-4) GlcNAc (b1-2)] Man (a1-6) [Gal (b1-4) GlcNAc (b1-2) [Gal (b1-4) GlcNAc (b1-4)] Man (a1-3)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ "+5xNeuAc (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)] GlcNAc+ "+Gal (b1-4) GlcNAc (b1-3) "

Glycan structures Gal (b1-4) GlcNAc (1-) [Gal (b1-4) GlcNAc (1-)] Man (a1-3) [Gal (b1-4) GlcNAc (1-) [Gal (b1-4) GlcNAc (1-)] Man (a1-6)] Man (b1-4) GlcNAc (b1-4) GlcNAc+ "+2xFuc+Gal (b1-4) GlcNAc (1-) "

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

In one embodiment, protein of the present invention or chimeric molecule comprise a following structure (P at least in the O-crosslink part ₂₀).In these charts, the different header structure of " u " or " " expression is a or b, and/or crosslinked position is 2,3,4 and/or 6.

rUe

Glycan structures Fuc

GlcuIu——Fuc

Glycan structures Glc (1-) Fuc

GlcNAc

Glycan structures GlcNAc

GalNAc

Glycan structures GalNAc

NeuAc a2—6 GaINAc

Glycan structures NeuAc (a2-6) GalNAc

GlcNAcb1—3 GalNAc

Glycan structures GlcNAc (b1-3) GalNAc

Luo Liang sugar structure GlcNAc (b1-3) [NeuAc (a2-6)] GalNAc

Galb1—3 GalNAc

Glycan structures Gal (b1-3) GalNAc

Gdl

Glycan structures Gal

NeuAca2——3 Gal

Glycan structures NeuAc (a2-3) Gal

Xyl u1—u Glc

Glycan structures Xyl (1-) Glc

NeuAc a2—3Gal b1—4 Xyl

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

Xyl ul-u Glc

Glycan structures Xyl (1-) Glc

Xgl ul-u Glc

+ Xyl

Glycan structures Xyl (1-) G1c+ "+xyl "

HeulAc a2-3 Gal b1 3 GalNAc

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

Fuo a1-2Gal b1-3 GalNAc

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

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

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

delta4，5GlcAbl-3 GalHAcb1-4 GlCltbl-3Galbi-3Gab1-4Xyl

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

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

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

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

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

Glycan structures Fuc (a1-4) GlcNAc (b1-6) [GlcNAc (b1-6) Ga ((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 al-2Gal bl-3GlcNAb1-3GalNAc

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

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

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

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

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

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

(Gal bl—4 GlcNAcb1—4 GlcNAcb1—3 Gal b1—3 GalHAc

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

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

Weary sugared structure GalNAc (b1-4) [NeuAc (a2-3)] Gal (b1-3) [NeuAc (the a2-6)] GalNAc of bridle

HeuAc u2—u Gal u1—u GalAcu1—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 (b-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc

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

NeuAc a2—u Galb1—u GlcNAu1—u Gal u1—u GalNAc

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

Practise weary sugared structure NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-6) [NeuAc (a2-3) Gal (bl-3)] GalNAc

Brave weary sugared structure 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)] GlcSAc (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

Weary sugared structure Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-6) [NeuAc (a2-3) Gal (the b1-3)] GalNAc of bridle

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

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

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

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

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

Glycan structures Gal (b1-4) GlcINAc (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 (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) GlcSAc (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) GlcSAc (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)] Ga1NAc

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) G1cNAc (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

Glycan structures NeuAc (2-3) Gal (1-) GlcNAc (1-3) Gal (1-3) [Gal (1-4) GlcNAc (1-6)] GalNAc+ "+Fuc.″

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

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

Weary sugared structure Fuc (1-) Gal (1-) [Fuc (1-)] GlcNAc (1-) Gal (1-) GlcNAc (1-) [NeuAc (2-) Gal (1-)] GalNAc of heap of stone

Glycan structures Gal (1-) GlcNAc (1-) Gal (1-) [Fuc (1-)] GlcNAc (1-) [NeuAc (2-) Gal (1-)] GalNAc+ "+Fuc "

Glycan structures Fuc (1-) Gal (1-) [Fuc (1-7)] GlcNAc (1-) Gal (1-) [Fuc (l-)] G1cNAc (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 (bl-6) [NeuAc (a2-3) Gal (b1-3)] GalNAc

Glycan knot Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-) Gal (b1-4) GlcNAc (b1-6) [NeuAc structure (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) GIcNAc (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

Weary sugared structure 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 (the b1-3)] GalNAc of bridle

Glycan structures NeuAc (a2-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Ga1 (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) Gl cNAc (b1-3) Gal (b1-3) Gal (b1-3) [Gal (b1-4) GlcNAc (b1-6)] GalNAc

Glycan structures Fuc (a1-2) [Gal (a1-3)] Gal (b1-) GlcNAc (b1-3) [Fuc (a1-2) [Gal (a1-3)] Gal (b1-) GlcNAc (b1-6)] Gal (b1-4) GlcNAc (b1-3) Gal (b1-3) Gal (b1-3) [Gal (b1-4) 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) G1cNAc (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) GlcSAc (b1-6)] GalNAc

Glycan structures NeuAc (a2-3) Gal (b1-4) [Fuc (a1-3)] GlcNAc (b1-3) Ga1 (b1-4) [Fuc (a1-3)] GlcNAc (b1-3) Gal (b1-4) [Fuc (a1-3)] G1cNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Gal (b-14) GlcNAc (b-13) Gal (b-14) GlcNAc (b-13) Gal (b-14) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [Gal (b1-3)] GalNAc

Glycan structures Gal (b1-4) G1cNAc (b1-3) Ga1 (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-3) Gal (b1-4) GlcNAc (b1-6) [Gal (b1-3)] GalNAc

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

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

Weary sugared structure 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) Ga1 (b1-3)] GalNAc of heap of stone

Glycan structures Fuc (a1-3) [Gal (b1-4)] GlcNAc (b1-) Gal (b1-4) GlcNAc (b1-) Ga1 (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

Can modify the biochemical form that host cell obtain protein of the present invention or chimeric molecule by several different methods known in the art, include but not limited in host cell, to introduce the transgenosis of one or more encode a kind of enzyme or plurality of enzymes, the required biochemical form of generation.This transgenosis comprises polytype sialytransferase, for example ST3Ga11, ST3Ga12, ST3Ga13, ST3Ga14, ST3Ga15, ST3Ga16, ST6Ga11, ST6Ga12, ST6GalNAc1, ST6GalNAc2, ST6GalNAc3, ST6GalNAc4, ST6GalNAc5, ST8Sia1, ST8Sia2, ST8Sia3, ST8Sia4, ST8Sia5, ST8Sia6; Galactotransferase, for example GalT1, GalT2; Fucosyltransferase is FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, FUT10, FUT11 for example; Sulfurtransferase; The GlcNAc transferring enzyme is GNT1, GNT2, GNT3, GNT4, GNT5 for example; Antenna nickase (antenna-cleaving enzymes) and endoglycosidase.

For example, the invalid terminal sialylation of N-glycan structures causes the expressed protein serum half-life to reduce, described protein is recombinant human AchE for example, can improve this situation (JBiochem 336:647-658 by adding rat beta galactose glycosides α-2,6-sialytransferase transgenosis in HEr 293 cells, 1998:J Biochem 363:619-631,2002).

Similarly, the invalid structure of the specific Lewis x group for example sialyl Lewis x structure of N-glycan structures causes expressing protein part bonded to reduce, described expressing protein is recombinant human PSGL-1 for example, can improve this situation people PNAS 95:12283-12288 such as (, 1998) Fritz by in HEK 293 cells, adding the fucosyltransferase transgenosis

In one embodiment, use to transform α-2 is arranged, 3 or α-2,6 sialic acids shift or α-2,3 and the human cell line of α-2,6 sialytransferases (" sialylated albumen ") produce protein or its chimeric molecule.Sialylated proteic example comprises sialylated-IFN-a2B, sialylated-IFN-a2B-Fc, sialylated-IFN-b1, sialylated-IFN-b1-Fc, sialylated-IFN-g, sialylated-IFN-g-Fc, sialylated-IFNAR2 and sialylated-IFNAR2-Fc.

Especially, with the biochemical parameter (P that comprises one or more plysiochemical parameters _x) feature characterize sialylated albumen of the present invention, comprise sialylated proteic monose (P ₉) and sialic acid content (P ₁₀), when being 1 during for standard: 0.1-100 NeuNAc with GalNAc; When being normalized into 3 times of seminoses, be 3: 0.1-100 NeuNAc.Sialylated proteic N-of the present invention connects the neutral percentage ratio (P of oligosaccharides ₁₃) be 0 to 99%, for example 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%.Sialylated proteic N one of the present invention connects the acid percentage ratio (P of oligosaccharides ₁₄) be 1 to 100%, for example 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%.

Sialylated proteic O-of the present invention connects the neutral percentage ratio (P of oligosaccharides ₁₅) be 0 to 99%, for example 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,2O, 21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98or99%.Sialylated proteic O-of the present invention connects the acid percentage ratio (P of oligosaccharides ₁₆) be 1 to 100%, for example 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%.

Compare transformation period (T in the sialylated proteic body with the transformation period that does not have genetically modified protein of the present invention or chimeric molecule ₁₁) 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 are crosslinked to be that α 2,6 in the N-crosslink part 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 are crosslinked to be that α 2,6 in the O-crosslink part is crosslinked.

In one embodiment, use to transform the human cell line that FUT3 (" marine alga glycated protein ", " fucosylated-protein ") is arranged and produce in protein or the invention its chimeric molecule.The proteic example marine alga of marine alga saccharification saccharification-IFN-a2B, marine alga saccharification-IFN-a2B-Fc, marine alga saccharification-IFN-b1, marine alga saccharification-IFN-b1-Fc, marine alga saccharification-IFN-g, marine alga saccharification-IFN-g-Fc, marine alga saccharification-IFNAR2, marine alga saccharification-IFNAR2-Fc, marine alga saccharification-IL-10, marine alga saccharification-IL-10-Fc.

Especially, with the biochemical parameter (P that comprises one or more plysiochemical parameters _x) feature characterize sialylated albumen of the present invention, comprise the proteic monose (P of marine alga saccharification ₉) and sialic acid content (P ₁₀), when being that standard is 1 to 0.1-100 NeuNAc with GalNAc; Be 3 to 0.1-100 NeuNAc when being standard with 3 times of seminoses.

In one embodiment, the marine alga glycated protein has the structure division that more contains Lewis structure (for example Lewis a, Lewisb, Lewis x or Lewisy) or sialyl Lewis structure (for example sialyl Lewis a or sialyl Lewis x).

In one embodiment, compare with the protein of the present invention that does not have genetically modified expression or the binding affinity of chimeric molecule, the marine alga glycated protein has the part binding affinity of change.

Use is from worker FN-a2B, IFN-b1, IFN-g, IFNAR2, IL-10, the forward primer separately and the reverse primer of the protein molecular of selecting among the IL-10Ra adopt the DNA of methods known in the art amplification coding associated protein by polymerase chain reaction (PCR) from EST, for example according to the PCR Super Mix High Fidelity (Cat.No.:10790-020) of Invitrogen company.Digest amplification also is connected on the corresponding restriction enzyme sites of suitable carrier, for example pIRESbleo3, pCMV-SPORT6, pUMCV3, pORF, pORF9, pcDNA 3.1/GS, pCEP4, pIRESputo3, pIRES puro4, pcDNA3.1/Hygro (+), pcDNA 3.1/Hygro (-), pEF6/V5-His.Connection carrier is transformed in the suitable e. coli host cell, for example XLGold, super competent cell (Strategene), XL-Blue, DH5 α, DH10B or other.

For the generation of chimeric molecule, from EST, adopt suitable forward and reverse primer dna sequence dna, for example IgG1, IgG2, IgG3, IgG4, IgGA1, IgGA2, IgGM, IgGE, IgGD by the Fc structural domain of pcr amplification immunoglobulin (Ig).Expansion is cloned on the corresponding restriction enzyme sites of suitable carriers, for example, pIRESbleo3, pCMV-SPORT6, pUMCV3, pORF, pORF9, pcDNA3.1/GS, pCEP4, pIRESpuro3, pIRESpuro4, pcDNA 3.1/Hygro (+), pcDNA3.1/Hygro (-), pEF6/V5-His.Towards order should proteic dna sequence dna be amplified and be cloned into separately be on the corresponding restriction enzyme sites of Fc-carrier of framework with Fc.

In a specific embodiments, the complement activation district in Fc receptor binding domain or Fc zone can be comprised one or more aminoacid insertion, deletion or the replacement of Fc region amino acid sequence by recombinant modified.In addition, the complement activation district in Fc receptor binding domain or Fc zone can be by chemically modified, change into its glycosylation form, adding or removal carbohydrate moiety, adding polyunsaturated fatty acid part or other part on the amino acid backbone or on the common translation entity of any association or the translation back entity based on fat.The Fc zone can also be the shortening form, cuts by a kind of enzyme enzyme and realizes, described enzyme comprises papoid, stomach en-or other any site-specific proteolytic enzyme.Dimer, tripolymer or more high-grade polymeric in conjunction with respective ligand or the stronger chimeric protein of acceptor ability can be passed through in the Fc zone, promote spontaneous conformation to form.

Use suitable Restriction Enzyme to differentiate that digestion is identified/separated and contain the bacterial clone that has corresponding gene.Separate also-70 ℃ glycerine storage of positive colony.Subsequently clonal expansion is contained in the aseptic LB liquid nutrient medium of penbritin (100 μ g/ml) 37 ℃ of wave and culture 16 hours to 75Oml.Extract plasmid according to methods known in the art, preferred, according to QiagenEndofree Plasmid Mega test kit (Qiagen Mega Prep Kit#12381).

The human host cell that suitable introducing contains the cloned dna sequence of protein of the present invention or chimeric molecule includes but not limited to HEK 293 and any redundant organism thereof, HEK 293 c18, HEK293-T, HEK 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene company), 293A (Invitrogen company), Hela cell and any redundant organism thereof, HepG2, PA-1 Jurkat, THP-1, HL-60, H9, HuT78, Hep-2, HepG2, MRC-5, PER.C6, SKO-007, U266, Y2 (Apollo company), WI-38, WI-L2.

Can modify the biochemical form that host cell obtain protein of the present invention or chimeric molecule by several different methods known in the art, include but not limited in host cell, to introduce the transgenosis of one or more encode a kind of enzyme or plurality of enzymes, the required biochemical form of generation.Can optimize cloned dna sequence and be incorporated in the host cell gene group by introducing special dna sequence dna, that dissimilar integration includes but not limited to be site-specific, target, directly or the integration of enzyme mediation.

Can be incorporated in the proper host cell by the DNA of multiple transfection method known in the art protein or its chimeric molecule, for example, use chemical reagent for example diethyllaminoethyl dextran (DEAE-dextran), calcium phosphate, artificial liposome or by direct microinjection, electroporation, biological particles transportation or infection or transfection virus structure, as described below.

DEAE-dextran is a kind of cationic polymers and electronegative nucleic acid combination.In the DNA/ polymer composite on the polymer too much positive charge can allow the more approaching association of mixture have the cytolemma of negative charge.The absorption of mixture relies on endocytosis by inference.Other synthetic cationic polymers that uses in the transfection comprises 1,5-dimethyl-1, and 5-phenodiazine 11 methylene radical gather Methobromide (polybrene), polymine and dendrimers.

Instantaneous and the stable transfection of various kinds of cell type can use coprecipitation of calcium phosphate.By certain controlled way DNA and calcium chloride are mixed, join then the buffered an alkali metal salt/phosphate solution in, mixed solution is at room temperature hatched.Produce and precipitate and absorbed by endocytosis or phagolysis by cell.

The most frequently used fat composition of liposome is the fat composition that has clean positive charge under physiology pH in the liposome-mediated gene transportation.Usually cationic lipid and neutral fat are mixed, for example L-dioleoyl phosphatidylethanolamine (DOPE).The cationic moiety of fat molecule and the negative charge of nucleic acid associate, and cause tightening in liposome/nucleic acid complexes amplifying nucleic acid.Absorb mixture by endocytosis.

Is a kind of technology of effectively still requiring great effort with the dna direct microinjection in cultured cells or nucleus, needing to be not suitable for the situation of a large amount of transfectional cells.

Electroporation uses a kind of electricimpulse to produce hole, allows nucleic acid enter cell, all needs the time length of paired pulses and intensity to carry out fine tuning and optimization for this technology of every type cell of using.The electroporation device that can buy comprise Amaxa Biosystems company the Nucleofector test kit (Amaxa Biosystems, Germany).This method depends on the nucleic acid microparticles rapid transit in recipient cell.

Comprise the use retrovirus, for example lentivirus, or dna virus, for example adenovirus with virus or retrovirus structure infections or transfection.Its process comprises that using a kind of virus or retroviral vector to transmit alien gene enters into host cell.

In some embodiments, produce protein or its chimeric molecule by instantaneous method or from stably transfected cell line.Use adhesion or suspension cell line to carry out transient transfection.For adherent cell system, cell is grown in containing the substratum of serum (2-10% serum), and described substratum is DMEM for example, DMEM/F12 (JRH).The serum that uses can be foetal calf serum (FCS), donor calf serum (DCS), newborn calf serum (NBCS) etc.By standard method known in the art plasmid vector is incorporated into cell.In a specific embodiment, use the DNA of DEAE dextran or calcium phosphate precipitation method transfection protein or its chimeric molecule.After the transfection, cell is transformed into and collects expressed proteins or its chimeric molecule in the suitable collection substratum (for example serum-free DMEM/F12).

Can carry out protein or the transient expression of its chimeric molecule from suspension cell by using top summary method to introduce plasmid vector.

Suspension cell can contain growth in blood serum medium or the serum free medium (for example Freestyle substratum (Invitrogen), CD293 substratum (Invitrogen), Excell substratum (JRH) etc.).Can carry out transfection under serum-free condition, carry out transfection by use suitable transfection method in a kind of suitable medium, for example, lipofectamine is in the OptiMEM substratum.

Transient expression causes 2-3 days peak expression after the transfection usually.Episomal vector duplicates in cell and continuous expression.Therefore, in order to obtain a large amount of products, with the free expression vector transfection in cell and amplifying cells.Protein or its chimeric molecule are expressed in the substratum, collect after several weeks at cell amplification.Express substratum and can contain serum or serum-free, cell can be adherent or suspend.

Enter cell by the transfection expression carrier and obtain stable clone, select with suitable reagent subsequently, for example phleomycin, homomycin, tetracycline, Xin Meisu G418, Rheumatrex etc.Stable clone can be survived in selection, because also contain resistant gene in the plasmid except the gene of coded protein or chimeric molecule.Introduced behind the gene 1 to 2 day, and began to whole cells (stabilization pond) or to selecting according to the cell of clone's density bed board.The non-transfected cell group is also selected to determine the cell effect of killing of selective reagents.For adherent cell, allow cell in tissue culturing plate, grow up to obtaining the isolating clone of visible.They are shifted out and are taped against (clone in each hole in 96 orifice plates) in the tissue culture hole with trysinization or physical method from flat board subsequently.For suspension cell, carry out limited dilution cloning after the selection, the clone obtains amplification subsequently, characterizes and/or carry out the limiting dilution analysis of next round subsequently.

Be grown in stable clone in the substratum that contains serum and can adapt to and reduce serum level gradually, come off subsequently and in low serum low suspension growth.Further reduce serum level subsequently up to the serum-free state.Some substratum can make adaptation faster (for example, directly replacing with the serum-free suspension growth from containing adherent condition), the CD293 substratum that example is an Invitrogen company.

After the growth, the clone can begin to carry out medium optimization in serum free medium.Detect clone's production characteristic in many different substratum, for example, complete have vigor cell quantity, up to obtaining the suitableeest composition.This depends on the production method of production.For example, cell may increase in a substratum, adds subsequently to strengthen the additive of expressing before product collection.

The albumen of overexpression or chimeric molecule may accumulate in host cell.The recovery of intracellular protein comprises with lysis buffer handles host cell, and described lysis buffer includes but not limited to contain the damping fluid of following composition: NP40, Triton X-100, Triton X-114, sodium lauryl sulphate (SDS), Glycocholate sodium, sodium deoxycholate, CHAPS, CHAPSO, Brij-35, Brij-58, Tween-20, Tween-80, octyl glucoside and Octylthioglucoside.Another kind of cracking host cell method can comprise sonication, homogenate, not Schwann Cells crushing and multigelation and handle cell with hypotonic solution.

Can in multiple different bio-reactors, produce final product,, comprise agitated pool, air lift type (airlift), packed bed perfusion, microcarrier, tubular fibre, bag technique, cell factory via the example of indefiniteness.Its method can be cultured continuously, batch, feeding culture (fed batch) or induce.Can in low blood serum medium, add the peptone class and increase the volume protein.

Use is in particular the purifying strategy purifying protein of the present invention or the chimeric molecule of protein of the present invention or chimeric molecule customization.Purification process includes but not limited to: tangential flow filtration (TFF): ammonium sulfate precipitation; SEC (SEC); Gel filtration chromatography (GFC); Affinity chromatography (AFC); The affine purification of A albumen; Receptor-mediated coordinate chromatograph (RMLC); Dyestuff coordinate chromatograph (DLC); Ion-exchange chromatography (IEC) comprises negatively charged ion or cation-exchange chromatography (AEC or CEc); Reverse-phase chromatography (RPC); Hydrophobic interaction chromatography (HIC); Metal chelate chromatography (MCC).

TFF is a kind of quick and effective bio-molecular separation method, is used to concentrate, desalination or fractionation sample.It is 10 milliliters that TFF can concentrate hundreds of samples that rise.Combine with suitable molecular weight mwco membrane, TFF can separate different sizes and molecular weight biomolecules (it is 5KDa that specified molecular weight is held back (NMWC), 10KDa, 30KDa, 100KDa).The diafiltration process comprises dilute sample reconcentration subsequently, can be used to desalination or replace sample buffer.

Saltout or ammonium sulfate precipitation can be used for the proteins concentrate diluting soln.Also be used for the fractionating proteins mixture.The ionic strength that increases proteinaceous solution causes that similar electrical charge rejection effect weakens between the protein molecule.It also reduces the protein molecule power of solvent shell on every side of keeping.When these power are reduced to enough degree, protein will precipitate; Compare with hydrophilic albumen, hydrophobin is precipitating than under the low salt concn.By progressively increasing ionic strength and carrying out centrifugal fractional separation of carrying out protein compound is a kind of very effective partial purification method of protein.

The SEC porous matrix of flowing through per sample is by size separation protein.SEC is identical with the GFC principle, and it is used to the molecule in the parting liquid phase system.In SEC, at first flow out together than molecule and solvent front that the hole in the packing material is big, be excluded fully.The molecule of middle size, between getting rid of fully and keeping, big or small by the hole in the substrate material according to it.The small molecules that frees in and out the hole is retained.Therefore, the protein of different sizes has different elution volumes and retention time.For the molecule of structural similitude, molecular size is big more, their flow processs more early.Before any sample of operation, should set up that typical curve is determined working limit and with reference to retention time.

When the protein shape is identical, can be according to the weighting material in different apertures in the post, by the molecular weight of uv-absorbing, fluorescence or scattering of light rapid screening post eluate.Photon correlation spectroscopy (PCS) is generally used in static sample and the liquid chromatographic detection.During the chromatogram that also is coupled to the low angle laser light scattering detects, directly detection molecules amount and proteinic shape irrelevant people AnalBiochem 175:492-499 such as (, 1988) Carr.SEC-HPLC is used to detect hGH degraded and assembles people Pharm Res 8:427-436 such as (, 1991) Pikal.It also is used to the pollution condition (Yoshioka et al.Pharm Res 10:103-108,1993) of the beta-galactosidase enzymes of Estimation Study.

AFC is according to specific interaction between the chemical structure between the biomolecules and suitable affinity ligand purifying biological molecule.By complementary immobilized the ligand specificity's and reversible absorption target molecule.Part can be a kind of inhibitor, substrate, stand-in or cofactor, perhaps a kind of antibody of energy specific recognition target molecule.Subsequently, molecule that will absorption by competitiveness displacement washes away, and perhaps allows conformational change by pH or ionic strength conversion.

A albumen affinity purification is to use an affinity purification example of the avidity of certain bacterioprotein, and described bacterioprotein energy broad incorporation antibody is no matter antibody is to antigenic specificity.A albumen, G albumen, L albumen all have the antibodies characteristic that checks on.These three kinds of albumen recombinant production and routine are used for from the crucial antibody type of multiple affinity purification.A albumen, the proteic genetic engineering recombinant forms of G are called as albumin A/G, also are operable.These antibody binding proteins can be immobilized on the supported matrix.This method has been modified the recombinant protein that is used for being connected with on the purification of target albumen antibody A protein binding zone (Fc zone).Under physiological condition, be attached on the immobilized A protein molecular, by changing pH or ionic strength wash-out.

RMLC is a kind of AFC of specific type, has used the intrinsic affinity of acceptor to its related objective molecule.Acceptor molecule is immobilized on the suitable chromatogram supported matrix by active amino, active hydrogen, carbonyl, carboxyl or mercapto groups.In a RMLC example, acceptor-Fc chimeric molecule molecule is fixed on the A albumen sepharose pearl the proteic avidity of A by acceptor Fc part.This method has directed sessile receptor, exposes the advantage of its ligand-binding site point to its corresponding cytokine.Under physiological condition, allow target molecule be adsorbed onto on the acceptor, by changing pH or ionic strength wash-out.

DLC is a kind of ALC, has used the conjugated protein ability of reactive dyestuffs selectivity and reversible.Dyestuff is the Soluran compound normally.Reactive chlorine group able person triasine dyes is easy to be immobilized on the supported matrix, for example sepharose (sepharose) or agarose, and can be immobilized on the nylon membrane recently.

The initial discovery of these dye-bond proteins comes from the blue dextran of observing as gel-filtration column void volume mark (conjugated compound of the blue FG-3A of cibacron) can delay some proteinic wash-out.So carried out dyestuff more specific researchs, most blue dyestuffs of prototype cibacron that use to specific protein.These dyestuffs demonstrate the most effective binding characteristic being used in combination Nucleotide aspect the protein of cofactor and enzyme, described albumen and enzyme be kinases and desaturase for example, though other albumen for example serum albumin also can combine closely.It is believed that aromatic series triasine dyes structure is similar with the nucleotide structure of nicotinamide-adenine (NAD), and the folding dependent interaction that takes place of the nicotinamide-adenine in dyestuff and these albumen.In many cases, under the competition form, can elute, and dyestuff has demonstrated the characteristic of competition substrate binding site in free solution conjugated protein by substrate or Nucleotide cofactor.Appearing these dyestuffs can be conjugated protein by " pseudo-affine " interaction of electrostatic and hydrophobic interaction and more specific and ligand-binding site point.Further simulate part (intending ecological dyestuff) by the specificity that modify to increase the dyestuff part and successfully be used for many desaturases of purifying and proteolytic enzyme (people such as McGettrick

Ion-exchange chromatography (IEC) has used albumen to come purifying protein because of the delay of the electrostatic interaction between ion exchange column matrix and the protein in pillar.When moving phase pH surpassed the pI of target protein, target protein was electronegative, and will and anion-exchange column (AEC) interact.When moving phase pH was lower than the pI of target protein, the target protein positively charged should use cationic exchange coloum (CEC).Come the wash-out target protein by the concentration of using the electric charge identical to increase counterion with target molecule.

RPC is according to the hydrophobic interaction separation of biomolecules between molecule and the chromatogram supported matrix.By the pH in the control separation, under the neutral form of ionogenic compound, their the easiest analyses.The moving phase additive, for example trifluoroacetic acid increases the albumen hydrophobicity by forming ion pair, and strong adsorption is to stationary phase.By changing the polarity of stationary phase, biomolecules elutes from the stratographic supported matrix.

HIC is similar with RPC, but has bigger specified aperture.In HIC, eluting solvent uses the water salts solution, replaces water or the mobile phase of organic phase used among the RPC.And it is opposite that the sample elution order is compared with RPC.Protein surface comprises hydrophilic residue and hydrophobic " sheet ", and the latter is usually located at the inside of folded protein and comes stabilizing protein.When hydrophobic flakes becomes when being exposed in the aqueous environment, they will destroy proteic normal solvent properties, and it is disadvantageous to become thermodynamics.In water moving phase, inorganic salt (for example ammonium sulfate) concentration is high more, and surface tension is big more, therefore increases the intensity of the hydrophobic interaction between HIC resin hydrophobic group and the albumen, adsorbs.But when gradient reduced salt concn, the surface tension of water moving phase descended, and therefore caused hydrophobic interaction to reduce, and caused albumen desorption from the pillar hydrophobic grouping.MCC is a kind of according to the technology of protein to the avidity protein isolate of chelated metal ions.Different metal ions includes but not limited to be fixed on by covalently bound chelating ligand (for example diglycinee) Cu2+, C2o+, zn2+, Mn2+, Mg2+ or the Ni2+ of chromatogram upholder stationary phase.The free hapto of metal ion is used in conjunction with different protein and polypeptide.By coming wash-out with competitive molecular replacement albumen or by changing pH.For example, reducing pH of buffer causes the binding affinity of protein one metal ion mixture to reduce the protein desorption.Perhaps, use gradient to reduce the protein of pH (adopting discontinuous gradient or linear gradient form) elution of bound from the pillar.

Can modify the biochemical form that host cell obtains protein of the present invention or chimeric molecule by several different methods known in the art.

The present invention has imagined after protein or chimeric molecule expression and purifying, and carbohydrate chemistry or enzyme are coupled on the peptide chain of protein or chimeric molecule.Can use chemistry and/or enzyme coupling process to modify, increase or reduce quantity or the feature that quantity or carbohydrate are obtained.Rely on employed coupling pattern, sugar can append to (a) arginic amide group, (b) free carboxyl group group, (c) mercapto groups, those in the halfcystine for example, (d) oh group those in Serine, Threonine, the oxylysine for example, (e) aromatic residue those in phenylalanine, tyrosine or the tryptophane for example, (f) amide group of glutamine, or (g) those in amino for example Histidine, arginine or the Methionin.Can add by chemistry or Enzymology method.For example, can use suitable reorganization glycosyltransferase continuously additional sugared unit on protein or its chimeric molecule.Can also use glycosyltransferase to increase the covalently bound substituent sugar that has.For example, can transfer to terminal galactose residues by the sialic acid that sialytransferase will have an additional polyoxyethylene glycol of covalency (PEG) and increase molecular size and serum half-life.

Can also chemistry or the carbohydrate side chain of enzymatic modification protein or chimeric molecule mix multiple functional group, comprise phosphate radical, sulfate radical, hydroxyl, carboxylate radical, O-sulfate radical and N-acetyl group.

All right chemistry or enzyme process are removed the carbohydrate in protein or its chimeric molecule.Can use trifluoromethanesulfonic acid or a kind of suitable compound to carry out chemical de-glycosylation.This processing can cause isolating of great majority or all sugar, except combination sugar, and keeps polypeptide complete.With can from protein or its chimeric molecule, remove discrete sugar or whole chain by multiple endoglycosidase and exoglycosidase.

Can be by coming the glycan composition of modifying protein or chimeric molecule with sialidase, or remove residual sialic acid with moderate acid treatment; With circumscribed-or inscribe-Glycosylase come the crosslinked oligose antenna of cutting N-or shorten O-to connect oligosaccharides.Can also handle with mycoside enzyme or sulfatase and remove side group, for example trehalose and sulfate radical.Can on amino acid backbone, add pseudo-glycan structures for example polyoxyethylene glycol or dextran by chemical method, perhaps can use the glycerine transferring enzyme cocktail that has sugar-dUDP precursor to increase sugared subunit to the glycan synthetic.

The present invention has imagined chemistry or enzyme process coupling protein matter or its chimeric molecule to radionuclide.This protein or chimeric molecule can be selected from the tabulation that contains IFN-a2B, IFN-a2B-Fc, IFN-b1, IFN-b1-Fc, IFN-g, IFN-g-Fc, IFNAR2 and IFNAR2-Fc, IL-10 and IL-10-Fc, IL-10Ra and IL-10Ra-Fc.

Can use iodization (for example to the additional iodine isotope of the peptide chain of protein or its chimeric molecule ¹²³I).Especially, isotropic substance can append on the phenol ring of (a) tyrosine of peptide chain of protein or its chimeric molecule, or (b) on the imidazole ring of Histidine.Can use the method for chloramine-T (Chloramine-T), iodine monochloride, triiodide, electrolytical, enzyme, combination, metallization removal, iodogen or iodine pearl to carry out iodization.

Can use the mtc labeled method to use methods known in the art that 99mTc is appended on protein of the present invention or the chimeric molecule, for example, carry out 99mTc labelled protein or chimeric molecule by bifunctional chelating agent (for example diethylenetriamine pentaacetic acid (DTPA)) subsequently by with going back original reagent (for example tin protochloride) reduction 99mTcO4-.

The present invention has imagined protein or its chimeric molecule chemistry or enzyme process are coupled on the chemotherapeutic.Can use methods known in the art that suitable reagent (for example Zoledronic acid) is attached on protein or its chimeric molecule, for example, by the linked reaction of N-hydroxysulfosuccinimide enhanced carbodiimide mediation.

The present invention has imagined protein or its chimeric molecule chemistry or enzyme process are coupled on the toxin.Can use methods known in the art that suitable toxin (Pseudomonas exotoxin, Ricin, gelonin and the diptheria toxin that comprise melittin, vanous toxin, shortening) is attached on protein or the chimeric molecule, described method is for example by maleimide or carbodiimide coupling chemical action.

Can isolating protein described herein or its chimeric molecule be transported in patient's body by the method that allows target recipient among isolating protein or chimeric molecule and the patient or part come in contact.In a specific embodiment, among the patient that protein or its chimeric molecule conduct " pharmaceutical composition " betransported.

On the other hand, the present invention has imagined a kind of isolating one or more protein mentioned above or pharmaceutical composition of chimeric molecule and pharmaceutically acceptable carrier or thinner of containing.

Be applicable to that the composition forms that injection is used comprises that aseptic aqueous solution (water soluble) and sterilized powder are used for preparing aseptic injectable solution temporarily.It must be stable under production and condition of storage, and must keep avoiding the pollution of microorganism (for example bacterium and fungi).Carrier can be that a kind of solvent or thinner comprise, for example, and water, ethanol, polyvalent alcohol (for example, glycerol, propylene glycol, liquid polyethylene glycol etc.), their suitable mixture and vegetables oil.Can keep suitable flowability, for example, by using tensio-active agent.Can pass through multiple antibacterium and anti-mycotic agent, for example metagin, trichloro-butyl alcohol, phenol, Sorbic Acid, thirmerosal etc. prevent the activity of microorganism.In many cases, the preferred grade oozed reagent, for example, and sugar or sodium-chlor.Can be by the absorption of in composition, using delayed absorption reagent to prolong Injectable composition, for example, aluminum monostearate and gel.

By in the suitable solvent that has required activeconstituents and optional other activeconstituents of desired number, mixing active compound, prepare aseptic injectable solution, subsequent filtration is sterilized or is adopted other suitable sterile method.For the sterilized powder that is used to prepare aseptic injectable solution, appropriate preparation method comprises vacuum-drying and freeze-drying, and described method produces the required composition that the activeconstituents powder adds any interpolation.

When promoting agent during, can be taken orally, for example by suitable protection; with the thinner of non-activity or and assimilable edible carrier; perhaps can be encapsulated in hard or soft shell capsule in, perhaps can the boil down to tablet, perhaps can directly be incorporated in the food of diet or by the breast milk administration.For oral therapeutic administration, activeconstituents can mix auxiliary material and use can absorb forms such as tablet, lozenge, capsule, elixir, suspension, syrup, wafer.This composition and preparation should contain the activeconstituents of at least 1% weight.The per-cent of composition and preparation can, certainly, be change and can be suitable between unit weight 5% to about 80% between.The quantity of active agent is the proper dosage that will obtain in the composition of this treatment usefulness.In a specific embodiment, preparation is according to composition of the present invention or preparation, and oral dosage unit form contains the conditioning agent between about 0.1 μ g and the 200mg.Other dosage comprises from about 1 μ g to about 1000mg, from about 10 μ g to about 500mg.These dosage can be each individuality or per kilogram of body weight.Can be per hour, every day, weekly, every month or annual administration.

Tablet, lozenge, pill, capsule etc. can also contain the composition of following tabulation.Can add tackiness agent for example resin, gum arabic, W-Gum, gelatin; Auxiliary material is Lin Suanergai for example; Disintegrating agent is W-Gum, yam starch, alginic acid etc. for example; Lubricant is Magnesium Stearate for example; Sweeting agent is sucrose, lactose or asccharin for example, perhaps adds sweetener for example peppermint, wintergreen oil or cherry food flavouring.When dosage unit form was capsule, it can contain the material and the liquid vehicle of the above-mentioned type.Multiple other material can be used to wrap by or the physical form of modifying dose unit in addition.For example, can use shellac, sucrose or both peridium patch agent together, pill or capsule.Syrup or elixir can contain active compound, and sucrose is as sweeting agent, and methyl alcohol and propylparaben are as sanitas, and dyestuff and food flavouring be cherry or oranges and tangerines food flavouring for example.Certainly, any material that uses in any dosage unit form in preparation should be pharmaceutical purity and be nontoxic substantially for the quantity of using.In addition, active compound can mix in sustained release preparation and the formulation.

The present invention has also imagined the topical formulation.In the topical formulation, active agent can suspend in emulsifiable paste or lotion or wax preparation or other liquor, so topical application emulsifiable paste or lotion or wax preparation or liquor cause active agent to be introduced into patient's biological surface.The vocabulary of Shi Yonging " biological surface " has been imagined on the body or inner any surface herein.The example of local mixture of the present invention adaptable " biological surface " comprises any epithelial surface, for example skin, respiratory tract, gi tract and genitourinary tract.

Except traditional emulsifiable paste, emulsion, paster or sprays, reagent of the present invention can also be used a series of methods based on iontophoresis or electroporation (poration) by the transportation of partial and/or transdermal.

" iontophoresis " causes the ability of charging particle movement based on electric current.Place on the skin a pair of close electrode skin and below capillary vessel between set up an electromotive force.At positive pole, positively charged drug molecule is expelled skin surface to capillary vessel.Opposite, will be pushed at the electronegative drug molecule of negative pole through skin.Because electric current can close and change, Iontophoretic device can start and close fast, and medicament transport is highly controlled and is sequencing.

Electroporation technology has used the high-frequency impulse energy, and (for example radio-frequency radiation, laser, heat or light) comes the of short duration stratum corneum of breaking in a variety of forms, and stratum corneum is to stop most medicines to enter into the skin layer of blood flow.Be important to note that differently with iontophoresis, the energy that electroporation technology uses is not used in the transportation medicine by skin, just makes things convenient for moving of it.Electroporation provides one " window ", compares the medicine substrate with normal circumstances and can pass through easier and fast.

Pharmaceutically acceptable carrier and/or thinner comprise any He all solvents, dispersion agent, and coating, antibacterium and anti-mycotic agent wait to blend delayed absorption agent etc.It is known in the art that pharmaceutically active substance is used this medium and reagent, and not have what conventional media or reagent and conditioning agent be inconsistent, and their application in pharmaceutical composition have been conceived to.The active compound that replenishes can also be incorporated in the composition.

Nephropathy syndrome Lowe ' s Syndrome; Lowe-BickelSyndrome; Lowe-Terry-MacLachlan Syndrome; low back pain; milk of sulfur; leukotrienes d; Lubs syndrome; Luft disease; the waist spinal canal stenosis; lumbar spinal stenosis; waist sacrum canalis spinalis is narrow; Lundborg-Unverricht Disease; Lundborg-UnverrichtDisease Included; Lupus; lupus; lupus erythematosus; Luschka-MagendieForamina Atresia; lyell's syndrome; the Lyell syndrome; lymphadenoid thyrocele; lymphangiectatic protein losing enteropathy; lymphangioleiomyoma; lymphangioleiomyoma; lymphangioma; the lymphatic vessel deformity; Lynch Syndromes; Lynch SyndromeI; Lynch syndrome II; Schindler type lysosome alpha-N-Acetylgalactosaminidase disappearance (Lysosomal Alpha-N-Acetylgalactosaminidase DeficiencySchindler Type); Lysosomal Glycoaminoacid StorageDisease-Angiokeratoma Corporis Diffusum; lysosome Glycosylase disappearance; MAA; Machado Disease; Ma-Yue disease; macrencephaly; megacephaly; the megacephaly hemihypertrophy; megacephaly is followed lipomatosis and Hemangiomata; megacephaly is followed pseudopapilloedema and Multiple (multiple) Hemangiomata; macroglobulinemia; macroglossia (disease); the loose syndromes of macroglossia-umbilical hernia-internal organ; Macrostomia (meloschisis) Ablepheron Syndrome; Bernard-Soulier Type familial megaloplastocyte reduces younger brother weary (Macrothrombocytopenia Familial Bernard-Soulier Type); the macula retinae sexual involution; macular amyloidosis; macular degeneration; disciform degeneration of macula; disciform degeneration of macula; disciform degeneration of macula; mottled corneal nutrition obstacle; MAD; Madelung ' s Disease; Maffucci syndrome (multiple chondroma companion internal organ cavernous hemangioma); epilepsy grand mal; malabsorption; malabsorption-ectodermal dysplasia-wing of nose underdevelopment; Maladie de Roger; Maladie de Tics; malaria; male four limbs and deformity of kidney; male gonad dysplasia disease; pernicious acanthosis; malignant acanthosis nigricans; pernicious astrocytoma; malignant atrophic papulosis; subtertian malaria; malignant hyperphenylalaninemia; malignant hyperthermia; pernicious hyperpyrexia; malignant melanoma; the central nervous system malignant tumour; mallory-Weiss tear; mallory-Weiss tear; Ma-Wei syndrome (orifice of the stomach mucous membrane lacerated wound syndrome); Mammary Paget ' s Disease; the mandibular bone ameloblastoma; faciomandibular dysostosis; mannosidosis; face-point-finger pattern cerneal dystrophy (Map-Dot-Fingerprint Type Corneal Dystrophy); maple syrup urine disease; marble bone; paroxysmal nocturnal hemoglobinuria (marchiafava-Micheli syndrome); Ma Kasigeen jaw winking syndromes; Ma Kasigeen (family name) phenomenon; the Ma Kasigeen ptosis is followed gunn syndrome; ridge grace syndrome is (during one-sided blepharoptosis; Ipsilateral upper eyelid and mandibular symphysis motion); Ma Kasigeen (gunn syndrome) syndromes; Ma Kasigeen sagging (following gunn syndrome); marden-Walker syndrome (the multiple arthrogryposis deformity of recessive hereditary); Ma-Wo Er Shi connective tissue disease (CTD); Malaysia side nutrition depletion; Malaysia side-Achard syndromes; the Malaysia side syndromes; Malaysia side syndromes I type; the Malaysia side varient; Malaysia side type supermotility syndromes; marginal corneal dystrophy; Mary's ataxia; Mary's disease; marie-Strumpell disease; Mariestrumpell Disease; ankylosing spondylitis (ankylosing spondylitis); marinesco-Sjogren syndrome (hereditary ataxia; cataract; dwarf and amentia syndrome); Ma-Si-Ge syndromes; Marker X syndrome; Ma Shaoer syndrome; Maroteaux LamySyndrome; Maroteaux Type Acromesomelic Dysplasia; Ma Shaoer is ectodermal dysplasia to follow the vision auditorily handicapped; Ma Shaoer-Smith's syndromes; the Ma Shaoer syndromes; the Ma Shaoer type is deaf-myopia-flood-saddle nose (Marshall TypeDeafness-Myopia-Cataract-Saddle Nose); Ma-Ao syndrome (false Parathyroid function is hanged down and subtracted disease); Ma-Bei syndromes; Martorell syndrome; MASA Syndrome; the big area myoclonia; mast cell leukemia; mastocytosis; mastocytosis is followed the blood disorder; Maumenee Corneal Dystrophy (cerneal dystrophy); the upper jaw bone ameloblastoma; the upper jaw bone dysostosis; maxillonasal dysplasia; the maxillonasal dysplasia adhesion type; maxillary sinus-eyelid synkinesis; unusual (the may-Hegglin anomaly of Mei-Hai Er Shi (thrombocyte); heredity nucleic acid is unusual); medium-chain acyl-coenzyme A dehydrogenase deficiency; McArdle (glycogenosis IV type); McCune-Albright; (kidney) MCD; McKusick type metaphysial chondrodysplasia; MCR; MCTD; U.S. syndrome; meckel-Gruber syndrome (internal organ tumour-head dysplasia syndrome); median cleft face syndrome; thalassemia; the medium chain ethylene reductase; medium chain ethylene reductase disappearance; medium chain ethylene reductase disappearance; (kidney) MCD; medullary sponge kidney; maximum expiratory flow volume; esophagectasis; macrencephaly; macrencephaly is followed the hyaloid inclusion body; Megalencephaly with Hyaline Panneuropathy; megaloblastic anemia; MAP; megalocornea-spiritual sluggish syndromes; basal cell naevus syndrome; MeigeShi lymphedema; the Meige syndrome (Meige ' s Syndromo); the 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; the mucous membrane lipodystrophy; Da Costa's syndrome (cardioneurosis); Meniere disease (auditory vertigo); Meniere disease; meninx capillary blood tuberculation; Menkes disease; menkes disease I; aphasia SOM and hallux valgus type backwardness; mental retardation-deafness-bone is unusual-coarse face and thick lip (Mental Retardation-Deafness-Skeletal Abnormalities-CoarseFace with Full Lips); the 5th thumb and toe underdevelopment type mental retardation; osteochondrodysplasia type mental retardation; the short and small type mental retardation of growthing lag-deafness-sexual organ (Mental Retradation-X-linked with GrowthDelay-Deafness-Microgeni-talism); Menzel type olivopontocerebellar atrophy; the mermaid syndrome; myoclonus epilepsy and ragged red fibrers syndrome; myoclonus epilepsy and ragged red fibrers syndrome; Merten-Singleton Syndrome; the limb injury syndromes; glomerular mesangium immunoglobulin-a type.Ephrosis, the mesentery lipodystrophy, Mesiodens-CataractSyndrome, Mesodermal Dysmorphodystrophy, MesomelicDwarfism-Madelung Deformity, metabolic acidosis, metachromatic leukodystrophy, metatarsus varus, the metatropic dwarfism syndromes, metatropic dysplasia, metatropic dysplasia I, metatropic dysplasia II, methylmalonic acidemia, methylmalonyl CoA carboxyl mutase disappearance disease, Meulengracht ' s Disease, faciomandibular dysostosis I, macroglobulinemia, malignant histocytosis, microangiopathic hemolytic anemia, microencephalon, primary nanocephalic dwarf I, microcephaly's deformity, microcephaly-ceasma hernia-.

In other embodiments, the pharmaceutical composition that contains separative IFN-a2B or its chimeric molecule can (as comprise hydroxyl glycosides, AZT, ribavirin separately or with other biological preparation, medicine, viramidine, zidovudine, dedeoycytidine, statin molecule antiviral) or therapy (therapies) (as operation, radiotherapy, vaccinotherapy, overheated, autologous peripheral blood stemcell transplant; The tradition chemotherapy, anthracycline chemotherapy, other combined chemotherapy comprises cis-platinum, chain left side star, Dx, Fluracil, ametycin, busulfan, turzolon, Etoposide, vincristine(VCR), endoxan, LY-188011, taxol, Qin Su, cytabarine; Use IFN-b, IFN-g, IL-2, TNF-α, IL-12, GM-CSF, Sch-39300, BCG, Thalidomide, Imatiniubmesylate, rhuMAb-VEGF, filgrastim, dexamethasone, isoretinoin, treretinoin, SU011248, treatment Gleevec)) following disease is treated in coupling, includes but not limited to: the treatment of tumour and chronic viral infection, comprising hairy cell leukemia, malignant melanoma, follicular lymphoma, Genital warts (comprising around sexual organ outside and the anus), the AIDS that Kaposi sarcoma is relevant, chronic type b and hepatitis C, non_hodgkin lymphoma, renal cell carcinoma, ovarian cancer, carcinoma of the pancreas, carcinoid tumor, neurospongioma, chronic granulocytic leukemia, mesothelioma, tumor of head and neck (comprising: the glossopharyngeum cancer; Laryngocarcinoma; Lip and oral carcinoma; The oropharynx cancer), T chronic myeloid leukemia, lymphoma, t cell lymphoma, lymphoma mantle cell, late period many focuses, disperse small lymphocyte/marginal zone lymphoma, folliculus SCC lymphoma, folliculus cell mixing lymphoma, T-cell lymphoma,cutaneous (mycosis fungoides, Sezary syndrome), liver cancer, unspecified solid tumor is (as being late period, shift unresectable tumour); Multiple myeloma, gastrointestinal tumor, eosophageal cancer, menigiomas, small cell lung cancer, children or adult HIV associated cancer, bladder cancer, lymphoma, the chronic lymphocytic leukemia that continues, the malignant tumour that takes place after the immunosuppression; Damage (SIL) on the rectum in intracutaneous tumorigenesis (AIN)/tesselated epithelium and infect fibromatosis, neuroblastoma, leukoencephalopathy as HIV, lymphomatoid granulomatosis, temporal fossa fibromatosis, vascular tumor, giant cell tumor of bone, carcinoma in situ, Zollinger Ellison syndrome, non-B islet-cell carcinoma, primary thrombocytosis, hepatitis A, hepatitis B, hepatitis C, hepatitis D, SARS, west Nile virus, HIV, sclerosing panencephalitis, simplexvirus 8, foot-mouth disease, Mediterranean fruit fly patient; Allergic disease comprises, atopic diseases (as atopic dermatitis, atopic eczema, high immunoglobulinlg E syndromes (HIES)), and asthma comprises the transformation reactions of nasal mucosa; Autoimmune disorder, type 1 diabetes for example, multiple sclerosis; Endometriod cysts, SA, Waldenstrom ' s macroglobulinemia is to the cognitive prevention that descends of alzheimer's disease, churg-Strauss syndrome, and be deleterious (as Japanese encephalitis) to infecting the reaction do not regulate proinflammatory cytokine; Systemic lupus erythematous.

In other embodiments, the pharmaceutical composition that contains separative IFN-b1 or its chimeric molecule can be independent, perhaps each medicine combines with one another, or with the other biological preparation, medicine or therapy coupling treatment squamous cell carcinoma of the head and neck, breast and cervical cancer, malignant melanoma, early stage neuroectodermal tumor, hepatocellular carcinoma, the metastasis of cancer, lung cancer, brain shifts, renal cell carcinoma, neurospongioma, mesothelioma of pleura or malignant pleural hydrops, Genital warts, chronic active hepatitis B virus (HBV), encephalitis (as west Nile virus) due to the hepatitis C virus (HCV), virus infection, multiple sclerosis (MS) comprises repeatability MS, cytomegalovirus CMV, foot and mouth disease, late period leishmaniasis, SARS, myocarditis due to the virus infection, ordinary sole of the foot flesh wart, herpes virus 6, Acute Stroke, ulcerative colitis, HIV, the AIDS that Kaposi sarcoma is relevant, HTLV-1 myelopathy (HAM), adrenoleukodystrophy, chronic inflammatory demyelination polyradiculoneuropathy (CIDP), marrow failure, optic neuritis, autonomic nervous dysfunction is with qualifying-Ba two syndromes.In another embodiment, drug component comprises isolating IFN-b1 or and chimeric molecule and EPO combination therapy MS.

In another embodiment, comprise the drug component of isolating IFN-g and chimeric molecule thereof can be separately or with other biological products, medicine (as the statin molecule) or therapy combination therapy pulmonary fibrosis (comprising the primary pulmonary fibrosis); Asthma; Osteopetrosis; Bacterium, fungi and virus infection (comprise systemic fungal infection, tuberculosis, leprosy, bird mycobacterium, human papillomavirus (HPV), chronic hepatitis C virus (HCV), hepatitis B virus (HBV), chronic HBV fibrosis, HIV, the AIDS that acute cryptococcal meningitis is relevant, chronic granulomatous disease (CGD) and relevant bacterium and fungi infestation, aspergillus tubigensis or other filamentary mould infect, cryptococcus property encephalitis, choamydiae infection, leishmaniasis); Atopic dermatitis, cyst fibrosis, chronic Postbronchiolitis air flue sequela, Qiu-Shi two syndromes (CSS), multiple sclerosis, rheumatoid arthritis, small cell lung cancer, malignant pleural mesothelioma (MPM), melanoma, cancer of the stomach, ovarian cancer, peritoneal cancer, renal cell carcinoma, colorectal carcinoma, non_hodgkin lymphoma and other solid tumor, CFA, granulomatous slack skin and multiple myeloma and leukocyte adhesion deficiency syndromes.

In another embodiment, comprise the drug component of isolating IFN-g and chimeric molecule thereof can be separately or with other biological products, those surgical patients that suppress in various damages back cellular immunization of medicine (as the statin molecule) or therapy combination therapy.

In another embodiment, the drug component that comprises isolating IFNAR2 and chimeric molecule thereof such as IFNAR2-Fc separately or with other biological products, medicine or therapy are united enhancing IFNalpha treatment chronic viral infection, hairy cell leukemia, malignant melanoma, follicular lymphoma, the AIDS that Kaposi sarcoma is relevant, or the effect of chronic type b and hepatitis C; Strengthen the IFNbeta treatment as chronic active hepatitis B, hepatitis C virus, cytomegalovirus and HIV and as squamous cell carcinoma of the head and neck, breast and cervical cancer, malignant melanoma, primary neuroectodermal tumors, hepatocellular carcinoma, the metastasis of cancer, lung cancer, brain shifts, renal cell carcinoma, the effect of neurospongioma and mesothelioma of pleura; Strengthen IFN omega treatment hepatitis C virus, parvovirus and other virus infection and the effect for the treatment of ovarian cancer, melanoma, myelocytic leukemia and other cancer as antitumor drug; Strengthen IFN tau treatment HIV-1 and papillomavirus, as the autoimmune disease of multiple sclerosis, and the effect that prevents allergic sensitization; Strengthen the effect of IFN kappa treatment encephalomyocarditis and other virus; Or the effect of enhancing IFN zeta treatment hepatitis virus, hsv, encephalomyocarditis and some cancer such as renal cell carcinoma and myelocytic leukemia.

In another embodiment, the drug component that comprises isolating IFNAR2 and chimeric molecule thereof can separately or unite other biological products, medicine or as 1 type Interferon, rabbit agonist alternative medicine or with 1 type Interferon, rabbit combination therapy disease described herein.

In another embodiment, the drug component that comprises isolating IFNAR2 and chimeric molecule thereof can separately or be united other biological products, medicine or therapy are treated the prevention of allograft rejection after autoimmune hepatitis, lupus, Sjogren's syndrome, psoriasis, atopic dermatitis and diabetes and type 1 diabetes and the bone marrow transplantation as the antagonist of 1 type Interferon, rabbit or inhibitor.

In another embodiment, the drug component that comprises isolating IL-10 and chimeric molecule thereof can separately or be united other biological products, medicine or therapy for treatment of cancer, autoimmune disease and chronic inflammatory diseases are for example treated component and can be prevented that body from prolonging and fierce immune response antigen and stimulator; Autoimmune disease (comprising thyroiditis, type 1 diabetes, inflammatory enteritis (as crohn), rheumatoid arthritis and psoriasis), comprises the virus infection of HIV at contact dermatitis, transplant rejection, GVHD, HEV infection, ulcerative colitis; Wegener ' s granulomatosis; Pancreatitis; Come from the acute pancreatitis (EREP) of endoscopic retrograde cholangiopancreatography; Blood vessel injury; Palsy symptom, multiple sclerosis, alzheimer's disease and meningitis in the CNS disease.In addition, the treatment component of inventing at present can separately or be united other medicines or therapy can suppress mastocyte to prevent the generation of acute myocarditis; Can promote the survival of neurone and neurogliocyte; Can be intra-uterine infection baby that mother gives birth to neuroprotective is provided; Can prevent to comprise the generation of the autoimmune disease of thyroiditis and type 1 diabetes; Can be used as anticoagulant and prevent gangrenosum acne and fibrotic liver injury.

In another embodiment, comprise that the drug component of isolating IL-10Ra and chimeric molecule thereof can separately or be united other biological products, medicine or therapy for treating comprise all kinds cancer (as melanoma, cancer, lymphoma); Eosinophilia, Sai Zhali syndromes; Lupus erythematosus; Systemic lupus erythematous; Sjogren's syndrome disease; The bleb disease; Rheumatoid arthritis; Atopic dermatitis; Virus infection and wound.

In another embodiment, comprise that the drug component of isolating IL-10Ra and chimeric molecule thereof can separately or be united other biological products, medicine or therapy start the antitumor action of expressing IL-10 tumour such as B cell lymphoma to crossing; Reverse is by the unicellular inactivation of the tumor inducing due to the cytokine, weaken the susceptibility and the function of neutrophilic granulocyte in lung of the secondary bacterial pneumonia that causes by too much IL-10 product, and prevent from GVHD liver injury by the autoimmunization mediation to strengthen delayed hypersensitivity (may be useful) for the curative effect that improves immunotherapy of tumors.

And medicinal drug composition of the present invention has higher drug effect, stronger thermostability, longer serum half-life or have higher blood dissolves when comparing with the protein that is expressed in non-human cell line or its mosaic.The present invention has also shown lower Ia scavenging(action) or related side effects.Because these improved characteristics, comparing pharmaceutical composition of the present invention with the protein that is expressed in non-human cell line or its mosaic can be with lower frequency administration.Thereby the reduction administration frequency can strengthen patient's adaptability helps treatment result.Patient's quality of life equally also can increase.

Therefore, in one embodiment, pharmaceutical composition of the present invention can be with the therapeutic dose administering mode administration identical with the protein that is expressed in non-human cell line or its mosaic.Therapeutic dose is meant the amount that produces the necessary pharmaceutical composition of activity in vivo.The accurate amount that gives mixture by such as other factors decisions that become to grade in accurate type, treatment patient's physical qualification and the pharmaceutical composition of treatment symptom.The pharmaceutical composition that contains albumen of the present invention or chimeric molecule isoform can be beneficial to the different formulated of administration has above-mentioned one or more symptoms in order to treatment patient.The mean treatment degree of functioning of pharmaceutical composition may be different.The effective dose of estimating in the 0.1ng/kg body weight between the 20 μ g/kg body weight; Or according to the suggestion or the prescription of qualified physicians.

The isolating protein or the chimeric molecule and the application of pharmaceutical composition in different therapies and/or diagnosis that comprise Partial Protein or its chimeric molecule have at least been the present invention further provides.

More specifically, the method that the invention provides treatment or prevent tested mammalian diseases, wherein can increase the albumen among the present invention or the amount or the activity of chimeric molecule and alleviate disease, this method comprise to described tested Mammals give effective dose isolating protein, comprise this proteic chimeric molecule, comprise this pulsating chimeric molecule in proteinic extracellular region territory or comprise the pharmaceutical composition of this isolating protein or chimeric molecule.

With non-restrictive example the present invention is further set forth below.

Embodiment 1

The preparation of carrier-Fc construct

(a) preparation of the DNA construct of expression Fc

The dna sequence dna of the Fc structural domain of coding human IgG l is by polymerase chain reaction (PCR) amplification, from EST cDNA storehouse (Clone ID 6277773, Invitrogen), the forward primer (SEQ ID NO:21) and the reverse primer (SEQ ID NO:22) in restriction enzyme BamH1 and BstX1 site have been mixed in use respectively.This amplicons cloned is gone among the pIRESbleo3 (Cat.NO.6989-1, BD Biosciences) corresponding restriction enzyme site with preparation construct pIRESb1eo 3-Fc.PIRESbleo 3-Fc discharges the big or small insertion fragment of 780bp expectation with BamH1 and BstX1 digestion, measures as gel electrophoresis.

(b) preparation of the DNA construct of marking protein

The dna sequence dna of coded protein from the ESTcDNA storehouse, uses forward primer and the reverse primer of having introduced restriction enzyme site according to table 8 by pcr amplification.After the amplification, amplicon is through suitable digestion with restriction enzyme and be cloned in the expression vector as shown in table 8.Suitable restriction enzyme is used to digest the carrier of the dna sequence dna that contains coded protein, to discharge the fragment of expectation size as shown in table 8.To the integrity of carrier-protein construct order-checking with definite clone's process described herein.

(c) Megaprep carrier-proteinic preparation

The aseptic LB liquid nutrient medium inoculation that contains penbritin (100 μ g/ml) of 750ml has transformed the overnight culture 75O μ l of carrier-protein or carrier-proteinic E.Coll.Culture was 37 ℃ of shaking culture 16 hours.Plasmid prepares according to Qiagen Endofree PlasmidMega Kit (Qiagen Mega Prep Kit #12381).

Table 8

Protein-Fc and relevant cloning information

Protein	The cDNA source	Forward primer	Reverse primer	Restriction endonuclease sites	Carrier	Size (bp)
							IFN-a2b	IFN-a pORF．IntegratedSciences	SEQ IDNO：25	SEQ IDNO：26	EcoRI.BamHI	pIRESbleo3(Cat. No.6989-1. BDBiosciences)	615
IFN-b1	pORF-IFNBl.IntegratedSciences	SEQ IDNO：37	SEQ IDNO：38	EcoRV.BamHI	pIRESbleo3(Cat. No.6989-1. BDBiosciences)	599
							IFN-g	pORF-hIFNg，Invitrogen	SEQ IDNO：57	SEQ IDNO：58	EcoRV.BamHl	pIRESbleo3(Cat. No.6989-1. BDBiosciences)	529
IFNAR2	Clone ID5494485.Invitrogen	SEQ IDNO：77	SEQ IDNO：78	BamHI.BamHI	pIRESbleo3-Fc	798

Perhaps, this proteinic nucleotide sequence of coding of being cloned in the carrier (for example pIRESbleo3 or pCEP4) can increase with primer, this primer has added the restriction enzyme site that the dna sequence dna that allows coded protein is cloned into Fc nucleotide sequence upstream among carrier-Fc, so that protein and Fc nucleotide sequence directly or by connexon ground meet the fusion of frame ground.

Embodiment 2

(a) preparation of IFN-a2b of the present invention, separation and purifying

(i) preparation of IFN-a2b of the present invention

At the 0th day, five 500cm of 3 * 10 ' cell inoculations that the embryo's human kidney cells that is used for transforming certainly is ²Tissue culturing plate (corning), described cell is HEK293, HEK293c18, HEK293T, 293CEN4, HEK293F, HEK293FT, HEK293E, AD-293 (stratagene) or 293A (Invitrogen) for example.Cell inoculation is in the EagleShi substratum/HamShi nutritional blend F12 (DMEM/F12) (JRH Biosciences) of every dull and stereotyped 90mlDulbeccoShi improvement, substratum has added the heat-inactivated calf serum (FCS of 10% (v/v), JRH Bioscienoes), 1OmM HEPES (Sigma), 4mM L-glutaminate (Amresco) and 1% (v/v) penicillin-Streptomycin sulphate (penicillin G 5000U/ml, Streptomycin sulphate 5000 μ gml) (JRH Biosciences).This flat board is at 37 ℃ and 5%CO ₂Overnight incubation under the condition.

First day, carry out transfection with calcium phosphate.Before the transfection, the substratum in each flat board has been used the fresh interpolation of 120ml heat-inactivated FCS, the 1OmM HEPES of 10% (v/v), the DMEM/F12 displacement of 4mM L-glutaminate and 1% (v/v) penicillin-Streptomycin sulphate.Preparation calcium phosphate/DNA precipitation carries pIRESbleo3 (Invitrogen) plasmid DNA of people IFN-a2b gene and the CaCl of 3720 μ l by adding 1200 μ g ₂(2.5M) in the sterilized water to final volume 30ml (solution A).With the 10ml transfer pipet solution A is joined in 2 * HEPES buffer saline (HBS) (solution B) of 30ml.In the adition process, blow and beat solution B gently.Mixture was 25 ℃ of vortex shaking culture 20 minutes.The mixture of 12ml is added drop-wise on each flat board.Culture plate is at 37 ℃ and 5%CO ₂Overnight incubation under the condition.

At the 2nd day, discard the cell cultures suspension.Each culture plate is washed the culture plate content 2 times with the DMEM/F12 substratum of 50ml.Add the fresh serum-free DMEM/F12 substratum of 100ml to each culture plate again, this culture medium supplemented has 40mM N-acetyl-D-mannosamine (NewZealand Pharmaceuticals), 10mM L-glutaminate, 4.1g/L seminose (Sigma), 15mM HEPES, 1% (v/v) penicillin-Streptomycin sulphate and ITS solution (5mg/L Sigma I8405, the human transferrin of 5mg/L fractional saturation and 5 μ g/ml selenium) are (Sigma).At 37 ℃ and 5%CO ₂Overnight incubation under the condition.

At the 3rd day, collecting cell was cultivated suspension, added the fresh serum-free DMEM/F12 substratum of 100ml to each culture plate and (contained 40 mM N-ethanoyl-D-mannosamines; 10mM L-glutamine; 4.1g/L seminose, 15mM HEPES, 1% (v/v) penicillin-Streptomycin sulphate and ITS solution.37 ℃ and 5%CO ₂Overnight incubation.100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) join in the cells and supernatant of collection 4 ℃ of preservations of mixed solution.

At the 4th day, collecting cell cultivation suspension was cultivated suspension to collecting cell and is added 100mMPMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) and the mixing of collection in the 3rd day together.Before the particle removal with 0.45 micron low albumen strainer (Durapore, Millipore).Mixture is stored in-70 ℃ or use at once.

The (ii) separation of IFN-a2b of the present invention and purifying

The process of dyestuff-part chromatogram (DLC) is as the first step of purifying IFN-a2b.For effectively combination and release in mass purification trace form, immobilized reactant dyestuff storehouse is used to screen IFN-a2b.Then, the suitable dyestuff-protein pH combination of check in small-scale cylindricality formula.

For extensive DLC, chemically-reactive dyes is numbered the selected conduct of 18 High (zymatrix) has best combination and elution property to IFN-a2b chemically-reactive dyes.Filtering cells and supernatant has 3ml or 6ml 50mMMES/5mM MgCl respectively by 4.0ml or 8.0ml under run by gravity ₂Pre-balance is to the cylinder (Alltech, ExtractClean Filter columns) of the DLC resin of pH6.Bulk flow by sample is stored in 4 ℃ and confirms the purifying success up to ELISA result.Pillar buffer A (20mM MES/5mM MgCl ₂PH6) flushing manifests clarification up to fraction.IFN-a2b with three kinds of elution buffers according to following sequentially eluting.

Wash-out 1: damping fluid C (50mM Tris-Cl/10mM EDTA pH8)

Wash-out 2:ENl.0 (50mM Tris-Cl/10mM EDTA/1.0M NaCl pH8)

(4-12%NuPAGE Bis-Tris gel, NuPAGE MES SDS electrophoresis elution damping fluid (Invitrogen) and anti--IFN-a2b ELISA (BenderMedSystems) measure elutriated fraction to the SDS PAGE that wash-out 3:EN2.0 (50mM Tfis-Cl/10mM EDTA/2.0M NaCl pH8) silver dyes.IFN-a2b be incorporated into reactive dyestuffs 18High and be eluted to the EN1.0 damping fluid and the EN2.0 damping fluid in.The DLC fraction that enrichment contains IFN-a2b is used for the size exclusive chromatography separation.

(16/70 preparative column of Superdex 75 (Pharmacia, Uppsala, Sweden)) separates the DLC component that merges to molecular-exclusion chromatography.Chromatographic condition: elutriant is 5OmMMES damping fluid (pH 6.5); Constant flow rate is 1.5ml/min.; Elution time is 115 minutes altogether, goes out the peak at 20～105 minutes wash-outs.SDS PAGE (the 4-12%NuPAGE Bis-Tris gel that uses silver to dye, NuPAGE MES SDS electrophoretic buffer (Invitrogen)) and anti--IFN-a2b ELISA (Bender Medsystems) measures elutriated fraction, discovery contains the chromatographic peak of IFN-a2b when wash-out is about 89-103 minute.

ELISA and ID SDS PAGE (4-12%NuPAGE Bis-Tris gel, NuPAGEMES SDS electrophoretic buffer (Invitrogen)) apparent molecular weight and the purity level of the resultant fraction of mensuration, anti--IFN-a2b ELISA (Bender MedSys tems) is used for quantitatively.HiPrep26/10 quick desalination post (Pharmacia) is to the fraction desalination of factor-containing.

The apparent molecular weight of the IFN-a2b of purifying is 18～24kDa, and silver dyes SDS PAGE and determines that its purity is at least 99%.Anti--IFN-a2b ELISA (Bender MedSystems) measures the IFN-a2b ultimate density and is at least 115 μ g/ml.

(b) preparation of IFN-b1 of the present invention, separation and purifying

(i) preparation of IFN-b1 of the present invention

In 0 day, with 3 * l0 ⁷The human embryonic kidney cell of individual conversion is that 5 500 cm are gone in cell (HEK 293, HEK293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene), or 293A (Invitrogen)) inoculation ²In the tissue culturing plate (Corning).Each culture plate adds 90ml DMEM/F12 (JRHBioscienceS), include 10% (v/v) calf serum (Dcs, JRH Biosciences), 4mM L-glutaminate (Amresco) and 1% (v/v) penicillin-Streptomycin sulphate (penicillin G 5000U/ml, streptomycin sulfate 5mg/ml) (JRH Biosciences).37 ℃ and 5%CO ₂Spend the night.

In the 1st day, use calcium phosphate to carry out transfection.Before the transfection, contain 10% (v/v) DES with 120ml, the fresh DMEM/F12 of 4mM L-glutaminate and 1% (v/v) penicillin-Streptomycin sulphate replaces former substratum.Add 1200 μ g and contain pIRESbleo3 (Invitrogen) plasmid DNA and the 3720 μ l 2.5M CaCl of people IFN-b1 gene ₂To aseptic H ₂Among the O, make that final volume is 30ml (solution A) preparation calcium phosphate/DNA throw out.Be added dropwise to solution A (HBS) (solution B) to 30ml 2 * HEPES buffer saline with 10ml transfer pipet point.In the process that drips, mixing solution B gently.Mixed solution was hatched 20 minutes in 25 ℃ of whirlpools.Each culture plate is selected and is added dropwise to the 12ml mixed solution.Remove the substratum contain the transfection mixed solution after 4 hours, each culture plate also adds 100ml and contains 10% (v/v) DCS, the 4mM L-glutaminate, and the DMEM/F12 and the final concentration of 1% (v/v) penicillin-Streptomycin sulphate are the HCl of 3.5mM, the substratum final pH is 7.37 ℃ and 5%CO ₂Overnight incubation.

In the 2nd day, abandon cells and supernatant.Add 100ml after every plate is washed 2 times with 50ml DMEM/F12 and contain 4OmM N-ethanoyl-D-mannosamine (New ZealandPharmaceuticals); 10mM L-glutamine, 4.1g/L seminose (Sigma) and fresh serum-free of 1% (v/v) penicillin-Streptomycin sulphate and phenol red DMEM/F12 substratum.37 ℃ and 5%CO ₂Overnight incubation.

In the 3rd day, the collecting cell culture supernatant.Each culture plate adds 100ml and contains 40mM N-ethanoyl-D-mannosamine, 10mM L-glutamine, the fresh serum-free of 4.1g/L seminose and 1% (v/v) penicillin-Streptomycin sulphate and phenol red DMEM/F12 substratum.37 ℃ and 5%CO ₂Overnight incubation.In the cell conditioned medium of collecting, add 100mM PMSF (1% (v/v)), 4 ℃ of preservations of mixed solution.

In the 4th day, the collecting cell culture supernatant added 100mM PMSF (1% (v/v)) in the lump therein together with the 3rd day collection liquid, then with the low protein binding strainer of 0.45 μ m filter (Durapore, Millipore).Mixed solution-70 ℃ preservation or use immediately.

The (ii) expression of high sialic acid IFN-b1

Use plRESbleo3-IFN-b1 and pCEP4-a2, the cotransfection of 6ST repeats the foregoing description 2 (b) (i).As structure pCEP4-a2 as described in implementing 8,6ST.PCEP4-a2,6ST and plRESbleo3-IFN-bl mix with 1: 3 ratio, 1200 these mixtures of μ g are joined 3720 μ l contain 2.5 M CaCl ₂Sterilized water in, final volume is 30ml (solution A).

(iii) use western blot analysis to identify IFN-b1

The (ii) middle substratum of collecting of embodiment 2 (b) is carried out 4-20%Tris-Glycine precast gel electrophoresis (Invitrogen), and 200V ran 1 hour in the Tris-Glycine SDS electrophoretic buffer (Invitrogen).

After being transferred to the albumen in the gel on the nitrocellulose membrane, seal nitrocellulose membrane to prevent non-specific binding with the TBS-T (containing 0.05%Tween-20) that contains 1% bovine albumin then.Use the monoclonal antibody (R﹠amp of anti--IFN-b1 then; D Systems) the room temperature vibration was hatched 1 hour.TBS-T flush away unconjugated one is anti-, uses alkaline phosphatase (AP)-bonded two of anti--mouse to resist (Promega) incubated at room 1 hour.Unconjugated two anti-on the flush away film once more.Preparation specification sheets according to AP coupling development test kit (Bio-Rad) makes the specificity of anti--IFN-b1 antibody in conjunction with development.

Find that the IFN-b1 spoon apparent molecular weight among the present invention is 20-35kDa.

(iv) use ion exchange chromatography and metal-chelate to close purification by chromatography IFN-b1

Utilize negatively charged ion or Zeo-karb (Amersham Biosciences Q or SPsepharose FF) that embodiment 2 (b) (i) is carried out purifying respectively with the (ii) middle supernatant of collecting of 2 (b).Use the IFN-b1 of 1M NaCl elution of bound from the post then.Apparent molecular weight and purity level with 1D SDS PAGE (4-2O% gradient Tris-Glycine gel (Invitrogen)) and anti--IFN-b1ELISA (PBL Biomedical Laboratories) resulting fraction of quantitative analysis.

(the HiTrap huge legendary turtle is closed HP post (1ml to use metal-chelate to close chromatography (MCC); AmershamBiosciences)) IFN-b1 of the present invention is further purified.According to manufacturer's suggestion, band Zn ²⁺Ionic HiTrap huge legendary turtle is closed the HP post and wants balance before use.The IEC fraction that contains IFN-b1 with syringe holder joins in the post, and binding buffer liquid (pH7) remove unconjugated albumen by the 0.02M sodium phosphate.According to following three kinds of elution buffer sequentially eluting IFN-b1:

Elutriant 1:0.02M sodium phosphate, 0.5M sodium-chlor pH7

Elutriant 2 0.02M sodium phosphates, 1M sodium-chlor pH7

Elutriant 3:0.02M sodium phosphate, 1M sodium-chlor pH3.5

Silver dyes the fraction of SDS PAGE (4-20% gradient Tris-Glycine gel (Invitrogen)) and anti--IFN-b1 ELISA (PBL Biomedical Laboratories) mensuration wash-out.IFN-b1 has Zn in conjunction with HiTrap huge legendary turtle crossed belt ²⁺On the high performance column, with 0.02M sodium phosphate 1M NaCl pH3.5 wash-out.

In addition, also can utilize MCC as the first step purification step, IEC came purifying IFN-b1 in second step.

(c) preparation of IFN-g of the present invention, separation and purifying

(i) preparation of IFN-g of the present invention

In 0 day, with 3 * 1O ⁷The human embryonic kidney cell of individual conversion is that 5 50Ocm are gone in cell (HEK 293, HEK293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene), or 293A (Invitrogen)) inoculation ²In the tissue culturing plate (Corning).Each culture plate adds 90ml DMEM/F12 (JRHBiosciences), include the heat-inactivated foetal calf serum (FCS of 10% (v/v), JRHBioscienoes), 4mM L-glutaminate (Amresco) and 10mM HEPES (sigma), 1% (v/v) penicillin-Streptomycin sulphate (penicillin G 5000U/ml, streptomycin sulfate 5mg/ml) (JRH Biosciences).37 ℃ and 5%CO ₂Spend the night.

In the 1st day, use calcium phosphate to carry out transfection.Before the transfection, contain the heat-inactivated foetal calf serum of 10% (V/V) with 120ml, the 4mM L-glutaminate, the fresh DMEM/F12 of 10mM HEPES and 1% (V/V) penicillin-Streptomycin sulphate replaces former substratum.Adding 12O0 μ g contains pIRESbleo3 (Invitrogen) plasmid DNA and the 3720 μ l 2.5MCaCl of people IFN-g gene ₂To aseptic H ₂Among the O, make that final volume is 30ml (solution A) preparation calcium phosphate/DNA throw out.Be added dropwise to solution A (HBS) (solution B) to 30ml 2 * HEPES buffer saline with 10ml transfer pipet point.In the process that drips, mixing solution B gently.Mixed solution was hatched 20 minutes in 25 ℃ of whirlpools.Each culture plate is selected and is added dropwise to the 12ml mixed solution.37 ℃ and 5%CO ₂Overnight incubation.

In the 2nd day, abandon cells and supernatant.Add 100ml after each culture plate is washed 2 times with 50ml DMEM/F12 and contain 40mM N-ethanoyl-D-mannosamine (New ZealandPharmaceuticals); 10mM L-glutamine; 15mM HEPES; 4.1g/L seminose (sigma); with 1% (v/v) penicillin-Streptomycin sulphate and ITS solution (5mg/L Sigma I8405, the human transferrin of 5mg/L fractional saturation and 5 μ g/ml selenium) fresh serum-free DMEM/F12 substratum (Sigma).37 ℃ and 5%CO ₂Overnight incubation.

In the 3rd day, the collecting cell culture supernatant.Each culture plate adds 100ml and contains 40mM N-ethanoyl-D-mannosamine, 10mM L-glutamine, 15mM HEPES, the fresh serum-free DMEM/F12 substratum of 4.1g/L seminose and 1% (v/v) penicillin-Streptomycin sulphate and ITS solution.37 ℃ and 5%CO ₂Overnight incubation.In the cell conditioned medium of collecting, add 100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)), 4 ℃ of preservations of mixed solution.

In the 4th day, the collecting cell culture supernatant added 100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) in the lump therein together with the 3rd day collection liquid, and then added 200mM MES (the Sigma)/50mM MgCl of 1/10 volume ₂(Sigma) pH6 adjusts pH to 6, at last with the low protein binding strainer filtration of 0.45 μ m (Durapore, Millipore).Preserve or use immediately for mixed solution-7O ℃.

The (ii) separation of IFN-g of the present invention and purifying

The first step of purifying IFN-g is used dyestuff-part chromatography (DLC).Use a fixed reactive dyestuffs storehouse to screen the IFN-g of effective combination and release in mass purification microtitre mode.Using on a small scale then, form detects suitable dyestuff-protein combination.

5ml dyestuff-part the post (pH6 or 7) of cell conditioned medium sample by O.5ml that thaw in small scale purification.This optimum step has been selected in large quantities of DLC of magnification ratio the optimal activity dyestuff-cytokine and the pH combination of high-recovery.

In extensive DLC, reactive dyestuffs 18High (zymatrix) is chosen as the reactive dyestuffs of IFN-g best combination and wash-out.Filtering cell conditioned medium liquid flows through the cylinder (Alltech, Extract Clean Filter columns) above 4.0ml or 8.0ml under gravity, and two cylinders are equipped with 3ml or 6mlDLC resin respectively, uses 50mM MES/5mM MgCl simultaneously before balance ₂Regulate pH to 6.Pillar buffer A (20mM MES/5mM MgCl ₂PH6) flushing 2 times is up to the fraction clarification (not being lark) that becomes.Use three kinds of elution buffer sequentially eluting IFN-g:

Elutriant 1: damping fluid C (50mM Tris-C1/10mM EDTA pH8)

Elutriant 2 EN1.0 (50mM Tris-Cl/10mM EDTA/1.0M NaCl pH8)

Elutriant 3 EN2.0 (50mM Tris-C1/10mM EDTA/2.0M NaCl pH8)

Season is blunt dyes the fraction that SDS PAGE (4-12%NuPAGE Bis-Tris gel and NuPAGE MESSDS electrophoretic buffer (Invitrogen)) and anti--IFN-g ELISA (R﹠D Systems) measure wash-out.The EN1.0 damping fluid that contains the IFN-g of wash-out carries out molecular-exclusion chromatography with the fraction that contains IFN-g to be separated.

Molecular-exclusion chromatography (16/70 preparative column of Superdex 75 (Pharmacia, Uppsala, Sweden)) or dextrane gel 200 (Pharmacia, Uppsala, Sweden) the DLC component of preparative column separation merging.Chromatographic condition: elutriant is a 50mM MES damping fluid (pH6.5); Constant flow rate is 1.5ml/min.; Elution time is 120 minutes altogether, goes out the peak at 20～100 minutes wash-outs.The SDS PAGE (4-12%NuPAGEBis-Tris gel, NuPAGE MES SDS electrophoretic buffer) and the anti--IFN-g ELISA that use silver to dye measure elutriated fraction.IFN-g chromatographic peak peak time is about 42-55 minute.

(Bio-Rad Laboratories UnoS12) is further purified the isolating component of molecular-exclusion chromatography through the cationic exchange coloum of 50mM MES pH5.6 pre-equilibration.Then with 50mM MESpH5.6 to the 50mM MES pH5.6 gradient elution bonded IFN-g that contains 1M NaCl.Apparent molecular weight and purity level with 1D SDS PAGE (4-12%NuPAGE Bis-Tris gel and NuPAGE MES SDS electrophoretic buffer (Invitrogen)) and anti--resulting fraction of IFN-g ELISA quantitative analysis.

The apparent molecular weight of the IFN-g of purifying is 18-28kDa, and coomassie brilliant blue staining determines that its purity is at least 95%.Anti--IFN-g ELISA estimates that the ultimate density of IFN-g is 55 μ g/ml.

(d) preparation of IFNAR2-Fc of the present invention and purifying

(i) preparation of IFNAR2-Fc of the present invention

In 0 day, with 3 * 10 ⁷The human embryonic kidney cell of individual conversion is that 5 500 cm are gone in cell (HEK 293, HEK293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene) or 293A (Invitrogen)) inoculation ²In the tissue culturing plate (Corning).Each culture plate adds 90ml DMEM/F12 (JRHBiosciences), include the heat-inactivated foetal calf serum (FCS of 10% (v/v), JRHBiosciences), 4mM L-glutaminate (Amresco) and 1% (v/v) penicillin-Streptomycin sulphate (penicillin G 5000U/ml, streptomycin sulfate 5mg/ml) are (Gibco).37 ℃ and 5%CO ₂Overnight incubation.

In the 1st day, use calcium phosphate to carry out transfection.Before the transfection, contain the heat-inactivated foetal calf serum of 10% (V/V) with 120ml, the fresh DMEM/F12 of 4mM L-glutaminate and 1% (v/v) penicillin-Streptomycin sulphate replaces former substratum.Add 1200 μ g and contain pIRESbleo3 (Invitrogen) plasmid DNA and the 3ml 2.5M CaCl of people IFNAR2-Fc gene ₂To aseptic H ₂Among the O, make that final volume is 30ml (solution A) preparation calcium phosphate/DNA throw out.Be added dropwise to solution A (HBS) (solution B) to 30ml 2 * HEPES buffer saline with 10ml transfer pipet point.In the process that drips, mixing solution B gently.Mixed solution is hatched 30 minutes vortex several seconds then in 25 ℃.Each culture plate is selected and is added dropwise to the 12ml mixed solution.Remove the substratum that contains the transfection mixed solution after 4 hours, each culture plate also adds 100ml and contains the heat-inactivated FCS of 10% (v/v), the 4mM L-glutaminate, the DMEM/F12 and the final concentration of 1% (v/v) penicillin-Streptomycin sulphate are the HCl of 3.5 mM, the substratum final pH is 7.37 ℃ and 5%CO ₂Overnight incubation.

In the 2nd day, abandon cells and supernatant.Add 100ml after each culture plate is washed 2 times with 50ml DMEM/F12 and contain 40mM N-ethanoyl-D-mannosamine (New ZealandPharmaceuticals); 10mM L-glutamine; 0.5g/L the fresh serum-free DMEM/F12 substratum of seminose (Sigma) and 1% (v/v) penicillin-Streptomycin sulphate.37 ℃ and 5%CO ₂Overnight incubation.

In the 3rd day, the collecting cell culture supernatant.Each culture plate adds 100ml and contains 40mM N-ethanoyl-D-mannosamine, 10mM L-glutamine, the fresh serum-free DMEM/F12 substratum of 0.5g/L seminose and 1% (V/V) penicillin-Streptomycin sulphate.37 ℃ and 5%CO ₂Overnight incubation.In the cell conditioned medium of collecting, add 100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)), 4 ℃ of preservations of mixed solution.

In the 4th day, the collecting cell culture supernatant, add 100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/V)) in the lump therein together with the 3rd day collection liquid, adding 2MTris-HCl pH8 (Sigma) is that the pH that 15mM regulates mixed collection liquid is 8 to final concentration, then with the low protein binding strainer of 0.45 μ m filter (Durapore, Millipore).Mixed solution-70 ℃ preservation or use immediately.

The (ii) purifying of IFNAR2-Fc of the present invention

With 1 liter of adjustment that contains IFNAR2-Fc the substratum of pH flow through an A albumen agarose column (Pharmacia) that the 1ml column volume is arranged, having used 100mMTris-HCl pH8 (Sigma) to equilibrate to pH before this post is 8.After the flushing of the post damping fluid (100mM Tris-HCl pH8) of 20 times of column volumes, IFNAR2-Fc is gone in the 1ml fraction by 0.1M citric acid (Sigma) pH4 wash-out, and adds 200 μ l 2M Tris-HCl pH8 immediately and be neutralized to pH8.The gained fraction is dyed SDS PAGE (4-20% gradient Tris-Glycine gel (Invitrogen)) by silver and is that standard uses the absorption value at spectrophotometry quantitative assay 280nm place to analyze with bovine serum albumin (New England Biolabs).

Dye SDS PAGE (4-20% gradient Tris-Glycine gel) by silver and determine that the apparent molecular weight of purifying IFNAR2-Fc is approximately between 50-80kDa.The final concentration of the IFNAR2-Fc of spectrophotometry is 145 μ g/ml.

(e) preparation of IL-10 of the present invention, separation and purifying

(i) preparation of IL-10 of the present invention

In 0 day, with 3 * 10 ⁷The human embryonic kidney cell of individual conversion is that 5 500cm are gone in cell (HEK 293, HEK293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene), or 293A (Invitrogen)) inoculation ²In the tissue culturing plate (Corning).Each culture plate adds 90ml DMEM/F12 (JRHBiosciences), include 10% (v/v) donor calf serum (DCS, JRHBiosciences), 4 mM L-glutaminate (Amresco) and 1% (v/v) penicillin-Streptomycin sulphate (penicillin G 5000U/ml, streptomycin sulfate 5000 μ g/ml) are (JRHBiosciences).

In the 1st day, use calcium phosphate to carry out transfection.Before the transfection, contain 10% (v/v) DCS with 120ml, the fresh DMEM/F12 of 4mM L-glutaminate and 1% (v/v) penicillin-Streptomycin sulphate replaces former substratum.Add 1200 μ g and contain pIRESbleo3 (Invitrogen) plasmid DNA and the 3720 μ l 2.5M CaCl of people IL-10 gene ₂To aseptic H ₂Among the O, make that final volume is 30ml (solution A) preparation calcium phosphate/DNA throw out.Be added dropwise to solution A (HBS) (solution B) to 30ml 2 * HEPES buffer saline with 10ml transfer pipet point.In the process that drips, mixing solution B gently.Mixed solution was hatched 2O minute in 25 ℃ of vortexs.Each culture plate is selected and is added dropwise to the 12ml mixed solution.Remove the substratum contain the transfection mixed solution after 4 hours, each culture plate also adds 100ml and contains 10% (v/v) DCS, the 4mM L-glutaminate, and the DMEM/F12 and the final concentration of 1% (vv) penicillin-Streptomycin sulphate are the HCl of 3.5mM, making the substratum final pH is 7.37 ℃ and 5%CO ₂Overnight incubation.

In the 2nd day, abandon cells and supernatant.Add 100ml after each culture plate is washed 2 times with 50ml DMEM/F12 and contain 40mM N-ethanoyl-D-mannosamine (New ZealandPharmaceuticals); 10mM L-glutamine; 4.1g/L fresh serum-free of seminose (Sigma) and 1% (v/v) penicillin-Streptomycin sulphate and phenol red DMEM/F12 substratum.37 ℃ and 5%CO ₂Overnight incubation.

In the 3rd day, the collecting cell culture supernatant.Each culture plate adds 100ml and contains 40mM N-ethanoyl-D-mannosamine, 10mM L-glutamine, the fresh serum-free of 4.1g/L seminose and 1% (v/v) penicillin-Streptomycin sulphate and phenol red DMEM/F12 substratum.37 ℃ and 5%CO ₂Overnight incubation.In the cell conditioned medium of collecting, add 100mM PMSF (1% (v/v)) and 500mMEDTA (1% (v/v)), 4 ℃ of preservations of mixed solution.

In the 4th day, the collecting cell culture supernatant added 100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)) in the lump therein together with the 3rd day collection liquid, adds the 200mM MES/50mM MgCl of 1/10 volume ₂The pH that pH6 regulates mixed collection liquid is 6, then with the low protein binding strainer filtration of 0.45 μ m (Durapore, Millipore).Mixed solution-70 ℃ preservation or use immediately.

The (ii) separation of IL-10 of the present invention and purifying

At first use dyestuff-part chromatography (DLC) purifying IL-10.Use of effective combination and the release of a fixed reactive dyestuffs storehouse with mass purification microtitre screening IL-10.On the small-scale post, detect corresponding dyestuff-protein combination then.

The 5ml cell conditioned medium sample that thaws has been selected in large quantities of DLC of magnification ratio the optimal activity dyestuff-cytokine and the pH combination of high-recovery by this optimum step of dyestuff-part post (pH6 or 7.3) of 0.5ml in small scale purification.

In extensive DLC, No. 8 High of reactive dyestuffs (Zymatrix) are chosen as the reactive dyestuffs of IL-10 best combination and wash-out.Filtering cell conditioned medium liquid flows through the cylinder (Alltech, Extract Clean Fi1tercolumns) above 4.0ml or 8.0ml under gravity, and two cylinders are equipped with 3ml or 6mlDLC resin respectively, shifts to an earlier date balance 50mM MES/5mM MgCl simultaneously ₂Regulate pH to 6.It is errorless that the total flux of sample is stored in 4 ℃ of results verifications up to ELIsA.Pillar buffer A (20mM MES/5mM MgCl ₂PH6) flushing is up to the fraction clarification that becomes.According to following three kinds of elution buffer sequentially eluting IL-10:

Elutriant 1: damping fluid C (50mM Tris-Cl/10mM EDTA pH8)

Elutriant 2 EN1.0 (50mMTris-Cl/10mM EDTA/1.0M NaCl pH8)

Elutriant 3 EN2.0 (50mMTris-Cl/10mM EDTA/2.0M NaCl pH8)

Blunt SDS PAGE (4-20%Tris-G1ycine gel (Invitrogen)) and the IL-10 ELISA (R﹠amp of dying of season; D Systems) fraction of mensuration wash-out.IL-10 combines and is eluted in EN1.0 damping fluid and the EN2.0 damping fluid with reactive dyestuffs 8High.HiPrep26/10 desalting column (Amersham Biosciences) will contain the DLC fraction of IL-10 and carry out quick desalination, and buffer exchange simultaneously is 50mM MES pH5.6.

The strong cat ion exchange column of 50mM MES pH5.6 (Sigma) pre-equilibration (Bio-RadLaboratories, Macro-Prep High S support) is further purified the fraction through desalting column.Then with 50mM MES pH6.5 to the 50mM MES pH 6.5 linear gradient elution post bonded IL-10 that contain 1M NaCl.Apparent molecular weight and purity level with 1D SDS PAGE (4-20% gradient Tris-Glycine gel (Invitrogen)) and the resultant fraction of IL-10ELISA quantitative analysis.

The IL-10 apparent molecular weight of purifying is 16～23kDa, and 1D SDS PAGE (4-20% gradient Tris-Glycine gel (Invitrogen)) estimates that its purity is at least 99%.The IL-10 final concentration that IL-10 ELISA estimates is 22.9 μ g/ml.

(f) preparation and the purifying of people's cell of expression IL-10Ra-Fc

(i) preparation of people's cell of expression IL-10Ra-Fc

In 0 day, with 3 * 10 ⁷The human embryonic kidney cell of individual conversion is that 5 500cm are gone in cell (HEK 293, HEK293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene), or 293A (Invitrogen)) inoculation ²In the tissue culturing plate (corning).Each culture plate adds 9Oml DMEM/F12 (JRHBiosciences), include 10% (v/v) heat-inactivated fetal bovine serum (FCS, JRHBieseiences), 4mM L-glutaminate (Amresco) and 1% (v/v) penicillin-Streptomycin sulphate (penicillin G 5000U/ml, streptomycin sulfate 500 μ g/ml) are (JRHBiosciences).

In the 1st day, use calcium phosphate to carry out transfection.Before the transfection, contain the hot deactivation FCS of 10% (v/v) with 120ml, the fresh DMEM/F12 of 4mM L-glutaminate and 1% (v/v) penicillin-Streptomycin sulphate replaces former substratum.Add 1200 μ g and contain plRESbleo3 (Clonetech, the BD Biosciences) plasmid DNA and the 3720 μ l CaCl of people IL-10Ra-Fc gene ₂To aseptic H ₂Among the O, make that final volume is 30ml (solution A) preparation calcium phosphate/DNA throw out.Be added dropwise to solution A (HBS) (solution B) to 30ml 2 * HEPES buffer saline with 10ml transfer pipet point.In the process that drips, mixing solution B gently.Mixed solution was hatched 20 minutes in 25 ℃ of vortexs.Each culture plate is selected and is added dropwise to the 12ml mixed solution.Remove the substratum that contains the transfection mixed solution after 4 hours, each culture plate also adds 100mL and contains the hot deactivation FCs of 10% (v/v), the 4mM L-glutaminate, the DMEM/F12 and the final concentration of 1% (v/v) penicillin-Streptomycin sulphate are the HCl of 3.5 mM, making the substratum final pH is 7.37 ℃ and 5%CO ₂Overnight incubation.

In the 2nd day, abandon cells and supernatant.Add 100mL after each culture plate is washed 2 times with 50mL DMEM/F12 and contain 40mM N-ethanoyl-D-mannosamine (New ZealandPharmaceuticals); 10mM L-glutamine (Amresco); 4.1g/L the fresh serum-free DMEM/F12 substratum of seminose (sigma) and 1% (v/v) penicillin-Streptomycin sulphate.37 ℃ and 5%CO ₂Overnight incubation.

In the 3rd day, the collecting cell culture supernatant.Each culture plate adds 100ml and contains 40mM N-ethanoyl-D-mannosamine, 10mM L-glutamine, the fresh serum-free of 4.1g/L seminose and 1% (v/v) penicillin-Streptomycin sulphate and phenol red DMEM/F12 substratum.Dull and stereotyped at 37 ℃ and 5%CO ₂Overnight incubation.In the cell conditioned medium of collecting, add 100mM PMSF (1% (v/v)) and 500mM EDTA (1% (v/v)), 4 ℃ of preservations of mixed solution.

In the 4th day, the collecting cell culture supernatant, add 100mM PMSF (1% (v/v)) and 500 mM EDTA (1% (v/v)) in the lump together with the 3rd day collection liquid, adding 2M Tris-HClpH8 (Sigma) is that 100mM is 8 with the pH that regulates mixed collection liquid to final concentration, then with the low protein binding strainer of 0.45 μ m filter (Durapore, Millipore).Mixed solution-70 ℃ preservation or use immediately.

The (ii) IL-10Ra-Fc of purifying people cell expressing

With 1 liter of adjustment that contains IL-10Ra-Fc the substratum of pH flow through an A albumen agarose column (Pharmacia) that the 1ml column volume is arranged by action of gravity, equilibrating to pH with 100mM Tris-HCl before this post is 8.After the flushing of the post damping fluid (100mMTris-HCl pH8) of 20 times of column volumes, IL-10Ra-Fc is gone in the 1ml fraction by 0.1M citric acid (Sigma) pH2.2 wash-out, and adds 300 μ l 2M Tris-HCl pH8 immediately and be neutralized to pH8.The gained fraction is dyed SDS PAGE (4-20% gradient Tris-Glycine gel (Invitrogen)) by silver and is that standard uses the absorption value at spectrophotometry quantitative assay 280nm place to analyze with bovine serum albumin (Pierce).Collection contains the purifying fraction of IL-10Ra-Fc and more than 4 days, changes liquid every day 2 times with 1L PBS pH7.4 dialysis.

The apparent molecular weight that silver dyes SDS PAGE (4-20% gradient Tris-Glycine gel) mensuration purifying IL-10Ra-Fc is approximately 65～80kDa, and purity is at least 95%.The ultimate density of the IL-10Ra-Fc of spectrophotometry is 100 μ g/ml.

Embodiment 3

(a) sign of IFN-a2b of the present invention

(i) two-way polyacrylamide gel electrophoresis

The sample of collecting by embodiment 2 by dialysis or desalting column (Pharmacia HR 10/10Fast Desalting column) exchange buffering liquid in (18 MOhm) water of repurity, and with SpeedVac thickener drying.Perhaps, use method well known in the art to precipitate this sample, for example the employed TCA precipitator method in the document.Then, sample is dissolved in the 240ml MSD damping fluid (5M urea, 2M thiocarbamide, 65mM DTT, 2% (w/v) CHAPS, 2% (w/v) thetine 3-10,0.2% (v/v) carrier ampholyte, 40mM Tris, 0.002% (w/v) tetrabromophenol sulfonphthalein, water) and under 15000g centrifugal 8 minutes again.

Isoelectrofocusing (IEF) is carried out with prefabricated 11cm or prefabricated 17cm pH of latex gel 3-10 solid phase pH gradient IEF adhesive tape (BioRad).Rehydration at least 6 hours at room temperature in the sample of IEF adhesive tape in sealed tube.The IEF adhesive tape places focus cell and covers with Liquid Paraffin.IEF to the 11cm adhesive tape 100V 1 hour, 200V 1 hour, 600V 2 hours, 1000V 2 hours, 2000V 2 hours, 3500V 12 hours and 100V carried out more than 12 hours, or 17cm adhesive tape 85kV a few hours are carried out (using identical V ramp-up process).

Ensuing isoelectrofocusing, adhesive tape are reduced and by alkylation, are being applied to second before gel.Adhesive tape was cultivated 20 minutes in 1 * Tris/HCl pH8.8,6M urea, 2% (w/v) SDS, 2% (v/v) glycerine, 5mM tributylphosphine oxide (TBP), 2.5% (v/v) acrylamide at least.

The 11cm adhesive tape is poured into the Tris glycine gradient gel (BioRad) of (11 * 8cm 1mm is thick) 10-20% in advance second to separation by Criterion.The 17cm adhesive tape is separated into 17 * 17cm, 1.5mm is thick, from the Tris of dabbling 10-20% glycine gradient gel.Precision or Kaleidoscope molecular weight marker (BioRad) also are used for gel.Adhesive tape is put into groove, uses 0.5% the agarose contain as the tetrabromophenol sulfonphthalein of trace dyestuff.

SDS-PAGE is used for the 11cm gel with Criterion or ProteanII electrophoresis system (BioRad) (200V1 hour (advance to be about to break away from the gel end up to damping fluid) and the every gel of 15mA constant current was used for the 17cm gel in 21 hours) carry out.Used damping fluid is the 192mM glycine, and 0.1% (w/v) SDS, 24.8mM Tris alkali are under pH8.3.

After finishing second spent the night in the methyl alcohol (MeOH) of gel sets 30 minutes-10% and 7% acetate (HAc).Then, gel decolours at least 30 minute with Sypro Ruby gel dyestuff (BioRad) dyeing 3 hours and with the HAc of 10% MeOH and 7% at least.Optionally, after fixing, gel Deep Purple fluorescent dyeing.Gel is at 300mM Na ₂CO ₃, 35mM NaHCO ₃Cultivated 2 * 3O minute, and then, cultivated in the dark at least 1 hour at the Deep Purple dyestuff of dilution in 1: 200.Then, gel is by cultivating decolouring in 2 * 15 minutes in 10% MeOH, 7% HAc.In two processes, gel FX laser light densometer (BioRad) and suitable spectral filter imaging.

Software I mageJ ( Http:// rsb.info.nih.gov/ij/) be used to analyze the relative intensity of protein spots on each gel.The spot of the selection area of gel is carried out optical densitometric method and carries out background subtraction with the appropriate area of the gel of protein spot.The target protein spot is carried out volume integral calculate spot mass center thus.Calculate the relative percentage intensity of each protein spots and make the associated value of the intensity of institute's spottiness reach 100% by stdn, with respect to other spots in the gel, the intensity of each protein spot is determined.

The molecular weight of each spot by spot and gel at the bottom of between the measurement of each distance and relatively measuring of the distance that shows with the Precision that also is used for gel or Kaleidoscope molecular weight marker.Have 4 ^ThPolynomial exponential function is applicable to that accurate mark is used for other protein spot location of branch and makes up the difference.Use this method, the molecular weight of each spot can be determined exactly.

The electric charge of isoform (pKa value) is determined by other distance of branch of measuring spot and each gel left side with ImageJ.Because the relation between the physical distance of the pI value of adhesive tape and gel is linear, be determined easily corresponding to the pI value of the different pKa values of isoform spot.

The major protein spot conforms to the isoform of IFN-a2b in the gained gel.The low strength spot may be IFN-a2b or low-level pollutent, yet, because intensity is low, so can not be determined by PMF.Inspection to gel shows that IFN-a2b of the present invention contains 2-22 isoform.Table 9 and 10 shows the main characteristic of these isoforms: pI value (± 1.0), apparent molecular weight (± 20%), and relative intensity (± 20% actual value or ± 2% sum, any one is all bigger).Gel is selected to be comprised in the zone of spot, has only reflected special a little proteic pI and the molecular weight read with the corresponding value in intensity weighted center.Consider the intrinsic otherness of size of protein spots in the 2D gel and position, determine that based on table 9 and 10 listed values the pI value of IFN-a2b of the present invention is between 4.5-7; Determine that based on table 9 and 10 listed values the apparent molecular weight of IFN-a2b of the present invention is between 13-24kDa.

The molecular weight and the pI value of table 9:IFN-a2b isoform

The spot period	Apparent iso-electric point (pI)		Apparent molecular weight MW (kDa)		Relative intensity (%) (standardized value)
									Scope		Scope		Scope
2	5.47	±1.00	17.41	±3.48	11.24	±2.25
							3	5.67	±1.00	17.25	±3.45	32.66	±6.53
4	5.88	±1.00	16.94	±3.39	43.96	±8.79
							5	6.11	±1.00	16.55	±3.31	12.14	±2.43

The molecular weight and the iso-electric point of table 10:IFN-a2b isoform

The spot period	Iso-electric point (pI)		Molecular weight MW (kDa)		Relative intensity (%) (standardized value)
									Scope		Scope		Scope
2	5.04	±1.00	19.40	±3.88	1.79	±2.00
								5.17	±1.00	±19.57	±3.91	4.51	±2.00
4	5.32	±1.00	19.58	±3.92	8.45	±2.00
							5	5.43	±1.00	19.65	±3.93	4.8O	±2.00
6	5.53	±1.00	19.7O	±3.94	8.78	±2.00
							7	5.65	±1.00	19.68	±3.94	8.60	±2.00
8	5.78	±1.00	19.79	±3.96	12.57	±2.51
							9	5.91	±1.00	19.46	±3.89	4.01	±2.00
10	5.98	±1.00	19.28	±3.86	4.72	±2.00
							11	6.06	±1.00	19.33	±3.87	5.96	±2.00
12	6.14	±1.00	19.57	±3.91	1.16	±2.00
							13	6.30	±1.00	19.61	±3.92	2.16	±2.00
14	5.16	±1.00	18.03	±3.61	1.40	±2.00
							15	5.29	±1.00	17.84	±3.57	3.81	±2.00
16	5.42	±1.00	17.70	±3.54	5.89	±2.00
							17	5.53	±1.00	17.80	±3.56	3.82	±2.00
18	5.63	±1.00	17.62	±3.52	5.57	±2.00
							19	5.76	±1.00	17.65	±3.53	6.27	±2.00
20	5.91	±1.00	17.62	±3.52	1.36	±2.00
							21	5.98	±1.00	17.48	±3.50	1.12	±2.00
22	6.07	±1.00	17.44	±3.49	1.46	±2.00
							23	6.30	±1.00	17.26	±3.45	1.81	±2.00

(ii) one dimension polyacrylamide gel electrophoresis

The dry sample of collecting by embodiment 2 (a), and then be dissolved in the 60 μ l 1D sample buffers (10% glycerine, 0.1%SDS, 10mM DTT, 63mM tris-HC1) and 100 ℃ of heating 5 minutes.For PNGaseF handles, 30 μ L parts of sample thief, and add NP40 to final concentration 0.5%.The PNGaseF that adds 5 μ L, sample was cultivated 3 hours at 37 ℃ then.For the Glycosylase cocktail facture of sample, get a part and add NP40 then to final concentration 0.5%.Add PNGase F 1 μ L, reach each 1 μ L of sialidase A (neuramidase), O-glycanase, β (1-4)-tilactase and B-N-acetylglucosaminedase.That handles cultivated 3 hours at 37 ℃ with untreated sample.Handle with untreated sample at prefabricated Tris gel, electrophoresis in Tris 4-20% gradient gel (BioRad) or the Tris HCl gradient gel (Invitrogen) for example.Accurate molecular weight mark (BioRad products catalogue coding 161-0363) also is used for gel.Criterion 4-20% or 18% gel are used for 1D SDS-PAGE (BioRad products catalogue coding: 345-0033 or 345-0024).SDS-PAGE uses MiniProtean II or standard electrophoresis system (BioRad), about 1 hour of 200V electrophoresis or go to the end of gel up to the buffering forward position.The damping fluid that uses as 192mM glycine, 0.1% (w/V) SDS, 24.8 mM Tris alkali under pH8.3.To fix at least 30 minutes among whole glue immersion 10%MeOH and the 7%HAc.Use Sypro Ruby gel dyestuff (BioRad) dyeing at least 3 hours then, and decoloured at least 30 minutes with 10%MeOH and 7%HAc.Perhaps use Deep Purple (Amersham) according to producing specification sheets dyeing.Select suitable spectral filter to make the gel video picture at last with FX laser intensity meter (Biorad).

N-connection oligosaccharides (handling through PNGase) release was connected oligosaccharides (handling through PNGase) and 0-connection oligosaccharides (Glycosylase mixture) release with N-after, the apparent molecular weight of IFN-a2b of the present invention (shown in observing among the SDS-PAGE) was determined.

(iii) protein N-end sequencing

Protein band downcuts and puts into the 0.5ml test tube and add 100ml and leaches damping fluid (100mM sodium acetate, 0.1%SDS, 50mM DTT pH 5.5) from the gel (two-way gel) that as above obtains.Gel slice was 37 ℃ of shaking culture 16 hours.Supernatant liquor is used for ProSorb film (ABI) and uses automatization 494 protein sequencers (Applied Biosystems) to check order according to the specification sheets of producer according to the specification sheets of producer.The sequence (CDLPQTH (S/F) LG) that obtains through evaluation is people IFN-a2b.

(iv) the peptide quality fingerprinting is composed

The protein band that cutting is as above prepared from gel (two-dimentional gel or one dimension gel), wash with 25 μ l lavation buffer solutions (the 50mM bicarbonate of ammonia that contains 50% acetonitrile), gel fragment at room temperature kept 1 hour at least, and vacuum centrifuge was used dry 30 minutes in the back.Gel fragment and 12 μ l trypsin solutions (being dissolved in the 12ng/ μ l order gradient pig trypsinase of bicarbonate of ammonia) suitably are positioned in each sample, hatch 1 hour for 4 ℃.Remove the bicarbonate of ammonia 20 μ l that remaining trypsin solution adds 50mM.Mixture is hatched to jiggle at 37 ℃ and is spent the night.The peptide sample is concentrated the back, and (Millipore, Bedford MA) desalt with C18 Zip-Tips.Binding peptide in 0.8 μ l matrix solution (α of 8 mg/ml-cyano-4-hydroxy styrenes acid (sigma), be dissolved in 70% acetonitrile/1% formic acid) directly on destination disk by wash-out.The peptide quality fingerprinting of tryptic peptide utilizes the biosystem navigation DE-STR of a sensitivity to analyze by peptide matrix assisted laser desorption/ionization time-of-fight mass spectrometry (MALDI-TOF Ms) and produces.Spectrogram obtains by the reflective-mode that uses the 20kV acceleration voltage.Mass calibration uses pancreatin from dissolved peak, and 2211.11Da and 842.51Da carry out as interior mark.The data that produced by peptide quality fingerprinting spectrum (PMF) are used for determining protein identity.Search (main Homo sapien (mankind) and Mammals clauses and subclauses) is carried out in database, for example the SWISS-PROT and TrEMBL, pass through PeptIdent ( WWW.expasy.ch/tools/peptident.html) program.Identification parameter comprises the peptide quality of 0.1Da permissible error, and each peptide is escaped trypsinase cracked maximum segment and methionine sulphoxide and halfcystine-acrylamide and modified.Identification is based on the peptide quality numbering of coupling and the percent of total of the aminoacid sequence of these peptides coverings, in other databases inputs relatively.General, having the peptide quality that at least 30% total sequence covers is necessary for definite identity, but very low and high-quality protein and the protein fragments that obtained by these albumen can not always satisfy these standards, therefore need further identification.

Inc or do not have albumen identity part and can analyze obtain by MALDI-TOF PMF, the peptide mixt of reservation or analyze through tryptic digestion and by electro-spray ionization series connection MS (ESI-MS/MS) from duplicating the identical spot that gel downcuts.For ESI-MS/MS, peptide is by using 1-2 μ l 70% acetonitrile on the Poros R2 micro-column, and 1% formic acid directly is eluted to borosilicate electron spray(ES) syringe needle (Micromass, Manchester, UK).Series connection MS quickens TOF mass spectrograph (Micromass) with three grades/quadrature of Q-Tof mixed type to carry out.The electron spray(ES) syringe needle that contains sample is embedded in source and the steady flow with capillary voltage 900-1200V acquisition.Carry out precursor ion scanning to detect the quality and the electric charge ratio (m/z) of peptide in the mixture.The m/z of each discrete precursor ion picks out and is used to rupture and with the argon gas collision of 18-30eV collision energy.Fragmention (corresponding amino acid whose disappearance from precursor peptide) is recorded and handles with MassLynx Version 3.4 (Micromass).Aminoacid sequence is by inferring out with the mass discrepancy of the y-of Mass Seq (Micromass) program or b-ion ' gradient ' series and determining by manual translation.Then, peptide sequence is used to search for NCBI and TrEMBL database, uses BLASTP program " shortnearly exact matches ".The minimum value of two pairing peptides be sure of it is necessary for what given identity was provided.

Acquired band is proved to be IFN-a2b.

(b) sign of IFN-b1 of the present invention

(i) two-dimentional polyacrylamide gel electrophoresis

The sample of collecting from embodiment 2 (b) is handled and is analyzed according to the foregoing description 3 (a) is (i) described.Spot is through being accredited as the IFN-b1 isoform of invention.

(ii) one dimension polyacrylamide gel electrophoresis

From embodiment 2 (b) (ii), 2 (b) (iii) or the sample collected in (iv) of 2 (b) handle and analyze according to the foregoing description 3 (a) is (ii) described.N-connection oligosaccharides (handling through PNGase) release is connected oligosaccharides (handling through PNGase) and O-connection oligosaccharides (through the Glycosylase mixture) release with N-after, determine the apparent molecular weight (shown in observing among the SDS-PAGE) of IFN-b1 of the present invention.

(iii) proteinic N-end sequencing

Check order according to the (iii) described N-end of the foregoing description 3 (a) to IFN-b1 of the present invention.

(iv) polypeptide quality fingerprinting

IFN-b1 polypeptide quality fingerprinting is measured according to the foregoing description 3 (a) is (iv) described.

The gel spot is through being accredited as IFN-b1.

(c) evaluation of IFN-g of the present invention

(ii) two-dimentional polyacrylamide

The sample that embodiment 2 (c) collects (i) is handled and is analyzed according to embodiment 3 (a).

The major protein spot conforms to the isoform of IFN-g in the gained gel.The low strength spot may be IFN-g or low-level pollutent, yet, because intensity is low, so can not be determined by PMF.Inspection to gel shows that IFN-g of the present invention contains 4-16 isoform.Table 11 and 12 shows the main characteristic of these isoforms: pI value (± 1.0), apparent molecular weight (± 20%), and relative intensity (± 20% actual value or ± 2% sum, any one is all bigger).In comprising the gel area of spot, the intensity weighted center is consistent with the value of listing, and has only reflected special a little proteic pI and the molecular weight read in the selection gel area.Because the intrinsic otherness of protein spots size and position in the 2D gel, so determine that according to table 11 and 12 listed values the pI value of IFN-g of the present invention is between 4-14; Determine that according to table 11 and 12 listed values the apparent molecular weight of IFN-g of the present invention is between 15-30 kDa.

The molecular weight and the pI value of the isoform of table 11:IFN-g

The spot period	Iso-electric point (pI)		Molecular weight (kDa)		Relative intensity (%) (standardized value)
									Scope		Scope		Scope
2	5.58	±1.00	21.94	±4.39	0.52	±2.00
							3	6.10	±1.00	22.42	±4.48	1.90	±2.00
4	6.88	±1.00	22.35	±4.47	10.86	±2.17
							5	8.19	±1.00	23.77	±4.75	9.46	±2.00
6	8.21	±1.00	21.21	±4.24	23.06	±4.61
							7	8.18	±1.00	16.95	±3.39	1.32	±2.00
8	9.64	±1.00	21.58	±4.32	46.44	±9.29
							9	9.93	±1.00	21.22	±4.24	6.46	±2.00

The molecular weight and the pI value of table 12:IFN-g isoform

The spot period	Iso-electric point (pI)		Molecular weight (kDa)		Relative intensity (%) (standard value)
									Scope		Scope		Scope
2	5.55	±1.00	22.92	±4.58	1.13	±2.00
							3	5.80	±1.00	23.72	±4.74	0.58	±2.00
4	5.81	±1.00	22.09	±4.42	0.85	±2.00
							5	6.17	±1.00	23.39	±4.68	0.49	±2.00
6	6.17	±1.00	22.02	±4.40	0.97	±2.00
							7	6.66	±1.00	24.18	±4.84	3.40	±2.00
8	6.66	±1.00	21.57	±4.31	4.33	±2.00
							9	7.37	±1.00	23.82	±4.76	6.78	±2.00
10	7.36	±1.00	21.09	±4.22	7.22	±2.00
							11	8.73	±1.00	23.59	±4.72	16.13	±3.23
12	8.74	±1.00	20.66	±4.13	11.49	±2.30
							13	9.24	±1.00	22.19	±4.44	4.08	±2.00
14	9.82	±1.00	23.07	±4.61	22.59	±4.52
							15	9.86	±1.00	20.67	±4.13	11.53	±2.31
16	10.05	±1.00	22.14	±4.43	4.96	±2.00
							17	10.04	±1.00	20.51	±4.10	3.46	±2.00

(ii) one dimension polyacrylamide gel electrophoresis

The sample that embodiment 2 (c) collects is (ii) handled according to embodiment 3 (a).Sloughing the apparent molecular weight that N-connects the IFN-g of the present invention that (PNGase processing) SDS-PAGE records behind the oligosaccharides is 12～20kDa.Slough N-connect oligosaccharides (PNGase processings) be connected with O-oligosaccharides (processing of Glycosylase mixture) afterwards the apparent molecular weight of the IFN-g of the present invention that records of SDS-PAGE be 12～20kDa.

(iii) protein N-end sequencing

(iii) carry out the N end order-checking of IFN-g of the present invention according to the foregoing description 3 (a).

(iv) peptide quality fingerprinting

The polypeptide quality fingerprinting spectrum of IFN-g of the present invention (iV) is carried out according to the foregoing description 3 (a).

It is IFN-g that the identity of gel spot confirms.

And 2 kinds of NX (S/T/C) motif l-asparagines (N) are modified to Aspartic Acid (D) in the theoretical aminoacid sequence of people IFN-g, and this quality that causes the tryptic peptide that records has changed 1Da.PNGase F can induce the residue that connects l-asparagine (N) in the oligosaccharides subtractive process and become Aspartic Acid (D) at relevant N-to modify, and this is consistent with above-mentioned phenomenon.Therefore, find that 2 definite N-glycosylation sites are respectively N-48 and N-120 (from signal peptide section start numbering) among the IFN-g of the present invention.

(d) sign of IFNAR2-Fc of the present invention

(i) Er Wei polyacrylamide gel electrophoresis

The sample of collecting from embodiment 2 (d) is handled and is analyzed according to the foregoing description 3 (a) is (i) described.

The major protein spot conforms to the isoform of IFNAR2-Fc in the gained gel.The low strength spot may be IFNAR2-Fc or low-level pollutent, yet, because intensity is low, so can not be determined by PMF.Inspection to gel shows that IFNAR2-Fc of the present invention contains 10-25 isoform.Table 13 shows the main characteristic of these isoforms: pI value (± 1.0), apparent molecular weight (± 20%), and relative intensity (± 20% actual value or ± 2% sum, any one is all bigger).In comprising the gel area of spot, the intensity weighted center is consistent with the value of listing, and has only reflected special a little proteic pI and the molecular weight read in the selection gel area.Because the intrinsic otherness of protein spots size and position in the 2D gel, so determine that according to the listed value of table 13 the pI value of IFNAR2-Fc of the present invention is between 4-7; Determine that according to the listed value of table 13 apparent molecular weight of IFNAR2-Fc of the present invention is between 50-105kDa.

Table 13

The molecular weight of IFNAR2-Fc isoform and pI value

The spot period	Iso-electric point (pI)		Molecular weight (kDa)		Relative intensity (%) (standard value)
									Scope		Scope		Scope
2	4.78	±1.00	80.49	±16.10	2.36	±2.00
							3	4.87	±1.00	78.96	±15.79	4.46	±2.00
4	4.96	±1.00	77.34	±15.47	5.58	±2.00
							5	5.04	±1.00	75.72	±15.14	6.96	±2.00
6	5.12	±1.00	74.61	±14.92	5.69	±2.00
							7	5.20	±1.00	73.83	±14.77	5.00	±2.00
8	5.27	±1.00	73.21	±14.64	4.73	±2.00
							9	5.34	±1.00	72.67	±14.53	4.61	±2.00
10	5.41	±1.00	72.05	±14.41	5.16	±2.00
							11	5.49	±1.00	71.25	±14.25	5.62	±2.00
12	5.57	±1.00	70.00	±14.00	3.92	±2.00
							13	5.65	±1.00	68.57	±13.71	6.36	±2.00
14	5.73	±1.00	67.76	±13.55	7.82	±2.00
							15	5.82	±1.00	66.99	±13.40	6.85	±2.00
16	5.91	±1.00	66.23	±13.25	6.O9	±2.00
							17	6.01	±1.00	65.19	±13.04	4.69	±2.00
18	6.10	±1.00	64.34	±12.87	3.40	±2.00
							19	6.20	±1.00	63.74	±12.75	2.08	±2.00
20	6.31	±1.00	63.30	±12.66	1.04	±2.00
							21	5.71	±1.00	52.17	±10.43	1.05	±2.00
22	5.83	±1.00	51.96	±10.39	1.02	±2.00
							23	5.97	±1.00	51.45	±10.29	1.51	±2.00
24	6.12	±1.00	51.01	±10.20	1.50	±2.00
							25	6.29	±1.00	50.42	±10.08	1.44	±2.00
26	6.44	±1.00	50.08	±10.02	1.05	±2.00

(ii) one dimension polyacrylamide gel electrophoresis

The sample that embodiment 2 (d) collects is (ii) handled according to embodiment 3 (a).Sloughing the apparent molecular weight that N-connects the IFNAR2-Fc of the present invention that (PNGase processing) SDS-PAGE records behind the oligosaccharides is 45～95kDa.After sloughing N-and connecting oligosaccharides (PNGase processing) and be connected oligosaccharides (processing of Glycosylase mixture) with O-, the apparent molecular weight of the IFNAR2-Fc of the present invention that SDS-PAGE records is 45～80kDa.

The order-checking of (iii) proteic N end

According to the (iii) described N end order-checking of carrying out IFNAR2-Fc of the present invention of embodiment 3 (a).

(iv) protein mass dactylogram

According to the (iv) described protein mass dactylogram order-checking of carrying out IFNAR2-Fc of the present invention of embodiment 3 (a).

Spot identity determines that it is IFNAR2-Fc.

(e) sign of IL-10 of the present invention

(i) two-dimentional polyacrylamide gel electrophoresis

The sample that embodiment 2 (e) collects (i) is handled and is analyzed according to embodiment 3 (a).

The major protein spot conforms to the isoform of IL-10 in the gained gel.The low strength spot may be IL-10 or low-level pollutent, yet, because intensity is low, so can not be determined by PMF.Inspection to gel shows that IL-10 of the present invention contains 4-28 isoform.Table 14 and 15 shows the main characteristic of these isoforms: pI value (± 1.0), apparent molecular weight (± 20%), and relative intensity (± 20% actual value or ± 2% sum, any one is all bigger).In comprising the gel area of spot, the intensity weighted center is consistent with the value of listing in the table, has only reflected special a little proteic pI and the molecular weight read in the selection gel area.Because the intrinsic otherness of protein spots size and position in the 2D gel, so determine that according to table 11 and 12 listed values the pI value of IL-10 of the present invention is between 6-10; Determine that according to table 14 and 15 listed values the apparent molecular weight of IL-10 of the present invention is between 10-23kDa.

The molecular weight and the pI value of table 14:IL-10 isoform

The spot period	Iso-electric point (pI)		Molecular weight (kDa)		Relative intensity (%) (standard value)
									Scope		Scope		Scope
2	6.35	±1.00	18.27	±3.65	0.62	±2.00
							3	6.51	±1.00	18.52	±3.70	1.83	±2.00
4	6.86	±1.00	17.73	±3.55	5.15	±2.00
							5	7.15	±1.00	17.54	±3.51	4.74	±2.00
6	7.43	±1.00	17.55	±3.51	0.71	±2.00
							7	7.50	±1.00	18.84	±3.77	0.69	±2.00
8	7.76	±1.00	17.57	±3.51	2.11	±2.00
							9	8.64	±1.00	18.17	±3.63	0.52	±2.00
10	8.81	±1.00	18.70	±3.74	1.36	±2.00
							11	6.38	±1.00	16.13	±3.23	0.85	±2.00
12	6.50	±1.00	15.91	±3.18	1.39	±2.00
							13	6.90	±1.00	15.12	±3.02	4.74	±2.00
14	7.21	±1.00	15.15	±3.03	9.62	±2.00
							15	7.44	±1.00	15.72	±3.14	0.71	±2.00
16	7.77	±1.00	14.96	±2.99	18.75	±3.75
							17	8.03	±1.00	14.78	±2.96	3.20	±2.00
18	8.29	±1.00	14.80	±2.96	1.74	±2.00
							19	8.47	±1.00	15.03	±3.01	1.96	±2.00
20	8.47	±1.00	13.77	±2.75	0.84	±2.00
							21	8.26	±1.00	13.28	±2.66	0.98	±2.00
22	8.62	±1.00	15.16	±3.03	2.10	±2.00
							23	8.87	±1.00	14.65	±2.93	24.08	±4.82
24	9.15	±1.00	14.38	±2.88	3.38	±2.00
							25	9.48	±1.00	15.26	±3.05	0.83	±2.00
26	6.91	±1.00	13.20	±2.64	1.83	±2.00
							27	7.14	±1.00	12.75	±2.55	2.68	±2.00
28	7.72	±1.00	11.96	±2.39	1.77	±2.00
							29	8.02	±1.00	12.43	±2.49	0.34	±2.00
30	9.06	±1.00	12.28	±2.46	0.49	±2.00

The molecular weight and the pI value of table 15:IL-10 isoform

The spot period	Iso-electric point (pI)		Molecular weight (kDa)		Relative intensity (%) (standard value)
									Scope		Scope		Scope
2	2.87	±1.00	15.97	±3.19	1.17	±2.00
							3	3.06	±1.00	15.97	±3.19	4.57	±2.00
4	3.28	±1.00	16.01	±3.20	4.73	±2.00
							5	3.48	±1.00	15.95	±3.19	5.52	±2.00

The spot period	Iso-electric point (pI)		Molecular weight (kDa)		Relative intensity (%) (standard value)
									Scope		Scope		Scope
6	3.67	±1.00	15.91	±3.18	4.17	±2.00
							7	3.86	±1.00	15.96	±3.19	3.13	±2.00
8	4.58	±1.00	16.34	±3.27	0.94	±2.00
							9	4.85	±1.00	16.87	±3.37	0.71	±2.00
10	6.01	±1.00	20.13	±4.03	2.12	±2.00
							11	6.00	±1.00	17.00	±3.40	9.17	±2.00
12	6.24	±1.00	17.04	±3.41	6.22	±2.00
							13	6.61	±1.00	17.04	±3.41	7.68	±2.00
14	7.11	±1.00	16.89	±3.38	14.79	±2.96
							15	7.58	±1.00	16.87	±3.37	3.7O	±2.00
16	7.79	±1.00	16.53	±3.31	11.95	±2.39
							17	7.98	±1.00	16.35	±3.27	3.15	±2.00
18	8.12	±1.00	16.51	±3.30	2.22	±2.00
							19	8.28	±1.00	16.47	±3.29	2.22	±2.00
20	8.46	±1.00	l6.46	±3.29	3.14	±2.00
							21	8.62	±1.00	16.41	±3.28	2.82	±2.00
22	8.79	±1.00	16.34	±3.27	5.91	±2.00

(ii) two-dimentional polyacrylamide gel electrophoresis

The sample that embodiment 2 (e) collects is (ii) handled according to embodiment 3 (a).Sloughing the apparent molecular weight that N one connects the IL-10 of the present invention that (PNGase processing) SDS-PAGE records behind the oligosaccharides is 10-23kDa.After sloughing N-and connecting oligosaccharides (PNGase processing) and be connected oligosaccharides (processing of Glycosylase mixture) with 0-, the apparent molecular weight of the IL-10 of the present invention that SDS-PAGE records is 10～23kDa.

(iii) protein N-terminal order-checking

N terminal sequence according to the (iii) described mensuration of the foregoing description 3 (a) IL-10 of the present invention.

(iv) polypeptide quality fingerprinting

According to the polypeptide quality fingerprinting of measuring IL-10 among the present invention with the foregoing description 3 (a) described in (iv).

Gel spot consistence is through being accredited as IL-10.

(f) evaluation of IL-10Ra-Fc of the present invention

(i) two-dimentional polyacrylamide gel electrophoresis

The sample that embodiment 2 (f) collects (i) is handled and is analyzed according to embodiment 3 (a).

The major protein spot conforms to the isoform of IL-10Ra-Fc in the gained gel.The low strength spot may be IL-10Ra-Fc or low-level pollutent, yet, because intensity is low, so can not be determined by PMF.Inspection to gel shows that IL-10Ra-Fc of the present invention contains 10-21 isoform.Table 16 shows the main characteristic of these isoforms: pI value (± 1.0), apparent molecular weight (± 20%), and relative intensity (± 20% actual value or ± 2% sum, any one is all bigger).In comprising the gel area of spot, the intensity weighted center is consistent with the value of listing in the table, has only reflected special a little proteic pI and the molecular weight read in the selection gel area.Because the intrinsic otherness of protein spots size and position in the 2D gel is so determine that according to the listed value of table 16 the pI value of IL-1ORa-Fc of the present invention is between 4.5-9.5; Determine that according to the listed value of table 16 apparent molecular weight of IL-10Ra-Fc of the present invention is between 50-100kDa.

The molecular weight and the pI value of table 16:IL-10Ra-Fc isoform

The spot period	Iso-electric point (pI)		Molecular weight (kDa)		Relative intensity (%) (standard value)
								Scope		Scope		Scope
	2	5.720	±1.00	78.929	±15.79	0.101								±2.00
3							5.821	±1.00	76.835	±15.37	0.177	±2.00
	4	5.916	±1.00	74.95	±14.99	0.319							±2.00
5							6.014	±1.00	73.713	±14.74	0.470	±2.00
	6	6.119	±1.00	72.833	±14.57	0.801							±2.00
7							6.226	±1.00	71.974	±14.39	1.758	±2.00
	8	6.329	±1.00	70.876	±14.18	2.214							±2.00
9							6.430	±1.00	69.696	±13.94	2.663	±2.00
	10	6.536	±1.00	68.708	±13.74	3.454							±2.00
11							6.648	±1.00	67.584	±13.52	5.859	±2.00
	12	6.757	±1.00	66.278	±13.26	5.485							±2.00
13							6.873	±1.00	65.218	±13.04	5.632	±2.00
	14	6.988	±1.00	64.301	±12.86	5.515							±2.00
15							7.114	±1.00	63.707	±12.74	7.698	±2.00
	16	7.254	±1.00	63.057	±12.61	8.326							±2.00
17							7.401	±1.00	63.416	±12.68	11.994	±2.40
	18	7.577	±1.00	62.111	±12.42	12.059							±2.41
19							7.766	±1.00	61.104	±12.22	10.309	±2.06
	20	7.978	±1.00	59.774	±11.95	8.582							±2.00
21							8.204	±1.00	59.000	±11.80	5.977	±2.00
	22	8.412	±1.00	58.931	±11.79	0.604							±2.00

(ii) one dimension polyacrylamide gel electrophoresis

The sample that example 2 (f) is collected is (ii) handled according to embodiment 3 (a).Sloughing the apparent molecular weight that N-connects the IL-10Ra-Fc of the present invention that (PNGase processing) SDS-PAGE records behind the oligosaccharides is 40～85kDa.After sloughing N-and connecting oligosaccharides (PNGase processing) and be connected oligosaccharides (processing of Glycosylase mixture) with O-, the apparent molecular weight of the IL-10Ra-Fc of the present invention that SDS-PAGE records is 36-85 kDa.

(iii) N-end order-checking

N-terminal sequence according to the (iii) described mensuration of the foregoing description 3 (a) IL-10Ra-Fc of the present invention.

(iv) polypeptide quality fingerprinting

According to the polypeptide quality fingerprinting of measuring IL-10Ra-Fc with the foregoing description 3 (a) described in (iv).

Gel spot consistence determines that it is IL-10Ra-Fc.

And 4 kinds of NX (S/T/C) motif l-asparagines (N) are modified to Aspartic Acid (D) in the theoretical aminoacid sequence of people IL-10Ra-Fc, and this quality that causes the tryptic peptide that records has changed 1Da.PNGase F can induce the residue that connects l-asparagine (N) in the oligosaccharides subtractive process and become Aspartic Acid (D) at relevant N-to modify, and this is consistent with above-mentioned phenomenon.Therefore, find that 4 definite N-glycosylation sites are respectively N-110, N-154, N-177 and N-323 (from signal peptide section start numbering) among the IL-10Ra-Fc of the present invention.

Embodiment 4

Proteinic sign of the present invention

(a) amino acid of IFN-a2b of the present invention, monose, oligosaccharides, phosphoric acid salt, vitriol and isoform analysis

(i) be used for the preparation of the sample that amino acid, monose, oligosaccharides, phosphoric acid salt and vitriol and isoform analyze

For the evaluation of monose and oligosaccharides glycosylation and phosphorylation and sulfation posttranslational modification, at first the method by hydrolysis or enzyme removes glycosyl from the polypeptide main chain.The sample buffer component also is removed, and water displacement is to eliminate the restraining effect of hydrolysis and enzyme digestion reaction before analyzing beginning.The IFN-a2b solution of purifying was dialysed 4 days on a large scale by 4 liters of deionization ultrafiltration water (18MOhm) among the PBS, changed liquid twice every day, with the regenerated cellulose dialysis membrane (Spectrapore) of specified molecular weight cut-off (NMWC) 5KDa.After the dialysis, with SaVant Speed Vac (New York, USA) drying solution.Then, the dry sample of crossing is resuspended and be divided into each several part and be used for various analyses with 2ml deionization ultrafiltration water (18Mohm).

(ii) pass through the amino acid composition analysis of vapor phase hydrolysis method

Amino acid in the sample is used by the column front derivation method of 6-quinolylamine base-N-hydroxy-succinamide base-carbamate (AQC) and is analyzed.The fluorescence amino acid derivative of separating stable and by anti-phase (C18) HPLC purifying.Operation is carried out according to Waters AccQTag amino acid analysis method.

Get the sample of 100 μ l IFN-a2b goods and drying in Speed Vac.Then, the exsiccant sample was 110 ℃ of hydrolysis 24 hours.After the hydrolysis, sample after drying before following deriving.Dry sample is dissolved in mark in the 10 μ L amino acid again, and (butyrine, AABA) solution add 35 μ L borate buffer solutions and add 15 μ L AQC derivative reagents afterwards.Reaction mixture heated 12 minutes at 50 ℃ in the constant temperature couveuse.The derivative amino sample changes the automatic sampler of HPLC system over to, and the HPLC system comprises placed in-line waters Alliance 2695 separation assemblies, Waters 474 fluorescent probes and Waters 2487 Dual λ light absorption ratio detectors.Control and analysis software are Waters Empower Pro Module (Waters Corporation, Milford.MA, USA).Sample uses suitable elutriant and gradient current by Waters AccQTag post (15cm * 3.9mm ID).

(iii) neutral and amino monose compositional analysis

Getting the sample of the IFN-a2b goods of two 100 μ l also handles to discharge monose with two kinds of different modes as described below.Three parts of every kind of treatment processs.

1. be heated to 100 ℃ of hydrolysis 4 hours to discharge neutral sugar (semi-lactosi, glucose, trehalose and seminose) with the trifluoroacetic acid (TFA) of 2M.

2. be heated to 100 ℃ of hydrolysis 4 hours to discharge aminosugar (N-acetyl-galn, N-acetyl-glucosamine) with the HCl of 4M.

All hydrolysates are dissolved in 200 μ l again and contain in the water of 0.8nmol internal standard substance with the freeze-drying of Speed Vac system.The internal standard substance that is used for neutral and aminosugar is 2-deoxidation-glucose.Then, sample under 10000g centrifugal 30 minutes to remove the deproteinize fragment.Supernatant liquor changes new test tube over to and by the analysis of high pH anion-exchange chromatography, uses LC 50 systems and GP50 pump and ED50 pulsed current detector (Dionex Ltd) together.Neutral and aminosugar analysis is carried out with Dionex CarboPac PA-20 post.With 10mM equal strength oxyhydroxide enriched material wash-out more than 20 minutes.This process is finished with Dionex EG50 wash-out generation systems.

(iv) acid monose compositional analysis

Get the sample of 100 μ l IFN-a2b goods and handle with the following method to discharge sialic acid monose.Three parts of disposal methods.

Sample with 0.1M TFA 80 ℃ of hydrolysis 40 minutes to discharge N-acetyl and N-glycolyl neuraminic acid.With Speed Vac freeze-drying hydrolysate, be dissolved in 200 μ l again and contain in the water of 0.8nmol internal standard substance.Being used to analyze sialic internal standard substance is lactobionic acid.Then, sample is 10, under the 000g centrifugal 30 minutes to remove the deproteinize fragment.Change supernatant liquor over to new test tube and, use Dionex LC 50 systems and GP50 pump and ED50 pulsed current detector together by the analysis of high pH anion-exchange chromatography.The sialic acid analysis is carried out with DionexCarboPac PA1, uses chromatography parameter known in the art (for example: suitable elutriant and gradient current).

(the v) analysis of oligosaccharides composition

Form in order to analyze oligosaccharides, get each three parts of the IFN-a2b product samples of two 300 μ l, and handle as follows.

1. finish the release that N-connects oligosaccharides with enzyme peptide-N4-(N-acetyl-B-D-glucose amido) l-asparagine acid amides enzyme (PNGase).At first, with 1/5 ^ThThe denaturing soln of volume (2%SDS (sigma)/1 M beta-mercaptoethanol (sigma)) adds in the sample.Sample be heated to 100 ℃ 5 minutes.1/10 ^ThThe 15%Triton-X100 of volume (Sigma) joins in the sample.Sample mixes gently and makes it be cooled to room temperature.Add the PNGase (sigma) of 25 units and hatch at 37 ℃.

2. the method for eliminating by B-is finished the release that O-connects oligosaccharides.At first, with the 4M Sodium Borohydride (new system) of 1/2 volume (Sigma) solution join in the sample.The 0.4M NaOH of 1/2 volume (BDH, HPLC grade) joins in the sample.Sample was cultivated 16 hours down at 50 ℃.Sample cools off in ice and the 0.4M acetate (Sigma) of 1/2 volume is joined in the sample.

N-connects with O-and is connected the further processing of sample to remove the damping fluid composition, uses CarboPac graphitised carbon SPE post.Column equilibration and elution requirement are as follows.

At first, post is used the water balance of two column volumes then with 1 column volume 80% acetonitrile (Sigma) pre-equilibration.Sample is adorned post and with the H of two column volumes under run by gravity ₂The O flushing.For the wash-out neutral oligosaccharides, 2ml 50% acetonitrile is used for post.For the wash-out acidic oligosaccharide, 2ml 50% acetonitrile/0.1% formic acid is used for post.The oligosaccharides of any reservation carries out wash-out by adding 2ml 80% acetonitrile/0.1% formic acid.

Contain neutrality or amino N-connection oligosaccharides are connected oligosaccharides with neutral or amino O-fraction usefulness Speed Vac drying by the SPE post is isolating.Sample is dissolved in 200 μ l water again, and analyzes by high pH anion-exchange chromatography, uses Dionex LC 20 systems and GP50 pump and ED50 pulsed current detector together.Analysis neutral and amino-oligosacchride is to carry out with CarboPac PA100 post and chromatography parameter known in the art (for example: suitable elutriant and gradient current).

(the vi) analysis of vitriol and phosphoric acid salt composition

Vitriol/phosphoric acid salt analysis is undertaken by the method for being described by Harrison and Packer (Harrison and Packer Methods Mol Biol 125:211-216,2000) basically.The sample of getting 100 μ l IFN-a2b goods is used for vitriol/phosphoric acid salt analysis, and in 4M HCl 100 ℃ of following hydrolysis 4 hours.Remove HCl by dry sample in Speed Vac.Then, sample is dissolved in 200 μ l H again ₂O.24 μ l samples inject the Dionex LC50 system that has GP50 pump and ED50 pulsed current detector.Separate by DionexIonPac ISll anion-exchange column, use chromatography parameter known in the art (for example: suitable elutriant and gradient current).

(the vii) further separation of protein isoform

Carry out the further separation of IFN-a2b isoform with the film anion-exchange column.Suitable sample volume, for example, 24 μ l separate by ProPac SAX-10 post (Dionex Ltd), use the Dionex SUMMIT system that has UV-Vis detector (Dionex Ltd).Separate and carry out with suitable elutriant and gradient known in the art.Find the TNF-a isoform in different peaks by wash-out.

(viii) result

Amino acid is formed

Use described reverse high performance liquid chromatography hydrolysis, derive and analyze IFN-a2b and form (table 17) so that following amino acid to be provided.The result is expressed as the weight of the every seed amino acid in the sequence and per-cent (comprising standard deviation (SD)) occurs.Glycine is a known contaminant in the amino acid analysis, has changed amino acid whose composition.If the amount of glycine is counted, the result is consistent with theoretical value.Other various amino acid whose per-cents also are slightly higher than the theoretical level of expectation.This may exist low-level pollutent to cause.

Table 17

Amino acid is formed
						Amino acid	The 1st takes turns	The 2nd takes turns	The 3rd takes turns	Mean value	Standard deviation
D	10.58	8.20	9.22	9.33	1.19
						S	8.73	9.17	7.02	8.31	1.14
E	16.83	l4.00	16.08	15.64	1.47
						G	6.72	14.72	9.82	10.42	4.03
H	1.26	1.49	1.67	1.47	0.21
						R	4.89	5.20	5.02	5.04	0.16
T	4.38	3.99	5.50	4.62	0.78
						A	6.84	6.89	6.37	6.70	0.29
P	3.62	3.71	3.10	3.47	0.33
						Y	2.11	2.57	1.97	2.22	0.31
V	4.70	4.50	4.65	4.62	0.11
						M	1.62	0.16	1.93	1.24	0.94
K	7.06	6.18	7.62	6.95	0.73
						I	4.66	4.63	4.51	4.60	0.08
L	11.52	10.68	11.23	11.14	0.42
						F	4.49	3.91	4.29	4.23	0.30
Amount to	100.00	100.00	100.00	100.00

The monose result

Each monose is obtained by hydrolysis on the amino acid backbone of IFN-a2b, and obtains following compositional analysis by high pH anion chromatographic (HP AEC) analysis as described.Glucose is common pollutent, is connected in the monose uncommon with O-in the N-connection.The result of sample is that standard (table 18～20) is passed through the GalNAc stdn by the result that sample obtains with GalNAc.Table 21 derives from the result's of three duplicate samples summary.

Table 18

The 1st monose of taking turns is formed
			Monose	Nmol/nmol albumen	With GalNAc is standard
Fucose	0.00	0.00
			GalNAc	2.56	1.00
GlcNAc	0.00	0.00
			Semi-lactosi	4.94	1.92
Glucose	16.86	6.57
			Seminose	0.00	0.00
NeuAC	— 0.00	0.00
			NcuGc	0.00	0.00

Table 19

The 2nd takes turns the composition of monose
			Monose	Nmol/nmol albumen	With GalNAc is standard
Fucose	0.00	0.00
			GalNAc	2.30	1.00
GlcNAc	0.00	0.00
			Semi-lactosi	2.62	1.14
Glucose	16.97	7.37
			Seminose	0.00	0.00
NeuAC	0.00	0.00
			NeuGc	0.00	0.00

Table 20

The composition of the 3rd monose of taking turns
			Monose	Nmol/nmol albumen	With GalNAc is standard
Fucose	0.00	0.00
			GalNAc	2.41	1.00
GlcNAc	0.00	0.00
			Semi-lactosi	8.26	3.42
Glucose	34.29	14.21
			Seminose	0.00	0.00
NeuAC	0.00	0.00
			NeuGc	0.00	0.00

Table 21

Monose is formed
					Monose	Nmol/nmol protein		With GalNAc is standard
	Minimum	Maximum	Minimum	Maximum
					Fucose	0.00	0.00	0.00	0.00
GalNAc	2.30	2.56	1.00	1.00
					GlcNAc	0.00	0.00	0.00	0.00
Semi-lactosi	2.62	8.26	1.14	3.42
					Glucose	16.86	34.29	6.57	14.21
Seminose	0.00	0.00	0.00	0.00
					NeuNAC	0.00	0.00	0.00	0.00
NeuGlv	0.00	0.00	0.00	0.00

Because the intrinsic otherness of above-mentioned chromatographic process, so as monose and sialic acid content among the IFN-a2b of the present invention is standard with GalNAc, measurement result is about 1: 0-3 trehalose, 1: 0-3 GlcNAc, 1: 0-6 semi-lactosi, 1: 0-3 seminose and 1: 0-5 NeuNAc; In specific embodiments, be 1: 0-1 trehalose, 1: 0-1GlcNAc, 1: 1-4 semi-lactosi, 1: 0-1 seminose and 1: 0-2 NeuNAc.

Because the intrinsic otherness of above-mentioned chromatographic process, so contents of monosaccharides percentage test scope is approximately 0～10% among the IFN-a2b of the present invention; O-connection oligosaccharides percentage test scope approximately is 0～20% among the IFN-a2b of the present invention.

(b) analysis of the amino acid of IFN-b1 of the present invention, monose, oligosaccharides, phosphoric acid salt, vitriol and isoform composition

(i) be used for the specimen preparation that amino acid, monose, oligosaccharides, phosphoric acid salt, vitriol and isoform are analyzed.

According to (i) the IFN-b1 solution of described processing purifying of the foregoing description 4 (a).

(ii) form with vapor phase hydrolysis methods analyst amino acid

According to the (ii) described processing of the foregoing description 4 (a) IFN-b1 formulation samples

The analysis that (iii) neutral and amino monose is formed

According to the (iii) described processing of the foregoing description 4 (a) IFN-b1 formulation samples

The analysis that (iv) acid monose is formed

According to the (iv) described processing of the foregoing description 4 (a) IFN-b1 formulation samples

(v) monose compositional analysis

According to the foregoing description 4 (a) (v) described processing IFN-b1 formulation samples

(vi) vitriol and phosphoric acid salt compositional analysis

According to the foregoing description 4 (a) (vi) described processing IFN-b1 formulation samples

(vii) the protein isoform is further purified

According to the foregoing description 4 (a) (vii) described processing IFN-b1 formulation samples.The various isoform elution peaks of IFN-b1 are obviously different.

(c) analysis of the amino acid of IFN-g of the present invention, monose, oligosaccharides, phosphoric acid salt, vitriol and isoform composition

(i) be used for the specimen preparation that amino acid, monose, oligosaccharides, phosphoric acid salt, vitriol and isoform are analyzed.

According to (i) the IFN-g solution of described processing purifying of the foregoing description 4 (a).

(ii) form with vapor phase hydrolysis methods analyst amino acid

According to the (ii) described processing of the foregoing description 4 (a) IFN-g formulation samples.

The analysis that (iii) neutral and amino monose is formed

(iii) handle described IFN-g formulation samples according to the foregoing description 4 (a).

The analysis that (iv) acid monose is formed

(iv) handle the sample of IFN-g preparation according to the foregoing description 4 (a).

(the v) analysis of oligosaccharides composition

(a) (v) handles the sample of IFN-g preparation according to the foregoing description 4.

(the vi) analysis of vitriol and phosphoric acid salt composition

(a) (vi) handles the sample of IFN-g preparation according to the foregoing description 4.

(the vii) further separation of albumen isoform

(a) (vii) handles the sample of IFN-g preparation according to the foregoing description 4.The various isoform elution peaks of IFN-g are obviously different.

(d) analysis of the amino acid of IFNAR2-Fc of the present invention, monose, oligosaccharides, phosphoric acid salt, vitriol and isoform composition

(i) be used for the specimen preparation that amino acid, monose, oligosaccharides, phosphoric acid salt, vitriol and isoform are analyzed.

According to (i) the PBS solution of the IFNAR2-Fc of described processing purifying of the foregoing description 4 (a).

(ii) form with vapor phase hydrolysis methods analyst amino acid

According to the (ii) described processing of the foregoing description 4 (a) IFNAR2-Fc formulation samples.

The analysis that (iii) neutral and amino monose is formed

(iii) handle described IFNAR2-Fc formulation samples according to the foregoing description 4 (a).

The analysis that (iv) acid monose is formed

(iv) handle the sample of IFNAR2-Fc preparation according to the foregoing description 4 (a).

(the v) analysis of oligosaccharides composition

(a) (v) handles the sample of IFNAR2-Fc preparation according to the foregoing description 4.

(the vi) analysis of vitriol and phosphoric acid salt composition

(a) (vi) handles the sample of IFNAR2-Fc preparation according to the foregoing description 4.

(the vii) further separation of albumen isoform

(a) (vii) handles the sample of IFNAR2-Fc preparation according to the foregoing description 4.The various isoform elution peaks of IFNAR2-Fc are obviously different.

(e) analysis of the amino acid of IL-10 of the present invention, monose, oligosaccharides, phosphoric acid salt, vitriol and isoform composition

(i) be used for the specimen preparation that amino acid, monose, oligosaccharides, phosphoric acid salt, vitriol and isoform are analyzed.

According to (i) the PBS solution of the IL-10 of described processing purifying of the foregoing description 4 (a).

(ii) form with vapor phase hydrolysis methods analyst amino acid

According to the (ii) described processing of the foregoing description 4 (a) IL-10 formulation samples.

The analysis that (iii) neutral and amino monose is formed

(iii) handle described IL-10 formulation samples according to the foregoing description 4 (a).

The analysis that (iv) acid monose is formed

(iv) handle the sample of IL-10 preparation according to the foregoing description 4 (a).

(the v) analysis of oligosaccharides composition

(a) (v) handles the sample of IL-10 preparation according to the foregoing description 4.

(the vi) analysis of vitriol and phosphoric acid salt composition

(a) (vi) handles the sample of IL-10 preparation according to the foregoing description 4.

(the vii) further separation of albumen isoform

(a) (vii) handles the sample of IL-10 preparation according to the foregoing description 4.The various isoform elution peaks of IL-10 are obviously different.

(f) analysis of the amino acid of IL-10Ra-Fc of the present invention, monose, oligosaccharides, phosphoric acid salt, vitriol and isoform composition

(i) be used for the specimen preparation that amino acid, monose, oligosaccharides, phosphoric acid salt, vitriol and isoform are analyzed.

According to (i) the PBS solution of the IL-10Ra-Fc of described processing purifying of the foregoing description 4 (a).

(ii) form with vapor phase hydrolysis methods analyst amino acid

According to the (ii) described processing of the foregoing description 4 (a) IL-10Ra-Fc formulation samples.

The analysis that (iii) neutral and amino monose is formed

(iii) handle described IL-10Ra-Fc formulation samples according to the foregoing description 4 (a).

The analysis that (iv) acid monose is formed

(iv) handle the sample of IL-10Ra-Fc preparation according to the foregoing description 4 (a).

(the v) analysis of oligosaccharides composition

(a) (v) handles the sample of IL-10Ra-Fc preparation according to the foregoing description 4.

(the vi) analysis of vitriol and phosphoric acid salt composition

(a) (vi) handles the sample of IL-10Ra-Fc preparation according to the foregoing description 4.

(the vii) further separation of albumen isoform

(a) (vii) handles the sample of IL-10Ra-Fc preparation according to the foregoing description 4.The various isoform elution peaks of IL-10Ra-Fc are obviously different.

(vii) result

Amino acid is formed

IL-10Ra-Fc obtains following amino acid with described reverse efficient liquid phase chromatographic analysis and forms (table 22) behind hydrolysis and derivatize.The result is expressed as the per-cent and the weight (comprising standard deviation (SD)) of the appearance of each in the sequence.Glycine is a known contaminant in the amino acid analysis, has changed amino acid whose composition.If the amount of glycine is counted, the result is consistent with theoretical value.

Table 22

Amino acid is formed
						Amino acid	The 1st takes turns	The 2nd takes turns	The 3rd takes turns	Mean value	Standard deviation
D	9.5	8.70	9.5	9.11	0.42
						S	8.8	9.05	8.8	8.91	0.14
E	10.4	9.98	10.4	10.21	0.21
						G	9.3	9.69	9.3	9.49	0.19
H	3.3	3.47	3.3	3.35	0.10
						R	4.3	4.61	4.3	4.41	0.18
T	7.1	7.32	7.1	7.17	0.14
						A	4.2	4.03	4.2	4.15	0.10
P	7.7	7.69	7.7	7.73	0.04
						Y	3.4	3.62	3.4	3.49	0.12
V	8.6	8.64	8.6	8.61	0.03
						M	0.9	0.64	0.9	0.83	0.16
K	6.9	6.57	6.9	6.82	0.22
						I	3.8	3.74	3.8	3.76	0.02
L	7.7	7.86	7.7	7.83	0.07
						F	4.0	4.38	4.0	4.13	0.22
Amount to	100.0	l00.00	100	100.00

Monose and vitriol

The single monose of IL-10Ra-Fc main chain hydrolysis and vitriol obtain down compositional analysis through aforesaid High pH value Zeo-karb (HP AEC) analysis.Sample result is standard (table xXx-xxx) with the seminose of Ga1NAc and 3 times of amounts respectively.Table xxx has summed up the result of 3 duplicate samples.Glucose is common pollutent, connects oligosaccharides at N-and is connected in the oligosaccharides uncommon with O-.The GlcNAc level is higher, may be that pollutent and GlcNAc co-elute cause in the analytic process.

Table 23

The 1st monose of taking turns is formed
			Monose	With GalNAc is standard	With the seminose is standard
Fucose	2.12	0.78
			GalNAc	1.00	0.37
GlcNAc	19.44	7.13
			Semi-lactosi	5.57	2.04
Glucose	1.15	0.42
			Seminose	8.18	3.00
NeuAc	0.23	0.09
			NeuGc	0.00	0.00
SO 4	1.45	0.53

Table 24

The 2nd monose of taking turns is formed
			Monose	With GalNAc is standard	With the seminose is standard
Fucose	1.06	0.80
			GalNAc	1.00	0.75
GlcNAc	22.41	16.89
			Semi-lactosi	2.58	1.95
Glucose	0.46	0.35
			Seminose	3.98	3.00
NeuAc	0.14	0.11
			NeuGc	0.00	0.00
SO 4	0.49	0.37

Table 25

The 3rd monose of taking turns is formed
			Monose	With GalNAc is standard	With the seminose is standard
Fucose	0.82	0.82
			GalNAc	1.00	1.00
GlcNAc	19.36	19.29
			Semi-lactosi	1.96	1.95
Glucose	0.36	0.36
			Seminose	3.01	3.00
NeuAc	0.15	0.15
			NeuGc	0.00	0.00
SO 4	0.13	0.13

Table 26

Monose is formed
					Monose	With GalNAc is standard		With the seminose is standard
	Minimum	Maximum	Minimum	Maximum
					Fucose	0.82	2.12	0.78	0.82
GalNAc	1.00	1.00	0.37	1.00
					GlcNAc	19.36	22.41	7.13	19.29
Semi-lactosi	1.96	5.57	1.95	2.04
					Glucose	0.36	1.15	0.35	0.42
Seminose	3.01	8.18	3.00	3.00
					NeuAc	0.14	0.23	0.09	0.15
NeuGc	0.00	0.00	0.00	0.00
					SO 4	0.13	1.45	0.13	0.53

Because the intrinsic otherness of above-mentioned chromatographic process, so as monose, sialic acid and sulphate content among the IL-10Ra-Fc of the present invention is standard with GalNAc, measurement result is about 1: 0.1-4 trehalose, 1: 2-34 G1cNAc, 1: 0.5-8 semi-lactosi, 1: 1-13 seminose, 1: 0-3NeuNAc and 1: 0-1.5 vitriol; Is standard as monose, sialic acid and sulphate content among the IL-10Ra-Fc of the present invention with 3 times of seminoses, and measurement result is 3: 0.1-2 trehalose, 3: 0.01-3 GalNAc, 3: 1-30GlcNAc, 3: 0.1-4 partly inspires confidence in biose, 3: 0-3NeuNAc and 3: 0-0.6 vitriol.

Content not of the same race comprises the relevant data (table 27) of amino acid composition, monose and vitriol.

Table 27

The content that calculates
		The weight percent of carbohydrate
Be expressed as the sulphating of the per-cent of contents of monosaccharides	1.8
		N-connects the neutral per-cent of oligosaccharides	64
N-connects the acid per-cent of oligosaccharides	36

Because the intrinsic otherness of above-mentioned chromatographic process approximately is 0～10% so be expressed as the sialic measurement range of the contents of monosaccharides per-cent of IL-10Ra-Fc of the present invention.Be expressed as the sulphating of the contents of monosaccharides per-cent of IL-10Ra-Fc of the present invention, measurement range approximately is 0～3%, and the measurement range that the N-of IL-10Ra-Fc of the present invention connects the acid per-cent of oligosaccharides is about 25～45%.

Embodiment 5

Saccharic amount dactylogram

Separate protein of the present invention and be adsorbed onto on polyvinylidene difluoride (PVDF) (PVDF) film with 2D gel electrophoresis technology as embodiment 3.Spot dyes with the protein dye (colloid Coomassie blue, Sypro Ruby or Deep Purple) of a normal content, and the relative quantity optical density standard measure of isoform.Cut off each spot and handle, depend on the circumstances with a series of deglycosylating enzymes and/or chemical process, removing the oligosaccharides that exists, the method for describing according to this specification sheets.In case oligosaccharides is removed, it is separated and analysis in the liquid chromatography one electrospray ionization mass spectrum system (LC-MS) that uses graphitized carbon post and organic solution (MeCN) gradient elution system.The peak shape that produces has produced " dactylogram " of the oligosaccharides that exists in the isoform.Further, mass spectrometer system produces signal to the quality of the various sugar that exist in the sample, its by with the pattern match of GlycoSuite database to discern their structure.In addition, each mass peak can many times of fragmentations to provide MS " spectrum.These fragments can obtain the prediction of structure, use methods known in the art, for example, and by the use of GlycosidIQ routine package.

The corresponding protein of expressing with inhuman cell system (such as intestinal bacteria, yeast and Chinese hamster ovary celI) has repeated above to separate, de-glycosylation and analytical procedure, and there is significant difference in their saccharic amount fingerprint.Say that on structure level this result shows that albumen of the present invention is different with the proteic glycan structures of corresponding inhuman cell expressing.

Embodiment 6

Fluorescence assisted carbohydrate electrophoresis

Obtain the oligosaccharides profile of target molecule with fluorescence assisted carbohydrate electrophoresis experiment (FACE protocol).From the oligosaccharides of purpose cytokine with ammonium hydroxide from the amino acid backbone hydrolysis, then with the amino naphthyl-1,3 of fluorescence 8-, 6-trisulfonic acid (ANTS) mark.The standard that polyacrylamide gel electrophoresis is used to separate kind and is used for the distinctive oligosaccharides profile of identifying purpose molecule.Further, discern oligosaccharides with the substance assistant laser desorpted and ionization-time of the flight mass spectrum (MALDI-TOF) that depends on fluorescence with to the special matrix of the ionization of various sugar.Measure the quality of various sugar and with GlycoSuite database identification of protein structure.Potential sugar constitutional features is further by the tandem mass spectrum technical description, and it obtains, and oligosaccharides exists and feature descriptions part or whole of their relative quantity.Further, repeated use is discerned the method for isoform to produce the profile of the oligosaccharides that exists in isolating each isoform by the 2D gel electrophoresis.

Embodiment 7

QCM and SPR

With quartz crystal microbalance (QCM) or surface plasma resonance (SPR) testing goal molecule in conjunction with feature and activity.In two kinds of methods, the chemical action that suitable molecular receptor is described with manufacturer is attached on the wafer.Molecules of interest is dissolved in the suitable biological buffer and makes it by the acceptor interaction on damping fluid and the chip.By the change that changes oscillation frequency (in the QCM method) or measure the gross protein quality on the wafer surface by the light scattering characteristic (in the SPR method) that changes chip.Then, only discharge back in the solution to observe molecules of interest with the biological buffer process chip.Then, acceptor reaches capacity and is used to calculate the binding curve of molecules of interest with complete isolating speed.

Embodiment 8

The generation of genetically modified host cell system

(a) contain α-2, the genetically modified host cell system of 6-sialytransferase

Coding for alpha-2, (α 2, and cDNA 6ST) is increased by poly (A)-primed cDNA by PCR for the 6-sialytransferase.The PCR product is connected in the suitable carriers, and for example pIREspuro4 or pCEP4 are to produce magnesium α 2,6ST plasmid.To clone's cDNA order-checking and by with disclosed α 2,6sT cDNA sequence relatively confirms its identity.Dna sequencing carries out with known method.

Mammalian host cell, the cell strain system (clone-molecules of interest) that comprises the identical pedigree of expressing high-level molecules of interest be with α 2, the 6ST plasmid transfection, and it also carries the antibiotics resistance mark.Existing down by microbiotic, culturing cell carries out the selection of stable transfectional cell; Show the cell strain system of antibiotic-resistant and be used for α 2 in the cell, the active check of 6ST after collecting transfection.In order to separate express alpha 2, the individual cells strain system of 6ST, the restriction dilution method clone of cell harvesting thing by describing as Kronman (Gene121:295-304,1992).One cell strain system is selected at random, the α 2 of cell amplification and detection strain system, 6ST activity.

Washed cell precipitates, and is resuspended in the dissolving damping fluid, and is prepended in the ice at ultrasonic degradation.Cellular lysate is centrifugal and clarifying supernatant liquor is used for protein concn (via currently known methods) and sialytransferase activation analysis.Sialytransferase is active in currently known methods, for example described methods analyst of Datta et al. (J Biol Chem 270:1497-1500,1995).

From high expression level α 2, the molecules of interest that purifying is expressed in 6ST clone-molecules of interest cell and carry out external and/or body in transformation period biological assay (seeing embodiment 10).From high expression level α 2, the molecules of interest of 6ST cell is compared with deriving from not through the molecules of interest of the identical parental cell line of any transgeneic procedure or the molecules of interest that derives from other clones, shows the transformation period in the external and/or body of increase.

(b) contain the genetically modified host cell system of mycose-base transferring enzyme

The cDNA of encoding trehalose based transferase (FT), for example FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, FUT10, FUT11 are increased by poly (A)～primed cDNA by PCR.The PCR product connects in the suitable carriers, and for example pIREspuro4 or pCEP4 are to produce instrument α 2,6ST plasmid.Clone's cDNA order-checking and by with the identity that relatively confirms it of disclosed FT cDNA sequence.Dna sequencing carries out with currently known methods.

The human host cell comprises the cell strain system FT plasmid transfection of the identical pedigree of expressing high-level molecules of interest molecule (clone-molecules of interest), and it also carries the antibiotics resistance mark.The selection of stable transfectional cell is carried out in cultivation by cell in the presence of microbiotic; Collect the strain system of the cell that shows antibiotic-resistant after the transfection and be used for the active check of FT in the cell.In order to separate the individual cells strain system of expressing FT, the cell harvesting thing is by restriction dilution method clone, such as Kronman 1992 description; Individual cells strain system is selected at random, the FT activity of cell amplification and detection strain system.

Washed cell precipitates, and is resuspended in the dissolving damping fluid, and is prepended in the ice at ultrasonic degradation.Centrifugal and the clarifying supernatant liquor of cellular lysate is used for protein concn (through currently known methods) and FT activation analysis.The FT activity is by the currently known methods analysis, and for example Mas et al. (Glycobiology 8 (6): 605-13,1998) is described.

The molecules of interest of expressing is by high expression level FT clone-molecules of interest cell purification.The Lewisx-specific antibody, for example L5 and sialylated Lewis x-specific antibody, KM93 for example, HECA493,2H5 or CSLEX, be used to detect the existence of Lewis x or sialylated Lewis x structure, according to methods known in the art, for example, (Glycobiology 15 (1): 87,2005) describe in detail as Lucka et al..Perhaps, the existence of Lewis x or sialylated Lewis x structure can be by handling sample detection and detecting the influence of Glycosylase on parameter with suitable Glycosylase, for example quality during usefulness Ms or the retention time during with HPLC.Saccharic amount dactylogram as describing among the embodiment 5, also can be used to predict the existence of Lewis x or sialylated Lewis x structure.

Embodiment 9

Differential gene expression

The difference of genetic expression can be analyzed with the purpose clone of molecules of interest.The purpose cell grows into proper density and with the molecules of interest of the concentration of certain limit or damping fluid control treatment a plurality of hours, for example, and 72 hours.

In the different time, carry out the RNA results, purifying and reverse transcription are according to the Affymetrix experimental program.Then, the cRNA (biological example plain mark) of preparation mark and with express matrix hybridization, for example U133 GeneChips.Washing and signal amplify then, and GeneChips is with GeneChip scanner (Affymetrix) scanning, and intensity for hybridization and obtain with GeneChip software (Affymetrix) at the multiple change information of different time points.

Molecules of interest induce unique genetic expression and cause with by different sources, for example E.coli, yeast or the cytokine of Chinese hamster ovary celI generation or the profile of receptor-inducible are compared different mRNA profiles.

Embodiment 10

The half life determination of molecules of interest of the present invention

The transformation period of molecules of interest is measured in vitro system.The composition that comprises molecules of interest is mixed in the human serum and in certain temperature cultivates certain hour (for example 37 degree were cultivated 12 hours etc. 4 hours).Measure by ELIsA method known in the art or dot blotting in this amount of handling the molecules of interest of back reservation.The biologic activity of the molecules of interest that keeps is measured by the suitable biological detection method selected by those skilled in the relevant art.Selected serum can come from multiple human blood group (for example A, B, AB, O etc.).

The transformation period of molecules of interest is also measured in the system in vivo.The composition that comprises molecules of interest by radioactive tracer mark (or additive method) and through vein, subcutaneous, back eye socket, intramuscular or peritoneal injection to for studying in the selected species, for example, mouse, rat, pig, primates or people.Time point blood sampling after injection and the existence of molecules of interest analyzed (by the ELISA method, dot blotting or by trichoroacetic acid(TCA) (TCA)-precipitation mark, for example radiocounting).Contain generation from different sources, E.coli for example, yeast, or the contrast that relatively can be used as of the composition of the molecules of interest of Chinese hamster ovary celI is carried out.

Embodiment 11

(a) in vivo test of molecules of interest of the present invention

The individual subjects of in vivo test described herein is a warm-blooded vertebrate, comprises the people.

Clinical trial is subjected to strictly controlling to guarantee that individuality is not interposing in the unnecessary risk and they are informed their effect under study for action fully.

Preferably, in order to compensate the psychological application of receiving treatment, treatment is carried out in the double blinding mode.Contrast or molecules of interest treatment group are gone in volunteer's random assignment.Further, it also is blind that relevant clinician gives receptor's treatment system for administration, to prevent there is prejudice in the observation after their treatment.Use this random fashion, each volunteer has the identical chance that is given new treatment or contrast.

Volunteers received molecules of interest or dummy for some time, simultaneously, the morbid state that associative list reveals or the biological parameter of healthy state be in beginning (baseline any treatment before), end (after final treatment) and studying during measured in the interval of rule.This measurement comprises the body fluid of comparing with level before the treatment, the level of molecules of interest in tissue or the organ.Other measurements include, but not limited to morbid state or by the index of the healthy state of being treated, body weight, and blood pressure, the serum titer of the pharmacology indicator of disease, for example special disease indicator or toxicity and ADME (absorb, distribute metabolism and drainage) measure.

Each patient's the information of record comprise the age (year), sex, height (cm), morbid state or sick family history (be/not), motivation grade (few/in/big) and number and for the kind of the therapy formerly of disease that shows or disease.

The volunteer who participates in this test is the grownup, and the age was at 18 to 65 years old and add the number approximate equality of the masculinity and femininity of test.Volunteer with some feature is evenly distributed for dummy and molecules of interest treatment.General, with the volunteer of dummy treatment to treatment have seldom or not reaction, yet when off-test, the morbid state or the sick index that demonstrate them with the volunteer of molecules of interest treatment trend towards the positive.

(b) with IFN-a2b treatment chronic hepatitis C of the present invention

The individual subjects of in vivo test as herein described is a warm-blooded vertebrate, comprising the people.

Strict control is carried out in this clinical trial guaranteed that with this individuality does not face unnecessary risk, and guarantee individual their effect under study for action of knowing fully.

In specific embodiments, consider the psychologic effect of receiving treatment, carried out the test of double blinding mode.The volunteer is assigned to recombinantinterferon 2-b (IFN-a2b) the treatment group and the IFN-a2b treatment group of the present invention of escherichia coli expression at random.And, in order to prevent the deviation of doctor in treatment, to the doctor do not inform yet IFN-a2b that the sort of E.coli of being of medicine that they used expresses the sort of be IFN-a2b of the present invention.Design has guaranteed that each routine experimenter has identical chance to accept existing therapy and new therapy at random.

The experimenter accepts treatment for some time that E.coli expresses IFN-a2b and IFN-a2b of the present invention, the certain hour interval measurement Biological indicators relevant with chronic hepatitis C before treatment during (accept any treatment before base measurement), treatment back (finishing the back in last treatment) and the treatment.Measurement index comprises the IFN-a2b level of body fluid, tissue and organ and the neutralizing antibody of anti-IFN-a2b, and compares with level before the treatment.Other mensuration not only is confined to the serum titre of the pharmacology index of body weight, blood pressure and chronic hepatitis C, glutamate transaminase) or toxicity for example specificity liver enzyme (for example:, comprise that also ADME (absorption, distribution, metabolism and drainage) is such as serum half-life and solubility.

The information of every patient record comprises the number of times and the type of age (year), sex, height (cm), family's medical history (be or not), motivation scoring (slight/moderate/severe), the past chronic hepatitis C treatment plan.

The volunteer who participates in this test is the grownup, and the age was at 18 to 65 years old and add the number approximate equality of the masculinity and femininity of test.The volunteer who possesses certain feature divides equally IFN-a2b treatment group and the IFN-a2b treatment group of the present invention that appointment enters escherichia coli expression.Substantially, research conclusion shows that IFN-a2b treatment group volunteer of the present invention expresses IFN-a2b treatment group with the enterobacteria of purchase and compares, and liver function is clearly better.

(c) IFN-b1 of the present invention adds determining of recombinant human erythropoietin (rhEPO) therapy combination therapy multiple sclerosis validity

The individual subjects of in vivo test as herein described is a warm-blooded vertebrate, comprising the people.

Strict control is carried out in this clinical trial guaranteed that with this individuality does not face unnecessary risk, and guarantee individual their effect under study for action of knowing fully.

On special embodiment, in order to eliminate the psychologic effect of receiving treatment, clinical trial designs for double blinding.The experimenter is randomized into best background (OB) dosage regimen combination therapy group and the placebo (the best background (OB) of recombinant human erythropoietin (EPO) therapy) that IFN-b1 of the present invention adds recombinant human erythropoietin (EPO) therapy.Design makes each routine experimenter have identical chance branch to go into treatment group and placebo at random.

The acute demyelination patient that multiple sclerosis causes appears in the clinical trial experimenter.The information of every patient record comprises the number of times and the type of age (year), sex, height (cm), family's medical history (be or not), motivation scoring (slight/moderate/severe), the past multiple sclerosis therapy scheme.

The experimenter accepts the OB dosage regimen treatment of IFN-b1 associating EPO therapy of the present invention, and IFN-b1 route of administration wherein of the present invention comprises intracutaneous, intramuscular and subcutaneous injection, but is not limited to above route of administration.

The experimenter that IFN-b1 of the present invention adds best background (OB) the dosage regimen combination therapy group of recombinant human erythropoietin (EPO) therapy and placebo accepts the treatment of for some time, should be before clinical trial be implemented (base measurement before the treatment), treatment stop the acute demyelination related biological parameter that the periodic measurement multiple sclerosis causes during back (final treatment finish after) and the clinical study.The parameter of measuring comprises the IFN-b1 and the EP0 level of body fluid, tissue, organ, and compares with the preceding level of treatment.Other testing index comprises that not only treatment morbid state index (for example: Multiple Sclerosis Functional Composite Score (MSFC)), the serum titre and the toxicity of body weight, blood pressure, anti-IFN β and anti-EP0 neutralizing antibody (NAbs), and comprise ADME (absorption, distribution, metabolism and drainage) and health-related quality of life, but not only be confined to above-mentioned parameter.

One of IFN-b1 treatment group patient of the present invention or multinomial multiple sclerosis index are lower, not only the serum titre of lower, anti-IFN β of Ju Xian MsFC score and anti-EP0 neutralizing antibody (NAbs) is lower, and the survival time, longer and overall general health situation was better.

Embodiment 12

(a) comparison of biologic activity between the IFN-a2b that expresses in IFN-a2b and the non-human cell system among the present invention

IFN-a2b has cytotoxicity to human erythroleukemia cell TF-1 clone.GM-CSF handles back TF-1 cell and breeds, and IFN-a2b then suppresses this propagation, causes necrocytosis.

In 96 orifice plates, 20000 the TF-1 cells in every hole are handled cell, 37 ℃, 68 hours with the IFN-a2b 0-100ng/ml among 0.2ng/mlGM-CSF (PeDrotech Cat.No 300-03 or R﹠D system, Cat. No 215-GM-010) and the present invention.Use CellTiter 96 aqueous solution analysis of cell proliferation (Promega) to measure cell quantity then.In this is analyzed, the tetrazolium salts compound MTS that in electron coupling reagent (phenazine methosulfate), exists (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) is reduced to formazan by cell biological and rises product.The concentration that formazan is risen generates the absorbance measurement of solution at 490nm by reading with spectrophotometer (E max precisionmicroplate reader, Molecular Devices).

Adopt the IFN-a2b that produces in E.coli (WHO 95/566) intestinal bacteria to repeat above-mentioned detection.ED50 value separately adopts the logarithmic value of absorption value to calculate than IFN-a2b concentration and one 4 parameter equation.

ED50 (4.4 to 6.6 ng/ml of the human IFN-a2b that produces in the ED50 value (0.011 to 0.017ng/ml) of IFN-a2b and the E.coli intestinal bacteria among the present invention; See Fig. 2) value has significant difference.Therefore, the inhibition strength of the TF-1 cel l proliferation that GM-CSF is caused of the IFN-a2b among the present invention be the E.coli expression in escherichia coli human IFN-a2b 250-600 doubly.

(b) comparison of biologic activity between the IFN-b1 that expresses in IFN-b1 among the present invention and the non-human system

IFN-b1 has cytotoxicity to human erythroleukemia cell TF-1 clone.GM-CSF handles back TF-1 cell proliferation, and IFN-b1 then suppresses this propagation, causes necrocytosis.

In 96 orifice plates, 20000 the TF-1 cells in every hole are with 0.2ng/mlGM-CSF (Peprotech Cat.No 300-03 or R﹠amp; D system, Cat.No 215-GM-010) and the IFN-b10-100ng/ml among the present invention handle cell, 37 ℃, 68 hours.Use CellTiter 96 aqueous solution analysis of cell proliferation (Promega) to measure cell quantity then.In this is analyzed, (3-(4,5-dime thylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) is reduced to formazan by cell biological and rises product the tetrazolium salts compound MTS that exists in electron coupling reagent (phenazine methosulfate).The concentration that formazan is risen generates the absorbance measurement of solution at 490nm by reading with spectrophotometer (E max precisionmicroplate reader, Molecular Devices).To matched curve between absorption value and the EP0 concentration value, calculate the ED50 value by 4 parameter equatioies.

The ED50 value of IFN-b1 among the present invention is 35-53ng/ml (Fig. 3)

Adopt the non-human cell system, E.coli intestinal bacteria for example, the human IFN-b1 of yeast or expressing cho cell repeats above-mentioned detection, finds that the ED50 value has marked difference.

(c) active comparison between the IFN-g that expresses in IFN-g among the present invention and the non-human system

IFN-g is known can to suppress the proliferation function of the human HT-29 cell that TNF-a causes.In 96 orifice plates, thin month bag of 4000 HT-29 in every hole handled cell, 37 ℃, 88 hours with the IFN-g0-20ng/ml among 1ng/ml TNF-a (Peprotech Cat.No 300-01A or R﹠D system, Cat.No 210-TA-010) and the present invention.Use CellTiter 96 aqueous solution analysis of cell proliferation (Promega) to measure cell quantity then.In this is analyzed, the tetrazolium salts compound MTS that in electron coupling reagent (phenazine methosulfate), exists (3-(4,5-dimethylthiazol-2-y1)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny1)-2H-tetrazolium) is reduced to formazan by cell biological and rises product.The concentration that formazan is risen generates the absorbance measurement of solution at 490nm by reading with spectrophotometer (E maxprecision microplate reader, Molecular Devices).To matched curve between absorption value and the IFN-g concentration value, calculate the ED50 value by 4 parameter equatioies.

Adopt the human IFN-g that produces in E.coli (the Peprotech Cat. No 300-02) intestinal bacteria to repeat above-mentioned detection.

The ED50 of the human IFN-g that produces in the ED50 value (0.04 to 0.06ng/ml) of IFN-g and the E.coli intestinal bacteria among the present invention (0.44 to 0.66ng/ml; See Fig. 4) value has significant difference.Therefore, the inhibition strength of the HT-29 cel l proliferation that TNF-a is caused of the IFN-g among the present invention is 11 to 17 times of human IFN-g of E.coli expression in escherichia coli.

(d) main shock in bright IFNAR2-Fc and the non-human system in the comparison of biological analysis between the solubility IFNAR2 molecule of expressing

IFN-a2B has cytotoxicity to human erythroleukemia cell TF-1 clone.GM-CSF handles back TF-1 cell and breeds, and IFN-a2B then suppresses this propagation, causes necrocytosis.Adding IFNAR2 can suppress the activity of IFN-a2B, causes GM-CSF inductive TF-1 cel l proliferation to produce.

In 96 orifice plates, 20000 the TF-1 cells in every hole are handled cell with the IFNAR2-Fc 0-5000ng/ml among 0.2ng/mlGM-CSF (Peprotech Cat.No 300-03 or R﹠D system, Cat.No 215-GM-010) and the present invention, 37 ℃, 24-48 hour.Use CellTiter 96 aqueous solution analysis of cell proliferation (Promega) to measure cell quantity then.In this is analyzed, the tetrazolium salts compound MTS that in electron coupling reagent (phenazine methosulfate), exists (3-(4,5-dimethylthiazo1-2-y1)-5-(3-carboxy met hoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) is reduced to formazan by cell biological and rises product.The concentration that formazan is risen generates the absorbance measurement of solution at 490nm by reading with spectrophotometer (E max precisionmicroplate reader, Molecular Devices).

To matched curve between the IFNAR2-Fc concentration value among absorption value and the present invention, calculate the ED50 value by 4 parameter equatioies.The ED50 value of IFNAR2-Fc is 0.14 to 0.18ng/ml (Fig. 5) among the present invention.

(e) active comparison between the IL-10 that expresses in IL-10 among the present invention and the non-human system

There is IL-10 inducing mouse mast cell line MC/9 propagation down in IL-4.

In 96 orifice plates, 21000 the MC/9 cells in every hole are handled cell, 37 ℃, 90 hours with the 0-50ng/ml IL-10 that contains 200pg/mlIL-4 (Peprotech Cat.No 200-04).Use CellTiter 96 aqueous solution analysis of cell proliferation (Promega) to measure cell quantity then.In this is analyzed, the tetrazolium salts compound MTS that in electron coupling reagent (phenazine methosulfate), exists (3-(4,5-dimethylthiazol-2-y1)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) is reduced to formazan by cell biological and rises product.The cured concentration of formazan generates the absorbance measurement of solution at 490nm by reading with spectrophotometer (E maxprecision microplate reader, Molecular Devices).ED50 value separately adopts the logarithmic value of absorption value to calculate than IL-10 concentration and one 4 parameter equation.

Adopt the human IL-10 that produces in E.coli (the Peprotech Cat.No 1-9276) intestinal bacteria to repeat above-mentioned detection.

The ED50 of the human IL-10 that produces in the ED50 value of IL-10 (O.012 to 0.018ng/ml) and the E.coli intestinal bacteria among the present invention (0.19 to 0.29ng/ml; See Fig. 6) value has significant difference.Therefore, to have the intensity that propagation takes place for IL-10 inducing cell down be 10 to 25 times of human IL-10 of E.coli expression in escherichia coli to the IL-4 among the present invention.

(f) comparison of biologic activity between the IL-10R α-Fc that expresses in IL-10R α-Fc among the present invention and the non-human system

Mouse hypertrophy cell is that MC/9 all reacts to many cytokines.The existing IL-4 that is reported in exists following IL-10 can induce MC/9 cell proliferation.IL-10R α-Fc is by suppressing combining of IL-10 molecule and cell IL-10 acceptor site with the IL-10 binding competition, thereby blocks the activity of IL-10, so IL-10 shows the biology inactivation.Therefore, IL-10 and IL-10R α-Fc are hatched jointly can suppress the short proliferation function of IL-10 the MC/9 cell.

In 96 orifice plates, 1ng/ml IL-10 (Peprotech Cat.No 1-9276) and the IL-10Ra-Fc 0-2500 ng/ml among the present invention were hatched 1 hour for 37 ℃ jointly.Every then hole adds the MC/9 cell that 21000 200pg/ml IL-4 (Peprotech Cat. No 200-04) handle, 37 ℃, 90 hours.Use CellTiter 96 aqueous solution analysis of cell proliferation (Promega) to measure cell quantity then.In this is analyzed, the tetrazolium salts compound MTS that in electron coupling reagent (phenazine methosulfate), exists (3-(4,5-dimethylthiazol-2-y1)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) is reduced to formazan by cell biological and rises product.The concentration that formazan is risen generates the absorbance measurement of solution at 490nm by reading with spectrophotometer (E max precision microplate reader, Molecular Devices).

Adopt E.coli (R﹠amp; D Systems Cat. No 274-R1) the solubility IL-10R alpha molecule of e. coli expression repeats above-mentioned detection.

Logarithmic ratio IL-10Ra concentration and 4 parameter equatioies calculating inhibition dosage (ND50) separately by light absorption value.

In the presence of 0.5ng/ml IL-10, the ND50 (18 to 121ng/ml of the human IL-10Ra of the solubility that produces in the ND50 value (0.8 to 1.0ng/ml) of IL-10Ra-Fc and the E.coli intestinal bacteria among the present invention; Fig. 7) value has significant difference.

Therefore, the IL-10Ra-Fc among the present invention is 18 to 150 times of the human IL-10Ra of solubility of E.coli expression in escherichia coli to the inhibition strength of IL-10 inductive MC/9 cell proliferation.

Embodiment 13

(a) comparison of external immunologic competence feature between the human IFN-a2b of IFN-a2b and non-human system expression among the present invention

The standard protein detection technique is adopted in the proteic evaluation and test of IFN-a2b among the present invention, as adopt A280 absorption peak method, calculate optical extinction coefficient (ε), or detect proteic molecular weight based on the SDS-PAGE analytical procedure, can also adopt Bradford method of protein detection (Bradford AhalBiochem 72:248-254,1976) in addition.

IFN-a2b among the present invention, adopt the standard protein detected result to carry out stdn, gradient dilution, adopt the quantitative immunoassay procedure of IFN-a2b of suitable commercialization purposes to detect, with the IFN-a2b of non-human cell expressing as standard substance, for example, instruct the anti-IFN-a2b ELISA test kit of employing to detect according to manufacturer.

In addition, can also adopt obtainable commercial original chemical to develop a kind of quantitative immunoassay procedure and detect IFN-a2b level among the present invention.For example adopt a human IFN-a Mab as a R﹠amp; The IFN-a Mab (Cat#21112-1) of d system develops an anti-IFN-a2bELISA method, as capture antibodies; Human IFN-a Pab such as R﹠amp; D system IFN-a Pab (Cat#331100-1) contains the molecular label of suitable detection as detecting antibody on it, as vitamin H, the method known in the available techniques detects a recombinant human IFN-a2b such as the R﹠amp that the E.coli Bacillus coli cells is expressed; D system recombinant human IFN-a2b (Cat#11105-1) is as protein standard substance.The concentration of IFN-a2b among the present invention is carried out stdn with the standard protein detected result, and the ELISA method in the employing technology utilizes above-mentioned described reactant to detect.

By commercialization ELISA test kit or by adopting the detected IFN-a2b protein concentration of the quantitative immunoassay of original chemical will be different with the stdn method of protein detection, this be because the ELISA test kit of commercialization purposes or the seizure in the immunoassay procedure and/or to detect antibody be the IFN-a2b protein antibodies of anti-non-human cell expressing among the present invention.

On structure level, such result will point out different immunologic competence feature between the IFN-a2b molecule of the IFN-a2b among the present invention and non-human cell expressing.

(a) comparison of external immunologic competence feature between the human IFN-b1 of IFN-b1 and non-human system expression among the present invention

The standard protein detection technique is adopted in the proteic evaluation and test of IFN-b1 among the present invention, as adopt A280 absorption peak method, calculate optical extinction coefficient (ε), or detect proteic molecular weight based on the SDS-PAGE analytical procedure, can also adopt in addition the Bradford method of protein detection (Bradford, 1976supra).

IFN-b1 among the present invention adopts the standard protein detected result to carry out stdn, and gradient dilution adopts the ELISA test kit of suitable commercialization purposes, for example a R﹠amp; The human IFN-b1 ELISA test kit of d system (Cat#41400-1) instructs according to manufacturer and detects, and above-mentioned described ELISA test kit adopts the human IFN-b1 of E.coli Bacillus coli cells expression as standard substance.

Protein concentration by IFN-b1 among the present invention of commercialization ELISA test kit detection will be different with the stdn method of protein detection, and this is because seizure in the ELISA test kit of commercialization purposes and/or detection antibody are the IFN-b1 protein antibodies of anti-non-human cell expressing.

On structure level, this presentation of results have different immunologic competence features between the IFN-b1 molecule of the IFN-b1 among the present invention and non-human cell expressing.

(c) comparison of external immunologic competence feature between the human IFN-g of IFN-g and non-human system expression among the present invention

The standard protein detection technique is adopted in the proteic evaluation and test of IFN-g among the present invention, as adopt A280 absorption peak method, calculate optical extinction coefficient (ε), or detect proteic molecular weight based on the SDS-PAGE analytical procedure, can also adopt in addition the Bradford method of protein detection (Bradford, 1976supra).

IFN-g among the present invention adopts the standard protein detected result to carry out stdn, and gradient dilution adopts the ELISA test kit of suitable commercialization purposes, for example, and a R﹠amp; The human IFN-gamma DuoSet of d system ^_ELISA kit (Cat#DY28s) or a R﹠amp; The human IFN-gamma QuantiKine of d system ^_ELISA kit (Cat#DIF50) detects according to manufacturer's guidance, and above-mentioned described ELISA test kit adopts the human IFN-g of E.coli Bacillus coli cells expression as standard substance.

Protein concentration by IFN-g among the present invention of commercialization ELISA test kit detection will be different with the stdn method of protein detection, and this is because seizure in the ELISA test kit of commercialization purposes and/or detection antibody are the IFN-g protein antibodies of anti-non-human cell expressing.

At structure level, has different immunocompetence features between the IFN-g in this this research of presentation of results and the IFN-g molecule of non-human cell expressing.

(d) comparison of external immunologic competence feature between the human IL-10 of IL-10 and non-human system expression among the present invention

The albumen assessment of IL-10 among the present invention is instructed according to manufacturer, adopts R﹠amp; The human IL-10 DuoSet of D ^_ELISA test kit (Cat.#DY217B) detects.

IL-10 among the present invention adopts the standard protein detected result to carry out stdn, and gradient dilution is at R﹠amp; The human IL-10 Duoset of d system ^_In the ELISA test kit (Cat.#DY217B), detect according to manufacturer's guidance, above-mentioned described ELISA test kit adopts the human IL-10 of E.coli Bacillus coli cells expression as standard substance.

R﹠amp; D system DuoSet ^_IL-10 ELISA test kit detected result draws the concentration curve (Fig. 8) of the IL-10 of IL-10 among the present invention that knows clearly and E.coli escherichia coli expression at the OD450nm place.Simulate logarithmic value-logarithmic value curve by Molecular Devices curve fitting software, data are analyzed, go out other result'ss (table 28) again.

The eigenwert (log (y)=A+B*log (x)) of table 28:IL-10 ELISA matched curve

	The DuoSet of R﹠D system (E.coli)	IL-10 among the present invention
			Relation conefficient	0.970	0.980
Slope (B)	0.482	0.704
			Y-axis is cut and is shown huge (A)	0.103	0.129

Pass through R﹠amp; The human IL-10 DuoSet of d system ^_The ELISA test kit, a commercial kit, the human IL-10 that E.coli expresses is as standard substance, adopt the protein concentration of the IL-10 among human IL-10 antibody test the present invention that anti-E.coli expresses, the result shows that IL-10 concentration is underestimated, and this test kit is in order to detect the human IL-10 concentration in laboratory sample and human patients expression itself.The relation conefficient of two curves of eigenwert demonstration of matched curve is very high (table 28) all.Yet the IL-10 rate of curve among the present invention is different from the human IL-10 (table 28) of E.coli escherichia coli expression.The Y-axis intercept of IL-10 among the present invention also is different from the human IL-10 (table 28) of E.coli escherichia coli expression in addition, illustrates that there is intrinsic difference in the immunologic competence between these two albumen.

In a word, these results have shown between the IL-10 molecule of the IL-10 among the present invention and non-human cell expressing to have different immunocompetence features.

(e) comparison of external immunologic competence feature between the IL-10Ra-Fc molecule of IL-10Ra-Fc and non-human system expression among the present invention

Among the present invention suitable method of protein detection is adopted in the assessment of IL-10Ra-Fc, Lowry albumen appraisal procedure for example, with IgG as standard substance.

IL-10Ra-Fc among the present invention adopts the standard protein detected result to carry out stdn, adopts quantitative immunology detection program of obtainable commercialization original chemical development.For example, adopt a human IL-10Ra-Fc Mab (R﹠amp; D system Cat#MAB274) as capture antibodies, a biotin labeled human IL-10Ra-FcPab (R﹠amp; D system Cat#BAF874) as detecting antibody, as protein standard substance, develops an anti-IL-10Ra-FcELISA method at the recombinant human IL-10Ra-Fc of Sf21 expressed in insect cells (Cat#874-RB-100 of R﹠D system).IL-10Ra-Fc concentration among the present invention adopts the standard protein detected result to carry out stdn, with the ELISA method known among the art, uses above-mentioned described reactant to detect.

The protein concentration of IL-10Ra-Fc (as a monomer) among the present invention that the quantitative immunodetection that develops by the employing original chemical detects will be different with the stdn method of protein detection, and this is because its seizure and/or detection antibody are the IL-10Ra-Fc protein antibodies of anti-non-human cell expressing.It should be noted that the IL-10Ra-Fc among the present invention expresses as dimer.

At structure level, this result will illustrate that the IL-10Ra-Fc among the present invention has different immunologic competence features with the mosaic type IL-10Ra-Fc molecule of non-human cell expressing.

Embodiment 14

Molecules of interest of the present invention being further purified and with the peptide quality fingerprinting of ESI-MS/MS spectrum

Except that embodiment 2 described purification process, the purifying of molecules of interest of the present invention is further undertaken by RP-HPLC, with commercial obtainable post.Elute protein is by 215 or 280nm absorbancy monitoring and being collected, simultaneously for since the delay that the conduit volume between wandering cells and the collecting terminal brings proofread and correct.

The gel piece that contains protein example from 1D or 2D gel digests in pancreatin solution, as described in embodiment 3.Perhaps, the solution that contains protein sample uses pancreatin to digest in ammonium bicarbonate buffers (10-25mM, pH 7.5-9).Solution is 37 ℃ of following incubated overnight.Then by add acetate up to pH in the 4-5 scope with stopped reaction.The peptide sample is concentrated and (Millipore, Bedford MA) or as described in the embodiment 3, contain Poros R2 chromatographic resin (Perspetive Biosystems, Framingham, prefabricated microtrabeculae desalination MA) with C18 Zip-Tips.

Protein sample (2-5 μ l) injects the C18 pre-column and washes under 30 μ l/min to concentrate and desalination with 0.1% formic acid.Wash after 3 minutes, pre-column is converted to into the analytical column that contains C18 RP silicon-dioxide (Atlantis, 75 μ m * 100mm, Waters Corporation) of row.Peptide with linear solvent gradient by wash-out in the post, step by step, from H ₂O: CH ₃CN (95: 5; + 0.1% formic acid) to H ₂O: CH ₃CN (20: 80 ,+0.1% formic acid) carries out more than 40 minutes with 200nl/min.LC is eluted in Micromass QTOF Ultima mass spectrograph, and (Micromass, Manchester analyze through positively charged ion nanoflow electron spray(ES) in UK).

Series connection MS mixes level Four/quadrature acceleration TOF mass spectrograph (Micromass) with Q-Tof to carry out.QTOF operates according to the data of drainage pattern (DDA).Obtain TOFMS and detect scanning (m/z 400-2000,1.0 seconds), and while three kinds of maximum multiplycharged ions (counting〉15) in detecting scanning, analyze through MS/MS continuously.The MS/MS spectrum is collected 8 seconds (m/z 50-2000).

The LC/MS/MS data (Micromass) are searched for Mascot (Matrix Science, London, UK) and ProteinLynx Global Server (" PLGS ").Protein sample is contemplated to molecules of interest.

Buy and execute example 15

(a) immunogenicity among the non-human animal

(i) animal immune of usefulness target protein matter

The non-human animal divides other group, for example, mouse with 1-100ug protein of the present invention and in inhuman cell expressed protein through subcutaneous, intraperitoneal or intramuscular (IP) immunity.Animal was accepted second immunisation in back one month in immunity.Before the immunity, protein is emulsification in adjuvant, for example, is used for the complete Freund's adjuvant and the incomplete Freund's adjuvant that is used for second immunisation of initial immunity.

(ii) to the evaluation of the antibody of target protein matter

In order to measure antibody response, get blood and collect serum from afterbody from the animal of each group.Protein specific antibody is by the of the present invention protein determination of solid phase ELISA with the 50ng/ hole.Different immune immunoglobulin (Ig) isotypes are by the detection TPPA with the anti-IgG1 of showing of mark, IgG2, IgG2b, IgG3, IgM, IgA, IgD.Perhaps, the resistance reaction is measured by the protein of the present invention that is adsorbed onto on the film that is used for dot blotting or Western blot.The mensuration of the isotype of different immunoglobulin (Ig)s is according to top described detection.Expect that protein of the present invention causes the antibody response that is different from expressed proteins in inhuman cell.

(iii) T analysis of cell proliferation

With the animal euthanasia of immunity and prepare splenocyte.The splenocyte of suitable quantity, for example, 5 * 10 ⁵Cell, the use by oneself animal of protein immunity of the present invention, cultivate for some time together with the protein of the present invention of different concns, and in the inhuman cell of using by oneself of equal amts in the inhuman cell of the splenocyte of the animal of expressed protein immunity and different concns expressed proteins cultivate together.For the T analysis of cell proliferation, spleen cell cultures 96 hours and in the end 16 hours are with 1 μ Ci[ ³H] thymus pyrimidine (6-7 μ Ci/umol) processing.Harvested cell to strainer and mix with standard method measure [ ³H] thymus pyrimidine.Compare with expressed protein in inhuman cell, expect that protein of the present invention causes different proliferative responses.

(iv) IFN gamma analyzes

For IFN gamma analyzes and the protein of protein of the present invention or inhuman cell expressing is cultivated together spleen cell cultures supernatant results and IFN gamma product 96 hours the time detect by sandwich ELISA, for example, R﹠amp; D system resists-IFN gamma Quantikine ^_ELISA test kit (Cat#DIF50) is according to the specification sheets of producer.Expection use by oneself albumen of the present invention together in IFN gamma product and the inhuman cell of using by oneself of the culture supernatant of cultured cells expressed protein together the IFN gamma product of cultured cells to compare be different.

(b) the vitro human immunogenicity is analyzed

(i) human T-cell's response analysis

Human dendritic cell and CD4 ⁺The T cell preparation is from people's whole blood, and as Stickler et al.Toxicological Sciences 77:280-289,2004 is described.Dendritic cell and CD4 ⁺The T cell places 96 orifice plates to cultivate altogether, comprises 2 * 10 ⁴Dendritic cell and 2 * 10 ⁵CD4 ⁺The T cell.Protein of the present invention and in inhuman cell expressed protein be peptide fragment through enzymic digestion, with the suitable enzyme of identifying by the cleavage site forecasting software, for example, PeptideCutter (http://au.expasy.org/tools/peptidecutter).The peptide fragment that obtains passes through the suitable technique purifying, for example, and liquid chromatography, and add in the coculture to final concentration 5 ug/ml.Culture was cultivated 5 days, then with 0.5uCi ³The H thymus pyrimidine adds in each culture.With cell harvesting in strainer and by [ ³H] thymus pyrimidine mixes mensuration cell proliferation.

Expection derives from protein peptide of the present invention and can cause more weak proliferative response, with respect to the peptide from expressed protein in the inhuman cell.

(ii) people's antibody response is analyzed

To through get blood and preparation blood plasma with people's donor of the protein therapeutic of inhuman cell expressing.The solid phase ELISA of expressed protein measures protein specific antibody in protein of the present invention by anti-50ng/well and the inhuman cell.Different immunoglobulin (Ig) isotypes is by the detection TPPA with the mark that shows anti-human IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD.

On the film that is adsorbed on during perhaps, by dot blotting or Western blot protein of the present invention and in inhuman cell expressed protein measure antibody response.The mensuration of different immunoglobulin (Ig) isotypes is according to detection described above.

Expection is present in the immunoglobulin (Ig) in the people's of the protein for treatment of inhuman cell expressing the serum can be combined in expressed protein in the inhuman cell, and with protein bound of the present invention a little less than or debond.

Embodiment 16

Of the present invention proteinic preparation from recombination group construct

The genome sequence of (SEQ ID NO is respectively 139,140,141,142) coding IFN-a2b, IFN-b1, IFN-g or IL-10 is cloned in the suitable expression after by pcr amplification among the present invention, for example pIRESbleo3, pCMV-SPORT6, pUMCV3, pORF, pORF9, pcDNA3.1/GS, pCEP4, pIRESpuro3, pIRESpuro4, pcDNA3.1/Hygro (+), pcDNA3.1/Hygro (-), pEF6/V5-His.These recombination structure bodies are used to prepare people's transformation then, and operation is according to embodiment 1 (C).Carry out IFN-a2b, IFN-b1, IFN-g or preparation of IL-10 recombinant DNA and the purifying of being selected from of the present invention as the above embodiments 2

It will be appreciated by those skilled in the art that invention described here can be made a variation or be modified to the different situation with those concrete descriptions.Should be understood to present invention includes all such variation or modification.The present invention has also comprised all steps, feature, component and the compound that relate in the specification sheets or point out and the arbitrary combination of two or more described steps or feature arbitrarily respectively or fully.

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＜110〉Apollo Life Sciences Ltd.

PRIEST, John D (only to the U.S.)

WATTS, Alan D (only to the U.S.)

WHITTAKER, Jason S (only to the U.S.)

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

CORBETT, Melissa (only to the U.S.)

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

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

JACKSON, Stuart (only to the U.S.)

LEE. Carol M Y (only to the U.S.)

LIDDELL. Catherine A (only to the U.S.)

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

＜120〉molecule and chimeric molecule thereof

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<212> DNA

＜213〉people

<400> 101

<210> 102

<211> 21

<212> PRT

＜213〉people

<400> 102

<210> 103

<211> 606

<212> DNA

＜213〉people

<400> 103

<210> 104

<211> 606

<212> DNA

＜213〉people

<400> 104

<210> 105

<211> 202

<212> PRT

＜213〉people

<400> 105

<210> 106

<211> 636

<212> DNA

＜213〉people

<400> 106

<210> 107

<211> 636

<212> DNA

＜213〉people

<400> 107

<210> 108

<211> 212

<212> PRT

＜213〉people

<400> 108

<210> 109

<211> 699

<212> DNA

＜213〉artificial sequence

<400> 109

<210> 110

<211> 699

<212> DNA

＜213〉artificial sequence

<400> 110

<210> 111

<211> 233

<212> PRT

＜213〉artificial sequence

<400> 111

<210> 112

<211> 1326

<212> DNA

＜213〉artificial sequence

<400> 112

<210> 113

<211> 1326

<212> DNA

＜213〉artificial sequence

<400> 113

<210> 114

<211> 442

<212> PRT

＜213〉artificial sequence

<400> 114

<210> 115

<211> 1356

<212> DNA

＜213〉artificial sequence

<400> 115

<210> 116

<211> 1356

<212> DNA

＜213〉artificial sequence

<400> 116

<210> 117

<211> 452

<212> PRT

＜213〉artificial sequence

<400> 117

<210> 118

<211> 1326

<212> DNA

＜213〉artificial sequence

<400> 118

<210> 119

<211> 1326

<212> DNA

＜213〉artificial sequence

<400> 119

<210> 120

<211> 442

<212> PRT

＜213〉artificial sequence

<400> 120

<210> 121

<211> 1356

<212> DNA

＜213〉artificial sequence

<400> 121

<210> 122

<211> 1356

<212> DNA

＜213〉artificial sequence

<400> 122

<210> 123

<211> 452

<212> PRT

＜213〉artificial sequence

<400> 123

<210> 124

<211> 1335

<212> DNA

＜213〉artificial sequence

<400> 124

<210> 125

<211> 1335

<212> DNA

＜213〉artificial sequence

<400> 125

<210> 126

<211> 445

<212> PRT

＜213〉artificial sequence

<400> 126

<210> 127

<211> 1365

<212> DNA

＜213〉artificial sequence

<400> 127

<210> 128

<211> 1365

<212> DNA

＜213〉artificial sequence

<400> 128

<210> 129

<211> 455

<212> PRT

＜213〉artificial sequence

<400> 129

<210> 130

<211> 1419

<212> DNA

＜213〉artificial sequence

<400> 130

<210> 131

<211> 1419

<212> DNA

＜213〉artificial sequence

<400> 131

<210> 132

<211> 473

<212> PRT

＜213〉artificial sequence

<400> 132

<210> 133

<211> 1419

<212> DNA

＜213〉artificial sequence

<400> 133

<210> 134

<211> 1419

<212> DNA

＜213〉artificial sequence

<400> 134

<210> 135

<211> 473

<212> PRT

＜213〉artificial sequence

<400> 135

<210> 136

<211> 1428

<212> DNA

＜213〉artificial sequence

<400> 136

<210> 137

<211> 1428

<212> DNA

＜213〉artificial sequence

<400> 137

<210> 138

<211> 476

<212> PRT

＜213〉artificial sequence

<400> 138

<210> 139

<211> 567

<212> DNA

＜213〉people

<400> 139

<210> 140

<211> 564

<212> DNA

＜213〉people

<400> 140

<210> 141

<211> 4260

<212> DNA

＜213〉people

<400> 141

<210> 142

<211> 3801

<212> DNA

＜213〉people

<400> 142

Claims (16)

1. isolating protein, it has measurable physical and chemical parameter feature, wherein said character representation, be associated with one or more distinctive pharmacological characteristics or form the basis of one or more distinctive pharmacological characteristics, wherein said isolating protein has and comprises a plurality of measurable physical and chemical parameter { [P _x] ₁, [P _x] ₂... [P _x] _n, physicochemical characteristics, P wherein _xRepresent that measurable physical and chemical parameter and " n " are 〉=1 integer, wherein [P _x] ₁To [P _x] _nIn each different measurable physical and chemical parameter of respectively doing for oneself, wherein the value of any measurable physicochemical property or more than one measurable physicochemical property a series of value representation, be associated with a distinctive pharmacological characteristics T _yPerhaps the pharmacological characteristics { [T of series of features _y] ₁, [T _y] ₂... .[T _y] _m, or form a distinctive pharmacological characteristics T _yPerhaps the pharmacological characteristics { [T of series of features _y] ₁, [T _y] ₂... [T _y] _mThe basis, T wherein _yRepresent that a distinctive pharmacological characteristics and m are 〉=1 integer, wherein [T _y] ₁To [T _y] _mIn each different pharmacological characteristic of respectively doing for oneself, wherein isolating protein is selected from IFN-a2B, IFN-b1, IFN-g, IFNAR2, IL-10 and IL-10Ra-Fc.

2. the isolating protein of claim 1, wherein said protein has the measurable physical and chemical parameter shown in one or more table 2.

3. the isolating protein of claim 1, wherein said protein has the distinctive pharmacological characteristics shown in one or more table 3.

4. chimeric molecule, it comprises isolating IFN-a2B, IFN-b1, IFN-g, IFNAR2 or IL-10 or its segment of the claim 1 that is fused on one or more peptides, polypeptide or the protein portion.

5. the chimeric molecule of claim 4, wherein peptide, polypeptide or protein portion comprise the constant region (Fc) or the framework region of human normal immunoglobulin.

6. the chimeric molecule of claim 4, wherein chimeric molecule is selected from IFN-a2B-Fc, IFN-b1-Fc, IFN-g-Fc, IFNAR2-Fc and IL-10-Fc.

7. pharmaceutical composition contains each isolating protein or chimeric molecule of claim 1 to 6.

8. in mammalian subject, treat or prophylactic method, wherein said disease can be improved by increasing proteinic amount or activity, described method comprise give described mammalian subject significant quantity according to claim 1 to 3 each isolating protein, claim 4 to 6 each chimeric molecule or the pharmaceutical composition of claim 7.

9. nucleotide sequence, it is to be selected from SEQ ID No:27,29,31,33,35,39,41,43,45,47,49,51,53,55,59,61,63,65 and, 67,69,71,73,75,79,81,83,85,87,89,91,93,95,99,101,103,105,107,111,113,115,116,118,119,121,122,124,125,127,128,130,131,133,134,136,137,139,140,142,143,145,146,148,149, nucleotide sequence, or the nucleotide sequence that has about at least 90% identity with above-mentioned arbitrary sequence of enumerating, or the nucleotide sequence that can under high stringent condition, hybridize with arbitrary above-mentioned sequence or their complementary type.

10. isolating protein or chimeric molecule, by being selected from SEQ ID NO:27,29,31,33,35,39.41,43,45,47,49,51,53,55,59,61,63,65,67,69,71,73,75,79,81,83,85,87,89,91,93,95,99,101,103,105,107,111,113,115,116,118,119,121,122,124,125,127,128,130,131,133,134,136,137,139,140,142,143,145,146,148,149 nucleotide sequence is coded, or by with above-mentioned arbitrary sequence of enumerating have about at least 90% identity nucleotide sequence or can be coded with the nucleotide sequence that any above-mentioned sequence or arbitrary their complementary type are hybridized under high stringent condition.

11. isolating nucleic acid molecule, coded protein or chimeric molecule or its funtion part, comprise NO:27 with SEQ ID, 29,31,33,35,39.41,43,45,47,49,51,53,55,59,61,63,65,67,69,71,73,75,79,81,83,85,87,89,91,93,95,99,101,103,105,107,111,113,115,116,118,119,121,122,124,125,127,128,130,131,133,134,136,137,139,140,142,143,145,146,148,149 have the nucleotide sequence of at least 90% similarity, or best comparison back and/or can with SEQ ID NO:27,29,31,33,35,39.41,43,45,47,49,51,53,55,59,61,63,65,67,69,71,73,75,79,81,83,85,87,89,91,93,95,99,101,103,105,107,111,113,115,116,118,119,121,122,124,125,127,128,130,131,133,134,136,137,139,140,142,143,145,146,148,149 or their one or more nucleotide sequences of under high stringent condition, hybridizing of complementary type.

12. isolating nucleic acid molecule, comprise that coding has basically as SEQ ID NO:28,30,32,34,36,40,42,44,46,48,50,52,54,56,60,62,64,66,68,70,72,74,76,80,82,84,86,88,90,92,94,96,100,102,104,106,108,112,114,117,120,123,126,129,132,135,138,141,144,147,150 one or more shown in the protein of aminoacid sequence or the nucleotide sequence of chimeric molecule, or the best comparison of coding back and SEQ ID NO:28,30,32,34,36,40,42,44,46,48,50,52,54,56,60,62,64,66,68,70,72,74,76,80,82,84,86,88,90,92,94,96,100,102,104,106,108,112,114,117,120,123,126,129,132,135,138,141,144,147, the protein of one or more aminoacid sequences with about at least 90% similarity of 150 or the nucleotide sequence of chimeric molecule.

13. test kit is used for measuring the human protein of people's cell expressing or the level that chimeric molecule is present in biological products, comprises (a) solid phase supported matrix; (b) one or more among the claim 1-3 each human protein or at the antibody of each chimeric molecule among the claim 4-6; (c) lock solution; (d) one or more substrate storage liquid; (e) substrate buffered soln; (f) normal man's protein or chimeric molecule sample; (g) working instructions.

14. the test kit of claim 13, wherein normal man's protein or chimeric molecule sample be claim 2 or 3 each isolating protein or the goods of the chimeric molecule of claim 4.

15. the test kit of claim 13 or 14, wherein this antibody or each antibody derived from comprise claim 2 or 3 each isolating protein or the goods of the chimeric molecule of claim 4 to mammiferous immunity.

16. each test kit of claim 13 to 15, wherein the human protein of people's cell expressing is naturally occurring people IFN-a2B, IFN-b1, IFN-g, IFNAR2, IL-10 or IL-10Ra.

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