CN109651514A

CN109651514A - P28-Fc fusion protein and its coding nucleic acid molecule, recombinant expression carrier, recombinant cell and application

Info

Publication number: CN109651514A
Application number: CN201910141315.0A
Authority: CN
Inventors: 王仁喜
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-02-26
Filing date: 2019-02-26
Publication date: 2019-04-19

Abstract

The present invention provides p28-Fc fusion protein and its coding nucleic acid molecule, recombinant expression carrier, recombinant cell and application, is related to technical field of pharmaceuticals, the p28-Fc fusion protein is made of hIL-27p28 and Fc.There is the base sequence for being unfavorable for mammal expression in the signal peptide coding region of human IL-2 7p28, in order to obtain the IL-27p28 of correct configuration, hIL-27p28 of the present invention contains Hongeybee melittin secretion signal peptide, to substitute human IL-2's 7p28 signal peptide, so that configuration correctly solubility IL-27p28 can not be obtained when overcoming the problems, such as heterogenous expression.The present invention also provides the application of the p28-Fc fusion protein and its coding nucleic acid molecule, recombinant expression carrier or recombinant cell in the drug of preparation treatment autoimmune disease.

Description

P28-Fc fusion protein and its coding nucleic acid molecule, recombinant expression carrier, recombinant cell And application

Technical field

The present invention relates to technical field of pharmaceuticals more particularly to p28-Fc fusion proteins and its coding nucleic acid molecule, recombination table Up to carrier, recombinant cell and application.

Background technique

Interleukin-27 (IL-27) is a member in IL-12 family (IL-12, IL-23, IL-27 and IL-35).IL-12 family Race's cell factor is all made of a α chain (p19, p28 or p35) and a β chain (p40 or EBI3), such as IL-27 is by p28 With [Pflanz, S.et al. (2002) the Immunity 16:779.] of EBI3 (Epstein-Barr virus induced gene 3) composition.IL-27p28 Also referred to as IL27, p28, IL30, IL-27, IL27p28 etc..

P28 is a polypeptide similar with the p35 of IL-12, and EBI3 is then one similar to the p40 of IL-12/IL-23 Albumen.For IL-27 in conjunction with the heterodimer receptor complex that TCCR/WSX-1 and gp130 is formed, gp130 is IL-6 family The shared receptor subunit [Pflanz, S.et al. (2004) J.Immunol.172:2225] of cell factor.

IL-27 is the main inhibitory cells factor, but may some pro-inflammatory effects.It is in local antigen when IL-27 is normal The paracmasis of the autoimmune response of delivery cell excitation generates.In addition, widely stimulate (including nTreg cell, IFN-β, Toll The ligand and statins of sample receptor) by the production of antigen presenting cell induction IL-27, to limit the development of inflammation.

Although IL-27 independent role is made without apparent direct pro-inflammatory effect in the joint of IL-12 and/or IL-2 Under, it can induce IFN-β to generate by T cell and natural killer cells.IL-2 limits the differentiation of nTreg and T cell, and IL-27 can inhibit the generation of IL-2, but IL-2 can pass through this effect of expression antagonism of inhibition IL-27R (WSX-1) again It answers.IL-27 can inhibit the development of Th17 cell and the development of induction IL-10 secretory Tr1 cell sample adjusting subgroup, partial action Be by induce c-Maf play a role [Batten, M.et al. (2006) Nat.Immunol.7:929.Stumhofer, J.S.et al.(2006)Nat.Immunol.7:937]。

Crabe etc. describe p28 subunit and another secretion compound of the cytokine-like factor 1 (CLF) composition [Crabe, S.et al.(2009)J.Immunol.183:7692].Similar to IL-27, p28/CLF is also divided by the Dendritic Cells activated It secretes, but it needs TCCR/WSX-1, gp130 and IL-6R α to transmit signal.Different from IL-27, p28/CLF can not only inhibit small Mouse naivety CD4⁺The proliferation of T cell can also induce the expression of IL-17 in the presence of TGF-β.Then PMA/ ion toxin weight is being used After new stimulation, the expression of IL-17 is suitable with the induction of TGF-β and IL-6, this shows that p28/CLF is promoting mouse Th17 points Alternative IL-6 in change.Above research thinks that IL-27p28 is co-secreted with formation heterodimers such as Ebi3 or CLF.

Now it has also been found that IL-27p28 can not depend on Ebi3 and can independently secrete, this display IL-27p28 has independent biology Learn function.In fact, the IL-27p28 of recombination can block cell factor (IL-6, IL-11 and IL- using receptor gp130 signal 27) biological function [Pflanz S, Hibbert L, Mattson J, RosalesR, Vaisberg E, et al.200； WSX-1and glycoprotein 130constitute a signal-transducing receptor for IL- 27.J.Immunol.172:2225–31；Stumhofer JS,Tait ED,Quinn WJ III,Hosken N,Spudy B, et al.2010；A role for IL-27p28as an antagonist of gp130-mediated signaling.Nat.Immunol.11:1119–26；Shimozato O,Sato A,Kawamura K,Chiyo M,Ma G, et al.2009；The secreted form of p28subunit of interleukin(IL)-27inhibits biological functions of IL-27and suppresses anti-allogeneic immune responses.Immunology 128:e816–25].The B for being overexpressed IL-27p28 energy antagonism dependence gp130 signal in vivo is thin Born of the same parents' reaction for example blocks hepatic injury, deletes antitumor reaction, inhibits rejection and Inhibition test autoimmune Portugal Grape film inflammation etc. reacts [Wang RX, Yu CR, Mahdi RM, Egwuagu CE.2012.Novel IL27p28/ IL12p40cytokine suppressed experimental autoimmune uveitis by inhibiting autoreactive Th1/Th17cells and promoting expansion of regulatory T cells.J.Biol.Chem.287:36012–21；Stumhofer JS,Tait ED,Quinn WJ III,Hosken N, Spudy B,et al.2010.A role for IL-27p28as an antagonist of gp130-mediated signaling.Nat.Immunol.11:1119–26；Shimozato O,Sato A,Kawamura K,Chiyo M,Ma G, et al.2009；The secreted form of p28subunit of interleukin(IL)-27inhibits biological functions of IL-27and suppresses anti-allogeneic immune responses.Immunology 128:e816–25；Dibra D,Cutrera J,Xia X,Kallakury B,Mishra L,Li S.2012.Interleukin-30:a novel anti-inflammatory cytokine candidate for prevention and treatment of inflammatory cytokine-induced liver injury.Hepatology 55:1204–14].Similar, the IL-27p28 of mutation cannot be combined with each other conduct with gp130 The antagonist of gp130 and hepatic injury [Rousseau F, Basset L, Froger J, the Dinguirard for limiting immune induction N,Chevalier S,Gascan H.2010.IL-27structural analysis demonstrates similarities with ciliary neurotrophic factor(CNTF)and leads to the identification of antagonistic variants.PNAS 107:19420–25].IL-27p28 deficient mice It is more susceptible to suffer from experimental autoimmune encephalomyelitis (EAE), and develops into a kind of much serious disease form.This disease is tight The increase of weight degree is increased with the expression of Th17 relevant molecule in central nervous system and the later stage experssion reduction of IL-10 is related [Diveu,C.et al.(2009)J.Immunol.182:5748].These research and propose a hypothesis: IL-27p28 may make For natural low-affinity receptor antagonist and then limit cytokine signaling [Yoshida H, HunterC.A.The Immunobiology of Interleukin-27.Annu.Rev.Immunol.2015.33:417–43.】。

However, nearest report is shown, the IL-27p28 recombinated in previous studies is the albumen not folded correctly, When it obtains correct conformation, it can generate suppression in conjunction with solubility IL-6R α and then in conjunction with gp130 homodimer Function [Garbers C, Spudy B, Aparicio-Siegmund S, Waetzig GH, Sommer J, et processed al.2013.An interleukin-6receptor-dependent molecular switch mediates signal transduction of the IL-27cytokine subunit p28(IL-30)via a gp130protein receptor homodimer.J.Biol.Chem.288:4346–54].These have researched and proposed a urgent problem It is how to give expression to soluble IL-27p28 albumen.Only giving expression to soluble IL-27p28 further could effectively grind Study carefully its function and application.

Summary of the invention

The present invention provides a kind of p28- to overcome the prior art that can not obtain the defect of soluble IL-27p28 albumen Fc fusion protein and its coding nucleic acid molecule, recombinant expression carrier, recombinant cell, the present invention is by human IL-2 7p28 albumen and Fc egg It is recombinated after white transformation, the fusion protein of acquisition can carry out solubility expression in insect cell, to obtain correct configuration The fusion protein containing IL-27p28.

The present invention also provides application of the p28-Fc fusion protein in the drug of preparation treatment autoimmune disease, originally Gp130 signal relational approach and Th17 cell can be effectively suppressed in the fusion protein of invention building, for autoimmune disease Occurrence and development have significant inhibiting effect.

In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:

The present invention provides a kind of p28-Fc fusion proteins, are made of hIL-27p28 and Fc；

The amino acid sequence of the hIL-27p28 is as shown in SEQ ID NO.1.

Preferably, the amino acid sequence of the p28-Fc fusion protein is as shown in SEQ ID NO.5.

The present invention also provides a kind of nucleic acid molecules of fusion protein described in coding above-mentioned technical proposal, the nucleic acid molecules Nucleotide sequence as shown in SEQ ID NO.6.

The present invention also provides a kind of recombinant expression carriers of fusion protein as claimed in claim 1 or 2, by above-mentioned technical side Nucleic acid molecules described in case are inserted into the multiple cloning sites of insect expression vector to get the weight of fusion protein described in preceding solution Group expression vector；

Contain Honeybee melittin secretion signal signal peptide in the insect expression vector.

Preferably, the insect expression vector is pMIB.

The present invention also provides a kind of recombinant cell of fusion protein described in expression preceding solution, the recombinant cells It is the insect cell for including recombinant expression carrier described in above-mentioned technical proposal.

The present invention provides nucleic acid described in p28-Fc fusion protein, preceding solution described in preceding solution point Recombinant cell described in recombinant expression carrier described in son, preceding solution or above-mentioned technical proposal itself is exempted from preparation treatment Application in the drug of epidemic disease disease.

Preferably, the autoimmune disease is the autoimmune disease cell-mediated by gp13 signal or Th17.

Preferably, the autoimmune disease includes multiple sclerosis, autoimmune encephalomyelitis, uveitis or ox Psoriasis.

The present invention also provides a kind of for treating the drug of autoimmune disease, and the drug includes aforementioned techniques side P28-Fc fusion protein and pharmaceutically acceptable auxiliary material described in case.

Compared with prior art, beneficial effects of the present invention:

(1) it the present invention provides a kind of p28-Fc fusion protein, is made of hIL-27p28 and Fc；The hIL-27p28's Amino acid sequence is as shown in SEQ ID NO.1.There is the alkali for being unfavorable for mammal expression in the signal peptide coding region of human IL-2 7p28 Basic sequence, in order to obtain the IL-27p28 of correct configuration, hIL-27p28 of the present invention eliminates the signal peptide of human IL-2 7p28 Part is changed to be expressed using the signal peptide in expression vector, so that it is correct to obtain configuration when overcoming heterogenous expression The problem of soluble IL-27p28.The effect of Fc segment in the present invention in the fusion protein is as follows:

A, be conducive to protein expression；

B, the half-life period of p28-Fc fusion protein when improving vivo applications；

C, the Fc segment can reduce fusion protein induction immunity of organism stimulation.

The present invention also provides the nucleic acid molecules for encoding above-mentioned fusion protein, the recombination for expressing the fusion protein Expression vector and recombinant cell, for fusion protein of the present invention after recombinant cell is expressed, what is obtained is soluble melt Hop protein, and can show that present invention obtains contain correct configuration by anti-p28 antibody and anti-Fc Identification of the antibodies The fusion protein of IL27p28.

(2) the present invention also provides the p28-Fc fusion protein and its coding nucleic acid molecules, recombinant expression carrier or again Application of the group cell in the drug of preparation treatment autoimmune disease.The embodiment of the present invention shows what the present invention constructed P28-Fc fusion protein is real for the generation of autoimmune encephalomyelitis (EAE) mouse, development and uveitis mouse model Generation, the development of the property tested Autoimmune uveitis (EAU) have significant inhibiting effect, that is, show provided by the invention P28-Fc and its coding nucleic acid molecule, recombinant expression carrier or recombinant cell have the function for the treatment of autoimmune disease, can It is used to prepare relative medicine.

Detailed description of the invention

Fig. 1 is the analysis result of codon preference in embodiment 1；

Fig. 2 is the electrophoretogram after the elution of p28-Fc fusion protein；Wherein, 1 is Marker, 2 fusion to elute for the first time Albumen p28-Fc, 3 for second of elution fusion protein p28-Fc, 4 for third time elution fusion protein p28-Fc, 5 be the The fusion protein p28-Fc of four elutions；

Fig. 3 is the result for carrying out Westeinblot identification to obtained p28-Fc fusion protein with anti-p28 antibody；Wherein, 1 is PBS, and 2 be p28-Fc fusion protein；

Fig. 4 is the result for carrying out Westein blot identification to obtained p28-Fc fusion protein with anti-igg Fc antibody； Wherein, 1 is PBS, and 2 be p28-Fc fusion protein；

Fig. 5 is the result that FACS detects STAT3 phosphorylation abilities in embodiment 1；

Fig. 6 is the result that Western biot detects STAT3 phosphorylation abilities in embodiment 1；

Fig. 7 is the result that MTS method detects ability of cell proliferation；

The testing result that Fig. 8 behaviour Th16 cell generates；

Fig. 9 is the knot that Western biot detects STAT3 and expression of STAT 3 phosphorylation in source EAE mouse CD4-T cell Fruit；

Figure 10 is the proliferative conditions result of the EAE mouse CD4-T cell after MTS hair detection different disposal；

Figure 11 be cell streaming technology detect different disposal after EAE mouse hydrocrania in Th17 cell percentages knot Fruit；

Figure 12 is the result that ELISA detects ILI-17 level in the EAE mouse cerebrospinal fluid of different disposal；

Figure 13 is the EAE mice clinical scoring event of different disposal；

Figure 14 is the EAU mouse CD4 that H-TdR incorporation methods detect different disposal⁺T cell proliferative conditions；

Figure 15 is the result that cell streaming technology detects the intraocular Th17 cell of EAU mouse；

Figure 16 is the EAU mouse Yan Di, also known as Shen Nong, a legendary ruler image and its disease development of different disposal.

Specific embodiment

The amino acid sequence of the hIL-27p28 is as shown in SEQ ID NO.1.

In the present invention, the Fc segment refers to human immunoglobulin(HIg) chain constant region, especially heavy chain immunoglobulin The c-terminus of constant region or in which a part.The nonantigenic combination activity of the Fc segment, be antibody molecule and effector molecule and The position of cell interaction.For example, immunoglobulin fc region may include two or more knots of heavy chain CH1, CH2, CH3, CH4 The combination in structure domain and immunoglobulin hinge region.According to the amino acid sequence of heavy chain constant region, immunoglobulin can be divided into not Same type, mainly there is 5 immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM.Some of them can also be further separated into subclass (isotype), such as IgG-1, IgG-2, IgG-3, IgG-4；IgA-1 and IgA-2 and different genotype." Fc of the present invention Segment " preferably includes at least one immunoglobulin hinge region and the area CH2 and CH3 of IgG.More preferably cut with scissors by IgG4 Sequence, constant region 2 and constant region 3 form, and the amino acid sequence of the hIgG4Fc is as shown in SEQ ID NO.3.

For the amino acid sequence of hIL-27p28 of the present invention as shown in SEQ ID NO.1, hIL-27p28 is people IL27p28 Segment after removing signal peptide sequence and terminator codon, to overcome conventional recombination IL27p28 that can not obtain the egg correctly folded White problem.

In a specific embodiment of the present invention, Fc segment of the present invention is to remove 1 group of constant region by hIgG4Fc segment section At.In the present invention, as shown in SEQ ID NO.3, the hIgG4Fc segment contains the amino acid sequence of the hIgG4Fc segment Hinge area (J), constant region 2 (CH2) and the constant region 3 (CH3) of someone IgG4Fc.The present invention removes constant region 1 in human IgG 4Fc Purpose be remove IgG CH1 dimerization function, that is, prevent p28-Fc fusion protein dimerization.The present invention is described in the building The purpose of Fc segment is selected to have three: a, be conducive to protein expression when p28-Fc fusion protein；B, p28-Fc when improving vivo applications The half-life period of fusion protein；C, the Fc segment can reduce fusion protein induction immunity of organism stimulation.

In the present invention, the amino acid sequence of the p28-Fc fusion protein is preferably as shown in SEQ ID NO.5:

FPRPPGRPQLSLQELRREFTVSLHLARKLLSEVRGQAHRFAESHLPGVNLYLLPLGEQLPDVSLTFQAW RRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERMQLWAMRLDLRDLQRHLRFQVLAAGFNLPEEEEEEEEEEE EERKGLLPGALGSALQGPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVWPLGFPTLSPQPPPCPSCPA PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVRVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPEDNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

The present invention also provides the nucleic acid molecules for encoding fusion protein described in above-mentioned technical proposal, the nucleic acid molecules Nucleotide sequence as shown in SEQ ID NO.6:

Wherein, thickened portion is the base sequence for encoding hIgG Fc；Horizontal line part is two terminator codons；From beginning to end two The base of section capitalization is SphI (GCATGC) and XbaI (TCTAGA) restriction endonuclease sites；It is added identical as two on carrier Base (TA) is in order to ensure correct coding p28-Fc amino acid prevents frameshit；Remainder is the alkali for encoding hIL-27p28 Basic sequence.

Nucleic acid molecules provided by the invention will not influence the function and the wherein base of regulating and expressing amount of IL-27p28, thus The p28-Fc fusion protein for obtaining coding has the correct folding configuration and biological function of IL-27p28, and p28-Fc melts Hop protein can the soluble high expression in host cell.

The present invention also provides a kind of recombinant expression carriers of fusion protein described in preceding solution, by above-mentioned technical side Nucleic acid molecules described in case are inserted into the multiple cloning sites of insect expression vector to get the weight of fusion protein described in preceding solution Group expression vector；Contain Honeybee melittin secretion signal signal peptide in the insect expression vector.

Due to human IL-2 7p28 signal peptide be based on codon preference the problem of, containing be not suitable for mammal express Base, the correct of recombination IL-27p28 is folded and solubility expression causes obstacle, the present invention is in order to overcome the problems, such as that this goes In addition to the signal peptide moiety of IL-27p28, and uses and contain Honeybee melittin secretion signal signal peptide Insect expression vector expresses the encoding gene of p28-Fc, to obtain, configuration is correct, fusion protein of solubility expression p28-Fc.In a specific embodiment of the present invention, the insect expression vector is preferably pMIB.In addition to this, the present invention may be used also It is constructed with selecting other to contain the insect expression vector of Honeybee melittin secretion signal signal peptide The recombinant expression carrier of p28-Fc fusion protein.

It is described heavy the present invention also provides a kind of recombinant cell of p28-Fc fusion protein described in expression preceding solution Group cell is the insect cell for including recombinant expression carrier described in above-mentioned technical proposal.In a specific embodiment of the invention In, the insect cell is preferably engineered High Five^TMCell strain.The present invention can also be suitable for recombinating elder brother using other The host cell that worm expression vector is expressed constructs the recombinant cell.

In the present invention, be conducive to the expression of fusion protein, thus the present invention using recombinant cell described in free serum culture It is preferably thin using recombination described in serum free medium culture when using recombinant cell expression p28-Fc fusion protein Born of the same parents.

The present invention also provides p28-Fc fusion protein described in preceding solution and its coding nucleic acid molecules, recombinant expression The application of carrier or recombinant cell in the drug of preparation treatment autoimmune disease.As shown in the embodiment of the invention, this hair The generation of autoimmune encephalomyelitis mouse model, development can be effectively suppressed in the p28-Fc fusion protein of bright offer, may also suppress Generation, the development of Autoimmune uveitis mouse model.

In the present invention, the autoimmune disease preferably itself is exempted from by gp13 signal or Th17 are cell-mediated Epidemic disease disease.As shown in the test of the embodiment of the present invention, the fusion protein p28-Fc that the present invention constructs can be as the short of money of gp130 Anti-agent inhibits its relevant IL-6, IL-27 signal, may also suppress human T lymphocyte's proliferation, inhibits the external production of people Th17 cell It is raw.

Specifically, autoimmune disease of the present invention includes but is not limited to multiple sclerosis, autoimmune meninx Scorching, uveitis or psoriasis.

The present invention also provides a kind of for treating the drug of autoimmune disease, and the drug includes aforementioned techniques side P28-Fc fusion protein and pharmaceutically acceptable auxiliary material described in case.The present invention is to the pharmaceutically acceptable auxiliary material without spy It is different to limit, using known in the art.

Drug of the present invention for treating autoimmune disease can be made into various dosage forms known in the art, including But it is not limited to tablet, oral solution, granule, capsule or pulvis.The present invention to the drug how to be prepared into corresponding dosage form without Particular determination, using methods known in the art.

Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood For limiting the scope of the present invention.

Embodiment 1

(1): the building of hp28-hIgG4Fc insect expression vector

Expression vector establishment process:

1. containing the base sequence and ammonia of signal peptide by Pubmed data base querying human IL-2's 7A, that is, IL-27p28 subunit Base acid sequence

Human IL-27p28cDNA(SEQ ID NO.7)

Wherein, the encoding base of the signal peptide of thickened portion behaviour IL-27p28.

Human IL-27p28 amino acid sequence (SEQ ID NO.8)

MGQTAGDLGWRLSLLLLPLLLVQAGVWGFPRPPGRPQLSLQELRREFTVSLHLARKLLSEVRGQAHRFA ESHLPGVNLYLLPLGEQLPDVSLTFQAWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERMQLWAMRLDLRD LQRHLRFQVLAAGFNLPEEEEEEEEEEEEERKGLLPGALGSALQGPAQVSWPQLLSTYRLLHSLELVLSRAVRELLL LSKAGHSVWPLGFPTLSPQP

Wherein, the amino acid sequence of the signal peptide of thickened portion behaviour IL-27p28.

2, the codon preference of IL-27A is analyzed by computer

Analysis result as shown in Figure 1, in display signal peptide coding region by be unfavorable for mammal expression coding (it is red and Pink colour).

3, using the insect expression vector pMIB for having Honeybee melittin secretion signal signal peptide:

Since human IL-2's 7p28 native signal peptide is unfavorable for lactation system expression, we are used with Honeybee The insect expression vector pMIB of melittin secretion signal signal peptide:

Insect expression vector pMIB (pMIB/V5-His A, B, and C Vector Kit, Catalog no.V8030- 01) it is purchased from Invitrogen company.

Honeybee melittin secretion signal signal peptide base sequence (SEQ ID NO.9):

ATG AAA TTC TTA GTC AAC GTT GCC CTT GTT TTT ATG GTC GTA TAC ATT TCT TACATC TAT GCC。

4, the base sequence, that is, hIgG Fc encoding mature ammonia for removing signal peptide and terminator codon in IL-27p28 subunit The base sequence (SEQ ID NO.2) and amino acid sequence (SEQ ID NO.1) of base acid.

5, pass through the base sequence (sequence 6) and amino of Pubmed data base querying human IgG 4Fc removal constant region 1 (CH1) Acid sequence (sequence 7) includes hinge area (J), constant region 2 (CH2) and constant region 3 (CH3).

6, the base sequence of coding hIL-27p28 peptide is connect with the base sequence of coding hIgG4, writes a Chinese character in simplified form p28-Fc (SEQ ID NO.6)。

7, the base sequence for encoding hIgG4Fc is abbreviated as Fc

The base sequence (SEQ ID NO.9) of Fc

Note: thickened portion is encoding human IgG4Fc hinge area (J), the base sequence of constant region 2 (CH2) and constant region 3 (CH3) Column；Dashed part is to have added two terminator codons；Both ends black base is SphI (GCATGC) and XbaI (TCTAGA) is limited Property restriction enzyme site；In addition base (italic TA) identical as two on carrier is in order to ensure correct coding Fc amino acid prevents Frameshit.

7, nucleic acid molecules shown in SEQ ID NO.6 are synthesized (for encoding by general biosystem (Anhui) Co., Ltd P28-Fc fusion protein) and SEQ ID NO.9 shown in Fc base sequence (the hIgG4Fc segment that can be expressed)；It recycles The method of SphI (GCATGC) and the site XbaI (TCTAGA) homologous recombination weighs SEQ ID NO.5 or SEQ ID NO.9 respectively Group arrives carrier pMIB carrier, obtains two recombinant expression carriers of pMIB/p28-Fc and pMIB/Fc (pair of the pMIB/Fc as test According to group).

8, pMIB/p28-Fc and pMIB/Fc two is identified by gene sequencing by general biosystem (Anhui) Co., Ltd A expression plasmid is correct

(2): utilizing the engineering High Five of free serum culture^TMCell strain expresses p28-Fc fusion protein.

1, prepare two expression plasmids of pMIB/p28-Fc and pMIB/Fc

We use PureLink^TMHQ Mini Plasmid isolated plasmid dna purification kit (Invitrogen company, Catalog number (Cat.No.) K2100-01), 10~15 g plasmid DNA are separated from 10~15 milliliters of culture bacteriums.

2, prepare High Five^TMInsect cell

For transfecting every time, 95% logarithmic phase cell is greater than using vigor.

(1) with serum free medium Express Five SFM (Invitrogen company, article No.: 10486-025) 60 2 × 10 are inoculated in millimeter culture dish⁶A High Five^TMCell (Invitrogen company, article No.: B855-02)；

(2) by cell incubation at least 15 minutes without shaking to allow cell to be adhering completely to the bottom of culture dish, formed Cell monolayer；

(3) cell is checked by being inverted, confirmation cell has adhered to.

3, plasmid transfection is to High Five^TMIn cell

By plasmid pMIB/p28-Fc or pMIB/Fc andTry (Invitrogen company, article No.: 10362- 010) agent mixes in the right way, and cultivates together with the insect cell being newly inoculated with.

A. prepare every kind of transfection mixture, use 1.5ml microcentrifugal tube.Add following reagent: 1 milliliter of free serum culture Base, 1-10 μ l pMIB/p28-Fc or pMIB/Fc (about 1 μ g/ μ l, pH8 in TE), 20 μ lReagent；

B. it is gently mixed transfection mixture 10 seconds.

C. transfection mixture is incubated at room temperature 15 minutes.When transfection mixture is incubated for, step 4 is carried out.

D. culture medium is carefully taken out from cell, not destroy cell monolayer.

E. entire transfection mixture is added dropwise in 60 millimeters of culture dishes.

4, stable cell line is obtained

A. it 48 hours after transfecting, takes out transfection liquid and fresh culture is added.

B. 80ug/ml blasticidin will be contained in selective medium after cell 1:5 (20% converges) dilution It is cultivated in Blasticidin S (Invitrogen company, article No.: R210-01) serum free medium Express Five SFM.

C. every the selective medium of replacement in 3 to 4 days, until observing that focus is formed.

D. clone cell is separated using dilution

F. the cell strain high using ELISA detection expression quantity

5, protein purification

Collect sample: the pMIB/p28-Fc or pMIB/Fc collected free serum culture 72 hours stablizes the culture of expression cell Supernatant.

Balance: with the equilibration buffer of 5~10CV, (20mM PB+0.15M NaCl, pH 7.0, adds debita spissitudo NaCl inhibits non-specific adsorption) balance chromatographic column, until efflux conductance and pH are constant (consistent with equilibrium liquid).

Charging: sample buffer should be as consistent with equilibrium liquid as possible.Solid sample can be dissolved with equilibrium liquid to be prepared；Low concentration Sample solution can be dialysed with equilibrium liquid；Enriched sample solution can be diluted with equilibrium liquid.In order to avoid blocking chromatographic column, sample is answered Through centrifugation or (0.45 μm) of micro-filtration processing.Inlet amount is calculated according to the content of target protein in the carrying capacity and feed liquid of medium.

Elution: continue to be eluted with equilibration buffer to baseline after loading.

Elution: being eluted with elution buffer (20mM sodium acetate, pH 3.0~4.0 or 0.1M glycine, pH 3.0), is collected Efflux.After elution, alkaline buffer (such as 1M Tris/HCl, pH 9.0) should be used to neutralize the protein solution being collected at once To neutrality.

In conjunction with p28-Fc fusion protein elute result as shown in Fig. 2, the results show that albumen, that is, third of the 4th swimming lane Secondary elution can obtain purer albumen.

2, we are that the purer albumen of third time elution acquisition is identified to the albumen of the 4th swimming lane.

(1) the anti-p28 antibody of application carries out Western blot identification

It collects protein sample (Protein sample preparation): collecting third time elution and obtain purer egg It is white, using 1X PBS diluted protein sample concentration to 0.1mg/ml, by isometric 2X SDS-PAGE albumen sample-loading buffer (green skies company, article No. P0015) is added in diluted protein sample or the not PBS of protein sample.100 DEG C or boiling water bath Heating 3-5 minutes, with abundant albuminate.

Electrophoresis: will directly be loaded in SDS-PAGE glue well, voltage 100V containing 10 μ g samples, and the time is 100 minutes, the bottom end that bromophenol blue reaches glue when electrophoresis can nearby stop electrophoresis.

Transferring film: it uses nitrocellulose filter (NC film) (green skies company, article No. FFN06/FFN09), uses Bio-Rad's Standard wet type membrane-transferring device can set transferring film electric current as 300mA, and the transferring film time is 50 minutes.

Closing: after transferring film, protein film is placed into preprepared Western cleaning solution immediately, and (the green skies are public Department, article No. P0023C) in, it rinses 1-2 minute, to wash away the transferring film liquid on film, addition Western confining liquid (green skies company, Article No. P0023B), it is slowly shaken on shaking table, room temperature is closed 60 minutes.

Primary antibody is incubated for: diluting rabbit-anti human IL-2 7p28 antibody (Invitrogen company, article No. by 1:1000 with 1X PBST PA5-20240), the primary antibody diluted is added immediately, room temperature slowly shakes incubation 1 hour on the side shaker.Western is added Cleaning solution (green skies company, article No. P0023C) slowly shakes washing 5-10 minutes on the side shaker.After exhausting cleaning solution, Cleaning solution is added to wash 5-10 minutes.It washs 3 times altogether.

Secondary antibody is incubated for: with 1X PBST by the goat anti-rabbit igg (connection of 1:5000 dilution horseradish peroxidase (HRP) label Biotech firm, section, article No. 70-GAR0072).The secondary antibody diluted is added immediately, room temperature slowly shakes incubation on the side shaker 45min.It is added Western cleaning solution (green skies company, article No. P0023C), slowly shakes 5-10 points of washing on the side shaker Clock.After exhausting cleaning solution, adds cleaning solution and wash 5-10 minutes.It washs 3 times altogether.

Protein Detection: albumen is detected using ECL class reagents such as BeyoECL Plus (green skies company, article No. P0018). Tabletting is carried out using dedicated tabletting magazine (FFC58/FFC83).With developing fixing kit (green skies company, goods when developing a film Number P0019/P0020) voluntarily preparing developer liquid and fixing solution develop a film by hand.The Bioexperiment that X-ray selects Kodak original-pack Dedicated Kodak X-OMAT BT film (green skies company, article No. FF057/FF081).

As a result as Fig. 3 shows that anti-p28 antibody can identify p28-Fc.

(2) Western blot identification is carried out using anti-igg Fc antibody

It collects protein sample: collecting third time elution and obtain purer albumen, using 1X PBS diluted protein sample concentration To 0.1mg/ml, isometric 2X SDS-PAGE albumen sample-loading buffer (green skies company, article No. P0015) is added to dilute The protein sample released or not in the PBS of protein sample.100 DEG C or boiling water bath heating 3-5 minutes, with abundant albuminate.

Primary antibody, that is, secondary antibody is incubated for: with 1X PBST by the mouse anti human of 1:5000 dilution horseradish peroxidase (HRP) label IgG4Fc antibody (SouthernBiotech company, article No. 9190-05).The secondary antibody diluted is added immediately, room temperature is shaken in side-sway It is slowly shaken on bed and is incubated for 45min.It is added Western cleaning solution (green skies company, article No. P0023C), delays on the side shaker It is slow to shake washing 5-10 minutes.After exhausting cleaning solution, adds cleaning solution and wash 5-10 minutes.It washs 3 times altogether.

As a result as Fig. 4 shows that anti-igg Fc antibody can identify p28-Fc.

3, protein quantification

We show using protein quantification method, obtain 0.8mg/ml p28-Fc albumen, we carry out using this albumen External functional experiment.

(3): external Function Identification.

1, p28-Fc fusion protein inhibition passes through IL-6 signal related to receptor gp13

Collect CD4⁺T cell: it collects health and donates blood the peripheral blood in people (307 hospital) source, exempt from using Mei Tian Ni company CD4 Epidemic disease magnetic bead (article No. 130-045-101) sub-elects CD4 to specifications⁺T cell.

Stimulate CD4⁺T cell: application anti-human CD3/CD28 magnetic bead (Gibco company, Cat.No.111.61D) stimulates people CD4⁺ T cell collects cell after stimulation 24 hours.

P28-Fc: according to purification process above, we have purified cell strain (the respectively 1# of two Expression of Plant Height P28-Fc And 2#) secretion P28-Fc, the P28-Fc of this two plants of cell origins is marked as 1# and 2#P28-Fc.

STAT3 activation: the CD4 of anti-human CD3/CD28 magnetic bead activation⁺Continue after T cell harvest in serum-free conditioned media Culture is 6 hours hungry, the addition μ of negative control Fc, 1 or 5 g/ml positive control MIL-45 (gp130 antagonist) or 1 μ g/ml 1# and 2#P28-Fc, 50ng/ml IL-6 (Gibco company, article No. PHC0064) is added immediately stimulates 30min.

PSTAT dyeing: 2% paraformaldehyde is added in the cell of processing, is kept for 10 minutes at 37 DEG C, application cell stream Formula buffer (PBS, pH 7.2, containing 0.2%BSA and 0.09% sodium azide) washed once, in 100% ice-cold methanol thoroughly Change, 30 minutes on ice.Application cell streaming buffer washes twice, then with anti-pY705-Stat3-PE (49/p-Stat3) Or isotype controls (BD Biosciences) 30 minutes, application cell streaming buffer washs 2 times

The analysis of cell stream data: cell is analyzed on FACScan (BD Biosciences).CellQuest version 3 .3 (BD Biosciences) is used for data collection and analysis.

As a result as shown in figure 5, p28-Fc fusion protein can effectively inhibit IL-6 signal.

2, p28-Fc fusion protein inhibition passes through IL-27 signal relevant to receptor gp13

P28-Fc: according to purification process above, we have purified cell strain (the respectively 1# of two Expression of Plant Height P28-Fc And 2#) secretion P28-Fc, the P28-Fc of this two plants of cell origins is marked as 1# and 2#P28-Fc

STAT3 activation: the CD4 of anti-human CD3/CD28 magnetic bead activation⁺Continue after T cell harvest in serum-free conditioned media Culture is hungry 6 hours, is added negative control Fc, 5 μ g/ml positive control MIL-45 (gp130 antagonist) or 1 μ g/ml1# and 2#P28-Fc, 10ng/ml IL-27 (R&D system company, article No. 2526-IL) is added immediately stimulates 30min.

Collect protein sample: collecting the cell of processing, be added 2X SDS-PAGE albumen sample-loading buffer (green skies company, Article No. P0015), 100 DEG C or boiling water bath heating 3-5 minutes, with abundant albuminate.

Primary antibody be incubated for: with 1X PBST by 1:1000 dilution rabbit-anti people STAT3 or pSTAT3 (R&D Systems, MAB1799, AF4607) antibody, the primary antibody diluted is added immediately, room temperature slowly shakes incubation 1 hour on the side shaker.Add Enter Western cleaning solution (green skies company, article No. P0023C), slowly shakes washing 5-10 minutes on the side shaker.It exhausts After cleaning solution, adds cleaning solution and wash 5-10 minutes.It washs 3 times altogether.

As a result as Fig. 6 shows that p28-Fc fusion protein can also effectively inhibit IL-27 signal.

3, p28-Fc fusion protein inhibits people CD4⁺T cell proliferation

Stimulate CD4⁺T cell: in 96 orifice plates, 200 μ L 10 are added in every hole⁶/ml CD4⁺It is negative that 50ng/ml is added in T cell Fc or 1#, 2#P28-Fc are compareed, is added anti-human CD3/CD28 magnetic bead (Gibco company, Cat.No.111.61D), people CD4 is stimulated⁺ T cell 72 hours.

MTT is added: after culture 72 hours, every hole adds 20 μ l MTT (3- (4,5)-dimethylthiahiazo (- z-y1)- 3,5-di-phenytetrazoliumromide, entitled 3- (4,5- dimethylthiazole -2) -2,5- diphenyl, four nitrogen of Chinese chemistry Azoles bromide, trade name: thiazolyl blue) (5mg/ml is prepared solution with PBS, pH=7.4, Trevigen company, article No. 4890-025- K), continue to be incubated for 4h.

Terminate: every hole adds 150ul DMSO, vibrates 10min, melts crystal sufficiently.

Colour developing: selection 490nm wavelength measures each hole absorbance value on enzyme linked immunological monitor, records result.

As a result such as Fig. 7 is shown, p28-Fc fusion protein can effectively inhibit human T lymphocyte's proliferation.

4, p28-Fc fusion protein inhibits people Th17 cell to generate

The generation of people's Th17 cell is that height relies on IL-6 signal, and p28-Fc fusion protein is by inhibiting IL-6 signal The generation of people's Th17 cell can be effectively suppressed.Steps are as follows for specific experiment:

P28-Fc: according to purification process above, we purify P28-Fc, obtain the P28-Fc that concentration is 0.8mg/ml and melt Hop protein.

Th17 is induced to generate: under 50ng/ml negative control Fc or P28-Fc existence condition, using anti-human CD3/CD28 magnetic Pearl (Gibco company, Cat.No.111.61D) combines 10 μ g/ml anti-IFN γ, 10 μ g/ml anti-IL-4,10ng/ml IL-6,10ng/ml TGF-β stimulates people CD4⁺T cell stimulates 72 hours.

Intracellular IL-17 dyeing: add 1ml Fixation/permeabilization working solution (eBioscience, Cat.No.00-5521,00-8333), fixed cell, room temperature is protected from light incubation 2 hours；Every pipe add 1ml 1 × Permeabilization buffer (eBioscience, Cat.No.00-4222), washes twice, then uses anti-IL- 17-PE (BD Biosciences, clone TC11-18H10) dye 30 minutes, every pipe add 1ml 1 × Permeabilization buffer (eBioscience, Cat.No.00-4222), washes twice.Appropriate streaming dyeing liquor weight It is outstanding, machine testing can be gone up.

As a result such as Fig. 8 is shown, p28-Fc fusion protein can effectively inhibit the people's Th17 cells in vitro for relying on IL-6 signal It generates.

(4): p28-Fc fusion protein inhibits multiple sclerosis (MS) mouse model, experimental autoimmune meningitis (EAE) mouse disease occurs, develops.

Multiple sclerosis (MS) is a kind of autoimmune disease mediated with Th17, and IL-6-gp130 signal path is The important deciding factor that Th17 is generated, and p28-Fc fusion protein can effectively inhibit gp130 signal path, it is possible to inhibit Th17 is generated and is inhibited MS.

Experimental autoimmune meningitis (EAE) mouse model and MS pathogenesis are quite similar, are the pole for studying MS Good animal model.

EAE induction:

Briefly, the C57BL/6 mouse of 9 week old gets an injection under the skin.Emulsification contains 4mg/ml mycobacterium tuberculosis CFA and 125 milligram of myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide 1:1 (v/v) of H37Ra；Injection emulsion arrives The bottom and two sides of mousetail.Intraperitoneal injection pertussis toxin (300ng pertussis toxin in PBS simultaneously；List Biological)。

It is weighed according to following step scale to animal, monitoring and clinical assessment:

0=does not have sign；The distal end 1=tail limp；1.5=tail weakness and some hind limb weakness；The complete tail fiber crops of 2= Numbness；The complete tail paralysis of 2.5=and partial hind are powerless；Weakness of limbs after the completion of 3=；The back 3.5=or it is important when can not it is correct before Myasthenia of limbs；4=euthanasia or spontaneous death.If losing the 20% of starting weight, will be euthanized.

It is small using myelin oligodendrocyte glycoprotein (MOG) 35-55peptide induction at the 0th day Mouse generates EAE, treats EAE mouse using every mouse 200ng control Fc or p28-Fc fusion protein at the 7th day, every group has 6 Mouse carried out following experiment at the 21st day:

1, p28-Fc fusion protein inhibits STAT3 activity in EAE mouse T cell

Go out mouse CD4 using CD4 immunological magnetic bead sorting⁺T cell detects source EAE mouse CD4 using Western blot⁺ STAT3 and pSTAT3 (pSTAT3) expression in T cell.Specific experiment process is as follows:

Sort mouse CD4⁺T cell: it is outstanding to take out mouse spleen grinding splenoblast for 21 days execution mouse after EAE induction Liquid, Application mouse spleen lymphocyte separation liquid kit (Solarbio company, article No.: P8860) separate monocyte, apply Mei Tian Ni company's mouse CD4 immunomagnetic beads (article No. 130-049-201) sub-elect CD4 to specifications⁺T cell.

It prepares protein sample: 2X SDS-PAGE albumen sample-loading buffer (green skies company, goods being added in the cell of collection Number P0015), 100 DEG C or boiling water bath heat 3-5 minutes, with abundant albuminate.

Primary antibody is incubated for: diluting rabbit-anti people STAT3 or pSTAT3 (Cell Signaling by 1:1000 with 1X PBST Technology company, article No. 4904and 9131) antibody, the primary antibody diluted is added immediately, room temperature is delayed on the side shaker Slow shake is incubated for 1 hour.It is added Western cleaning solution (green skies company, article No. P0023C), slowly shakes on the side shaker Washing 5-10 minutes.After exhausting cleaning solution, adds cleaning solution and wash 5-10 minutes.It washs 3 times altogether.

As a result such as Fig. 9 is shown, p28-Fc fusion protein can also effectively inhibit STAT3 activation and pSTAT3 (pSTAT3) it expresses.

2, p28-Fc fusion protein inhibits reaction of the EAE mouse T cell to autoantigen peptide MOG33-55

The EAE mouse CD4 of various processing is stimulated using MOG33-55⁺T cell detects cell Proliferation using MTS method.Specifically Steps are as follows:

Sort mouse CD4⁺T cell: 21 days execution mouse after EAE induction take out various processing mouse spleens and are ground into Splenocyte suspension, Application mouse spleen lymphocyte separation liquid kit (Solarbio company, article No.: P8860) separate monokaryon Cell sub-elects CD4 using Mei Tian Ni company's mouse CD4 immunomagnetic beads (article No. 130-049-201) to specifications⁺T cell.

Stimulate cell: in 96 orifice plates, 200 μ L 10 are added in every hole⁶/ml CD4⁺50ng/ml MOG33-55 is added in T cell Peptide (SBS Genetech Genentech, Inc. (US) 460 Point San Bruno Blvd, South San Francisco, CA, 94080 synthesizes by Beijing) stimulates CD4⁺T cell 72 hours.

MTS is added: every hole adds 20 μ l MTS solution, continues to be incubated for 4h.

As a result as Figure 10 shows that p28-Fc fusion protein can effectively inhibit EAE mouse T cell to the anti-of autoantigen It answers.

3, p28-Fc fusion protein inhibits the generation of EAE mouse Th17

Mouse hydrocrania is collected, application cell streaming technology detects Th17 cell percentages in hydrocrania.Specific experiment step It is rapid as follows:

It collects mouse hydrocrania: wiping rat the nape of the neck skin with wet gauze, cut off dorsal body setae exposed skin.Two ear lines are cut ((1.5cm or so) puts caudal ward along subcutaneously 2cm is cut off wherein, skin is separated two sides, expands the visual field one transverse incision.It is close to big Mouse skull successively successively shears each muscle layer, and the broken ends of fractured bone successively pulls to caudal and expands the visual field.Note that being pressed when having bleeding with dry gauze Hemostasis keeps art mouth clear.Close to after neck when ligamentum flavum, the muscle of attached lid, exposure extensive region pillow are carefully separated with No. 7 injection needles Film.With l ml syringe (syringe needle haemostatic clamp makes needle point and needle body at 150 degree of obtuse angles), needle inclined-plane is upward, and needle tip is closely horizontal It is pierced into cavum subarachnoidale, fixed needle body slowly extracts cerebrospinal fluid, and general collection capacity is 100-200 microlitres.

Intracellular IL-17 dyeing: 1500rpm X 6min is centrifuged mouse hydrocrania, obtains cell precipitation, adds 1ml Fixation/permeabilization working solution (eBioscience, Cat.No.00-5521,00-8333), fixed cell, Room temperature is protected from light incubation 2 hours；Every pipe adds 1ml 1 × permeabilization buffer (eBioscience, Cat.No.00- 4222) it, washes twice, then dyes 30 points with anti-IL-17-PE (BD Biosciences, clone TC11-18H10) Clock, every pipe add 1 × permeabilization of 1ml buffer (eBioscience, Cat.No.00-4222), wash twice. Appropriate streaming dyeing liquor is resuspended, and can go up machine testing.

As a result as Figure 11 shows that p28-Fc fusion protein can effectively inhibit the generation of EAE mouse Th17.

4, p28-Fc fusion protein inhibits the generation of EAE mouse IL-17

Mouse cerebrospinal fluid is collected, it is horizontal using IL-17 in ELISA detection mouse cerebrospinal fluid.

Steps are as follows for specific experiment:

It collects mouse cerebrospinal fluid: wiping rat the nape of the neck skin with wet gauze, cut off dorsal body setae exposed skin.Two ear lines are cut ((1.5cm or so) puts caudal ward along subcutaneously 2cm is cut off wherein, skin is separated two sides, expands the visual field one transverse incision.It is close to big Mouse skull successively successively shears each muscle layer, and the broken ends of fractured bone successively pulls to caudal and expands the visual field.Note that being pressed when having bleeding with dry gauze Hemostasis keeps art mouth clear.Close to after neck when ligamentum flavum, the muscle of attached lid, exposure extensive region pillow are carefully separated with No. 7 injection needles Film.With l ml syringe (syringe needle haemostatic clamp makes needle point and needle body at 150 degree of obtuse angles), needle inclined-plane is upward, and needle tip is closely horizontal It is pierced into cavum subarachnoidale, fixed needle body slowly extracts cerebrospinal fluid, and general collection capacity is 100-200 microlitres.

Coating: buffer is coated with by anti-mouse IL-17 coated antibody (Invitrogen, goods with 0.05M PH9. carbonate Number 88-7371) to be diluted to protein content be 5 μ g/ml.Add 0.1ml in the reacting hole of each polystyrene board, 4 DEG C overnight. Next day discards solution in hole, is washed 3 times, every time 3 minutes with washing buffer.

Sample-adding: adding certain diluted measuring samples 0.1ml in the above-mentioned reacting hole being coated with, and sets 37 DEG C and is incubated for 1 hour. It is washed out.

Detection antibody is added: adding certain diluted anti-mouse IL-17 detection antibody (Invitrogen, article No. 88-7371) 0.1ml sets 37 DEG C and is incubated for 1 hour in the above-mentioned reacting hole being coated with.It is washed out.

Enzyme labeling antibody: in each reacting hole, the enzyme labelled antibody 0.1ml of diluted fresh is added.37 DEG C are incubated for 0.5 hour, Washing.

Add substrate solution to develop the color: being added the tmb substrate solution 0.1ml of Extemporaneous in each reacting hole, 37 DEG C 10~30 points Clock.

It terminates reaction: 2M sulfuric acid 0.05ml being added in each reacting hole.

Result judgement: on ELISA detector, at 450nm, each hole OD value is surveyed after returning to zero with blank control wells.In Standard items compare, and calculate IL-17 concentration.

As a result as Figure 12 shows that p28-Fc fusion protein can effectively inhibit the generation of EAE mouse IL-17.

5, p28-Fc fusion protein inhibits EAE mice clinical score

Standard is fallen ill [with reference to Stromnes, I.M., and J.M.Goverman.2006.Active by EAE induction of experimental allergic encephalomyelitis.Nat.Protoc.1:1810– 1819.Butzkueven,H.,B.Emery,T.Cipriani,M.P.Marriott,and T.J.Kilpatrick.2006.Endogenous leukemia inhibitory factor production limits autoimmune demyelination and oligodendrocyte loss.Glia 53:696–703.Gresle, M.M.,G.Shaw,B.Jarrott,E.N.Alexandrou,A.Friedhuber,T.J.Kilpatrick,and H.Butzkueven.2008.Validation of a novel biomarker for acute axonal injury in Experimental autoimmune encephalomyelitis.J.Neurosci.Res.86:3548-3555.] determine EAE Clinical scores.As a result such as Figure 13 is shown, p28-Fc fusion protein can effectively inhibit the generation of EAE mouse disease, development.

(5): p28-Fc fusion protein inhibits uveitis Experimental model of small mice Autoimmune uveitis (EAU) Mouse disease occurs, develops.

Uveitis (uveitis) is a kind of autoimmune disease mediated with Th17, and IL-6-gp130 signal is logical Road is the important deciding factor that Th17 is generated, and p28-Fc fusion protein can effectively inhibit gp130 signal path, it is possible to Th17 is inhibited to generate and inhibit uveitis.

Experimental autoimmune uveoretinitis (EAU) mouse model and uveitis pathogenesis are quite similar, are research The fabulous animal model of uveitis.

Induce EAU and histology

Vitamin A acid binding protein (IRBP) and 300 grams of people's IRBP peptide (amino acid residues between containing 150g ox photoreceptor 0.2 milliliter of solution 1-20) and the complete Freund's adjuvant 1:1 (v/v) for containing M. tuberculosis strains H37Ra (2.5mg/ml) Emulsification, active immunity C57BL/6 mouse is induction of EAU.Receive Bordetella pertussis toxin (0.2 gram/mouse) simultaneously.

Bovine interphotoreceptor retinoid-binding protein (IRBP) was used at the 0th day And human IRBPpeptide (amino acid residues 1-20) inducing mouse generates EAU, at the 7th day using every Mouse 200ng control Fc or p28-Fc fusion protein treats EAU mouse, and every group has 6 mouse, carries out at the 21st day following Experiment:

1, p28-Fc fusion protein inhibits reaction of the EAU mouse T cell to autoantigen

The EAE mouse CD4 of various processing is stimulated using IRBP⁺T cell detects cell Proliferation using H-TdR incorporation methods.

As a result as Figure 14 shows that p28-Fc fusion protein can effectively inhibit EAU mouse T cell to the anti-of autoantigen It answers.

2, p28-Fc fusion protein inhibits the generation of EAU mouse Th17

Application cell streaming technology detects the intraocular Th17 cell of EAU mouse.Steps are as follows for specific experiment:

Obtain intraocular cell: the mouse of all kinds of processing is put to death in excessive anesthesia, wins eyeball, and ocular tissue is ground and is filtered, and is received Collect cell suspension, PBS is washed twice, carries out cell inner dyeing.

As a result as Figure 15 shows that p28-Fc fusion protein can effectively inhibit the generation of EAU mouse Th17 cell.

3, p28-Fc fusion protein inhibits EAU mice clinical score

We have checked eyes in 21 days after immune by funduscopy；Receive the control EAU mouse or EAU mouse of Fc Eye fundus image show serious papilledema, retinal vasculitis and choroid infiltration.And p28-Fc display suppression EAU processed, relative to control mice, the significant reduction of EAU scoring.

As a result as Figure 16 shows that p28-Fc fusion protein can effectively inhibit the development of EAU mouse disease.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Sequence table

<110>Wang Renxi

<120>p28-Fc fusion protein and its coding nucleic acid molecule, recombinant expression carrier, recombinant cell and application

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 215

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<213>artificial sequence (Artificial Sequence)

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Phe Pro Arg Pro Pro Gly Arg Pro Gln Leu Ser Leu Gln Glu Leu Arg

1 5 10 15

Arg Glu Phe Thr Val Ser Leu His Leu Ala Arg Lys Leu Leu Ser Glu

20 25 30

Val Arg Gly Gln Ala His Arg Phe Ala Glu Ser His Leu Pro Gly Val

35 40 45

Asn Leu Tyr Leu Leu Pro Leu Gly Glu Gln Leu Pro Asp Val Ser Leu

50 55 60

Thr Phe Gln Ala Trp Arg Arg Leu Ser Asp Pro Glu Arg Leu Cys Phe

65 70 75 80

Ile Ser Thr Thr Leu Gln Pro Phe His Ala Leu Leu Gly Gly Leu Gly

85 90 95

Thr Gln Gly Arg Trp Thr Asn Met Glu Arg Met Gln Leu Trp Ala Met

100 105 110

Arg Leu Asp Leu Arg Asp Leu Gln Arg His Leu Arg Phe Gln Val Leu

115 120 125

Ala Ala Gly Phe Asn Leu Pro Glu Glu Glu Glu Glu Glu Glu Glu Glu

130 135 140

Glu Glu Glu Glu Arg Lys Gly Leu Leu Pro Gly Ala Leu Gly Ser Ala

145 150 155 160

Leu Gln Gly Pro Ala Gln Val Ser Trp Pro Gln Leu Leu Ser Thr Tyr

165 170 175

Arg Leu Leu His Ser Leu Glu Leu Val Leu Ser Arg Ala Val Arg Glu

180 185 190

Leu Leu Leu Leu Ser Lys Ala Gly His Ser Val Trp Pro Leu Gly Phe

195 200 205

Pro Thr Leu Ser Pro Gln Pro

210 215

<210> 3

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<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

ttcccaaggc ccccagggag gccccagctg agcctgcagg agctgcggag ggagttcaca 60

gtcagcctgc atctcgccag gaagctgctc tccgaggttc ggggccaggc ccaccgcttt 120

gcggaatctc acctgccagg agtgaacctg tacctcctgc ccctgggaga gcagctccct 180

gatgtttccc tgaccttcca ggcctggcgc cgcctctctg acccggagcg tctctgcttc 240

atctccacca cgcttcagcc cttccatgcc ctgctgggag ggctggggac ccagggccgc 300

tggaccaaca tggagaggat gcagctgtgg gccatgaggc tggacctccg cgatctgcag 360

cggcacctcc gcttccaggt gctggctgca ggattcaacc tcccggagga ggaggaggag 420

gaagaggagg aggaggagga ggagaggaag gggctgctcc caggggcact gggcagcgcc 480

ttacagggcc cggcccaggt gtcctggccc cagctcctct ccacctaccg cctgctgcac 540

tccttggagc tcgtcttatc tcgggccgtg cgggagttgc tgctgctgtc caaggctggg 600

cactcagtct ggcccttggg gttcccaaca ttgagccccc agccc 645

<210> 3

<211> 224

<212> PRT

<213> human

<400> 3

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

1 5 10 15

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

20 25 30

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

35 40 45

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

50 55 60

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

65 70 75 80

Arg Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

85 90 95

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

100 105 110

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

115 120 125

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

130 135 140

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

145 150 155 160

Asn Gly Gln Pro Glu Asp Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

165 170 175

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

180 185 190

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

195 200 205

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215 220

<210> 4

<211> 678

<212> DNA

<213> human

<400> 4

cccccatgcc catcatgccc agcacctgag ttcctggggg gaccatcagt cttcctgttc 60

cccccaaaac ccaaggacac tctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 120

gtggacgtga gccaggaaga ccccgaggtc cagttcaact ggtacgtgga tggcgtggag 180

gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgta ccgtgtggtc 240

agggtcctca ccgtcctgca ccaggactgg ctgaacggta aggagtacaa gtgcaaggtc 300

tccaacaaag gcctcccgtc ctccatcgag aaaaccatct ccaaagccaa agggcagccc 360

cgagagccac aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtc 420

agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 480

aatgggcagc cggaggacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 540

ttcttcctct acagcaggct aaccgtggac aagagcaggt ggcaggaggg gaatgtcttc 600

tcatgctccg tgatgcatga ggctctgcac aaccactaca cacagaagag cctctccctg 660

tctccgggta aatgataa 678

<210> 5

<211> 439

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 5

Phe Pro Arg Pro Pro Gly Arg Pro Gln Leu Ser Leu Gln Glu Leu Arg

1 5 10 15

Arg Glu Phe Thr Val Ser Leu His Leu Ala Arg Lys Leu Leu Ser Glu

20 25 30

Val Arg Gly Gln Ala His Arg Phe Ala Glu Ser His Leu Pro Gly Val

35 40 45

Asn Leu Tyr Leu Leu Pro Leu Gly Glu Gln Leu Pro Asp Val Ser Leu

50 55 60

Thr Phe Gln Ala Trp Arg Arg Leu Ser Asp Pro Glu Arg Leu Cys Phe

65 70 75 80

Ile Ser Thr Thr Leu Gln Pro Phe His Ala Leu Leu Gly Gly Leu Gly

85 90 95

Thr Gln Gly Arg Trp Thr Asn Met Glu Arg Met Gln Leu Trp Ala Met

100 105 110

Arg Leu Asp Leu Arg Asp Leu Gln Arg His Leu Arg Phe Gln Val Leu

115 120 125

Ala Ala Gly Phe Asn Leu Pro Glu Glu Glu Glu Glu Glu Glu Glu Glu

130 135 140

Glu Glu Glu Glu Arg Lys Gly Leu Leu Pro Gly Ala Leu Gly Ser Ala

145 150 155 160

Leu Gln Gly Pro Ala Gln Val Ser Trp Pro Gln Leu Leu Ser Thr Tyr

165 170 175

Arg Leu Leu His Ser Leu Glu Leu Val Leu Ser Arg Ala Val Arg Glu

180 185 190

Leu Leu Leu Leu Ser Lys Ala Gly His Ser Val Trp Pro Leu Gly Phe

195 200 205

Pro Thr Leu Ser Pro Gln Pro Pro Pro Cys Pro Ser Cys Pro Ala Pro

210 215 220

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

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

245 250 255

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

275 280 285

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

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

305 310 315 320

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

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

405 410 415

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

420 425 430

Leu Ser Leu Ser Pro Gly Lys

435

<210> 6

<211> 1337

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

gcatgctatt cccaaggccc ccagggaggc cccagctgag cctgcaggag ctgcggaggg 60

agttcacagt cagcctgcat ctcgccagga agctgctctc cgaggttcgg ggccaggccc 120

accgctttgc ggaatctcac ctgccaggag tgaacctgta cctcctgccc ctgggagagc 180

agctccctga tgtttccctg accttccagg cctggcgccg cctctctgac ccggagcgtc 240

tctgcttcat ctccaccacg cttcagccct tccatgccct gctgggaggg ctggggaccc 300

agggccgctg gaccaacatg gagaggatgc agctgtgggc catgaggctg gacctccgcg 360

atctgcagcg gcacctccgc ttccaggtgc tggctgcagg attcaacctc ccggaggagg 420

aggaggagga agaggaggag gaggaggagg agaggaaggg gctgctccca ggggcactgg 480

gcagcgcctt acagggcccg gcccaggtgt cctggcccca gctcctctcc acctaccgcc 540

tgctgcactc cttggagctc gtcttatctc gggccgtgcg ggagttgctg ctgctgtcca 600

aggctgggca ctcagtctgg cccttggggt tcccaacatt gagcccccag ccccccccat 660

gcccatcatg cccagcacct gagttcctgg ggggaccatc agtcttcctg ttccccccaa 720

aacccaagga cactctcatg atctcccgga cccctgaggt cacgtgcgtg gtggtggacg 780

tgagccagga agaccccgag gtccagttca actggtacgt ggatggcgtg gaggtgcata 840

atgccaagac aaagccgcgg gaggagcagt tcaacagcac gtaccgtgtg gtcagggtcc 900

tcaccgtcct gcaccaggac tggctgaacg gtaaggagta caagtgcaag gtctccaaca 960

aaggcctccc gtcctccatc gagaaaacca tctccaaagc caaagggcag ccccgagagc 1020

cacaggtgta caccctgccc ccatcccagg aggagatgac caagaaccag gtcagcctga 1080

cctgcctggt caaaggcttc taccccagcg acatcgccgt ggagtgggag agcaatgggc 1140

agccggagga caactacaag accacgcctc ccgtgctgga ctccgacggc tccttcttcc 1200

tctacagcag gctaaccgtg gacaagagca ggtggcagga ggggaatgtc ttctcatgct 1260

ccgtgatgca tgaggctctg cacaaccact acacacagaa gagcctctcc ctgtctccgg 1320

gtaaatgata atctaga 1337

<210> 7

<211> 732

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

atgggccaga cggcaggcga ccttggctgg cggctcagcc tgttgctgct tcccttgctc 60

ctggttcaag ctggtgtctg gggattccca aggcccccag ggaggcccca gctgagcctg 120

caggagctgc ggagggagtt cacagtcagc ctgcatctcg ccaggaagct gctctccgag 180

gttcggggcc aggcccaccg ctttgcggaa tctcacctgc caggagtgaa cctgtacctc 240

ctgcccctgg gagagcagct ccctgatgtt tccctgacct tccaggcctg gcgccgcctc 300

tctgacccgg agcgtctctg cttcatctcc accacgcttc agcccttcca tgccctgctg 360

ggagggctgg ggacccaggg ccgctggacc aacatggaga ggatgcagct gtgggccatg 420

aggctggacc tccgcgatct gcagcggcac ctccgcttcc aggtgctggc tgcaggattc 480

aacctcccgg aggaggagga ggaggaagag gaggaggagg aggaggagag gaaggggctg 540

ctcccagggg cactgggcag cgccttacag ggcccggccc aggtgtcctg gccccagctc 600

ctctccacct accgcctgct gcactccttg gagctcgtct tatctcgggc cgtgcgggag 660

ttgctgctgc tgtccaaggc tgggcactca gtctggccct tggggttccc aacattgagc 720

ccccagccct ga 732

<210> 8

<211> 63

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

atgaaattct tagtcaacgt tgcccttgtt tttatggtcg tatacatttc ttacatctat 60

gcc 63

<210> 9

<211> 692

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

gcatgctacc cccatgccca tcatgcccag cacctgagtt cctgggggga ccatcagtct 60

tcctgttccc cccaaaaccc aaggacactc tcatgatctc ccggacccct gaggtcacgt 120

gcgtggtggt ggacgtgagc caggaagacc ccgaggtcca gttcaactgg tacgtggatg 180

gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagttcaac agcacgtacc 240

gtgtggtcag ggtcctcacc gtcctgcacc aggactggct gaacggtaag gagtacaagt 300

gcaaggtctc caacaaaggc ctcccgtcct ccatcgagaa aaccatctcc aaagccaaag 360

ggcagccccg agagccacag gtgtacaccc tgcccccatc ccaggaggag atgaccaaga 420

accaggtcag cctgacctgc ctggtcaaag gcttctaccc cagcgacatc gccgtggagt 480

gggagagcaa tgggcagccg gaggacaact acaagaccac gcctcccgtg ctggactccg 540

acggctcctt cttcctctac agcaggctaa ccgtggacaa gagcaggtgg caggagggga 600

atgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca cagaagagcc 660

tctccctgtc tccgggtaaa tgataatcta ga 692

Claims

1. a kind of p28-Fc fusion protein, which is characterized in that be made of hIL-27p28 and Fc segment；

The amino acid sequence of the hIL-27p28 is as shown in SEQ ID NO.1.

2. fusion protein according to claim 1, which is characterized in that the amino acid sequence of the p28-Fc fusion protein is such as Shown in SEQ ID NO.5.

3. encoding the nucleic acid molecules of fusion protein as claimed in claim 1 or 2, which is characterized in that the nucleotide of the nucleic acid molecules Sequence is as shown in SEQ ID NO.6.

4. a kind of recombinant expression carrier of fusion protein as claimed in claim 1 or 2, which is characterized in that will be as claimed in claim 3 Nucleic acid molecules are inserted into the multiple cloning sites of insect expression vector and carry to get the recombinant expression of fusion protein as claimed in claim 1 or 2 Body；

5. recombinant expression carrier according to claim 4, which is characterized in that the insect expression vector is pMIB.

6. a kind of recombinant cell for expressing fusion protein as claimed in claim 1 or 2, which is characterized in that the recombinant cell is packet Include the insect cell of recombinant expression carrier described in claim 4 or 5.

7. described in p28-Fc fusion protein as claimed in claim 1 or 2, nucleic acid molecules as claimed in claim 3, claim 4 or 5 Recombinant expression carrier or recombinant cell as claimed in claim 6 preparation treatment autoimmune disease drug in answering With.

8. application according to claim 7, which is characterized in that the autoimmune disease is by gp13 signal or Th17 Cell-mediated autoimmune disease.

9. application according to claim 7 or 8, which is characterized in that the autoimmune disease include multiple sclerosis, Autoimmune encephalomyelitis, uveitis or psoriasis.

10. a kind of for treating the drug of autoimmune disease, which is characterized in that the drug includes claims 1 or 2 institute The p28-Fc fusion protein and pharmaceutically acceptable auxiliary material stated.