CN104628870A

CN104628870A - Human IL12Rbeta1-CHR protein and Fc fusion protein thereof

Info

Publication number: CN104628870A
Application number: CN201510066730.6A
Authority: CN
Inventors: 姚文兵; 郭薇; 王辰; 高向东; 王欣; 郁冬梅; 雷雯
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2015-02-04
Filing date: 2015-02-04
Publication date: 2015-05-20

Abstract

The invention discloses a human IL12Rbeta1-CHR protein and an Fc fusion protein of the human IL12Rbeta1-CHR protein, a gene encoding the protein and use of the protein in treating autoimmune diseases. The protein disclosed by the invention has high affinity on hIL-12/IL-23 and can antagonize differentiation and development of IL-12/IL-23 induced Th1/Th17 cells in vitro and in vivo and moreover, the protein can inhibit secretion of inflammatory factors such as inflammatory cytokines IFN-gamma, IL-17A, IL-22 and the like and can be used for treating chronic diseases associated with Th1/Th17 dysfunction such as autoimmune diseases or chronic infection and the like. Compared with the IL12Rbeta1-CHR/Fc protein, the IL12Rbeta1-CHR/Fc protein provided by the invention has the advantages of good stability, high activity, long half-life in vivo and the like. The invention includes a molecular design, expression and purification, treatment of diseases and the like of the protein.

Description

A kind of people's IL12R β 1-CHR albumen and Fc fusion rotein thereof

Technical field

The invention belongs to biological technical field, be specifically related to a kind of can in conjunction with IL-12/IL-23 and recombinant human IL12R β 1-CHR albumen of its effect of antagonism and Fc fusion rotein and this proteinoid purposes in treatment autoimmune disease thereof.

Background technology

Autoimmune disorder (autoimmune disease) is the most common disease of the 3rd class after cardiovascular disorder and cancer, be one of major chronic disease of world wide serious threat public health, its overall incidence accounts for 3% ~ 5% of world population.The pathogenesis of autoimmune disorder is not yet completely clear, and the immunocyte caused with the reason such as virus infection, heredity, environment, medicine is abnormal, immunoloregulation function is disorderly relevant.CD4 ⁺the exception of regulatory T cells (T helper cell, Th) is one of most important pathogenesis of autoimmune disorder, in the past in Two decades years, constitutes people understand CD4 to the classification of Th1 and Th2 cell ⁺the basis of T cell immunologic function and their effects in the natural immunity and adaptive immunity regulate.Th1 cell, by the cytokine such as secretion of gamma-IFN, IL-12, plays a significant role in anti-infective, antitumor reaction, and simultaneously, the active abnormal rising of Th1 is also one of important mechanisms of organ specific autoimmune disease morbidity.Th2 cell then promotes the humoral immunoresponse(HI) of body by cytokines such as secretion IL-4, IL-5, IL-13, also take part in the pathogenic process of asthma, Autoimmune diseases on the other hand.T regulates cell (Treg) then to play vital role safeguarding in body immunologic balance, by the functional classification of above-mentioned T cell, enhances the understanding of people to Immune Regulative Network, also provides foundation for intervening disease simultaneously.But going deep into along with research, it is found that above-mentioned T cell functional classification fully can not explain the pathogeny of the some diseases such as some autoimmune diseases, infection and anaphylaxis.

Nearest research finds, a group is different from the cell subsets of Th1, Th2, Treg, and they do not express IL-4 or IFN-γ, but high-level secretory IL-17, is named as Th17 cell.The discovery of Th17 cell is more relevant to the research of autoimmune disorder, and it is a kind of special cell subsets in T cell atomization, plays immunologic function by secretion characteristic cytokines IL-17.IL-17 is a kind of pro-inflammatory cytokine, can raise and activated neutral granulocyte, induce other inflammatory Cytokines Expressions, and transmitting inflammation cell also finally causes tissue injury in the infiltration of lesions position.Research shows, the unbalance of the unsuitable activation of Th17 cell and function is there is in many autoimmune disease patient's bodies, show as that IL-17 and transcription factor ROR γ t are abnormal to raise, if the expression of the differentiation of Th17 cell in patient body, amplification and relevant cell factor therefore effectively can be blocked, can prevent, delay even to stop generation, the development of autoimmune disorder.There is a large amount of clinical and experimental study to find to suppress the function of Th17 cell, can significantly improve the autoimmune disorder symptoms such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematous and psoriatic.Hepatic tissue has differentiation advantage to Th17 cell, in liver autoimmunization and other inflammation disease, the induction of liver microenvironment to Th17 cell is extremely important, and in Patients with Hepatitis B Virus Infection and liver cancer patient body, Th17 cell significantly increases, and these phenomenons all cause the extensive concern of researchist to Th17 cell.

IL-23, belongs to IL-12 family, shares IL-12p40 subunit with IL-12, also has a unique p19 subunit simultaneously, p40 and p19 subunit can be combined with the IL-12R β 1 of DCRS5 mixture and IL-23R subunit and play a role respectively.IL-23 is the important factor in Th17 development and cell differentiation and function maintenance process, short inflammatory effect is played by stimulating T cell secretion IL-17, the disappearance of IL-23 significantly can reduce Th17 Hemapoiesis, the incidence of model mouse autoimmune disorder can be reduced simultaneously, alleviate the severity of its morbidity.Numerous evidence proves that IL-23/Th17/IL-17 axle plays an important role in autoimmune disorder generation evolution, points out us using IL-23/Th17 as a new drug target, may obtain better disease treatment prospect.In long-term evolution process, acceptor and part is very high in conjunction with general avidity, and due to soluble receptors be the extracellular region of cell receptor, its site of closing is the region that is combined with cell receptor of part just.The outstanding performance of medicine Enbrel (TNF α R-Fc fusion rotein) in clinical of severe rheumatoid arthritis in the treatment of FDA approved listing, demonstrates this receptor protein and can obtain even better curative effect the same as antibody equally.

Protein drug be treatment autoimmune disorder new focus, such medicine is with strong points, good effect, but due to the transformation period short, in experimentation on animals or clinical study, repetitively administered of having to, application is restricted.How to extend the focus that recombinant protein plasma half-life and enhanced stability are field of biological pharmacy, wherein by building Fc fusion rotein, to realize this object be the strategy that feasibility is higher.Fc fusion rotein refer to technology such as utilizing genetically engineered certain is had biologic activity functional protein molecule (soluble ligand, acceptor or other need the biologically active substance of prolong half-life) with Fc segment composition and the novel recombinant protein that produces.Such fusion rotein not only remains the biologic activity of functional protein molecule, also has many special propertys: Fc fragment can be that pH dependency is combined with FcRn, can extend the fusion rotein transformation period and improve stability, making it be more suitable for playing a role in human body; The interchain disulfide bond of Fc fragment is conducive to fusion molecule and forms polymer, thus strengthens ligand binding capacity and improve biological activity; The introducing of Fc fragment is conducive to improving the expression level of fusion molecule in mammalian cell.

To sum up, the present invention is directed to IL-12/IL-23p40 subunit, by genetic engineering means synthesis people's IL12R β 1-CHR albumen and Fc fusion rotein thereof, avidity experiment confirms that this proteinoid can be combined with people IL-12/IL-23, and then by setting up mouse and people Th1/Th17 development and cell differentiation platform and associated autoimmune disease animal model, in the body studying this proteinaceous molecule and external activity.People IL12R β 1-CHR albumen of the present invention and Fc fusion rotein thereof, can specific inhibition IL-12/Th1 and IL-23/Th17 signal path, reduces the abnormal Th1/Th17 cell levels increased of vivo and vitro, improves disease symptoms.And the IL12R β 1-CHR/Fc fusion rotein in the present invention comparatively IL12R β 1-CHR albumen has good stability, active high, the advantages such as Half-life in vivo is long.

Summary of the invention

The present invention selects p40 subunit to be target molecule, build and have expressed the people IL12R β 1 extracellular region recipient cell factor in conjunction with homology region (CHR) and Fc fusion rotein thereof, to playing the effect being similar to antibody, for the treatment of autoimmune disorder and chronic infectious diseases provides new selection.

The first problem that the present invention solves is to provide the gene order of coding hIL12R β 1-CHR, and be connected into PGEX-4T-2, the prokaryotic expression carriers such as pet 22b, pet 32a, transform BL21 (DE3), the bacterial strains such as ROSETTA are expressed.

The Second Problem that the present invention solves is that the albumen of PGEX-hIL12R β 1-CHR vector expression obtains gst fusion protein by GST affinity column purifying, specific position is cut further by zymoplasm enzyme, answer GST affinity column collection stream and wore acquisition containing the hIL12R β 1-CHR albumen of label, detect albumen by SDS-PAGE and Western blot.

The 3rd problem that the present invention solves is to provide the gene order of the IL12R β 1-CHR/Fc comprising IL-12R β 1 signal peptide, is connected into pcDNA3.1, the carrier for expression of eukaryon such as pngfpN, PEE144.

The four problems that the present invention solves utilizes cheap linear PEI to carry out transfection, reduces the transient transfection cost that short-term collects eukaryotic expression albumen in a large number.

The 5th problem that the present invention solves be by liposome transfection or electrotransfection technology by eukaryotic expression vector transfection to Chinese hamster ovary celI, G418 pressurize the CHO monoclonal cell strain of screening, acquisition stably express IL12R β 1-CHR/Fc fusion rotein.

The 6th problem that the present invention solves is the strain of suspension culture stable transfected cells, collects secretory protein in a large number, separation and purification, and Analysis and Identification freeze-drying are preserved.

The 7th problem that the present invention solves is that IL12R β 1-CHR/Fc fusion rotein can obtain highly purified protein molecular by Protein A, Protein G affinity column and other column chromatography, is detected the fusion rotein of purifying by SDS-PAGE and Western blot.

The Eight problems that the present invention solves be avidity experiment prove to express the hIL12R β 1-CHR albumen that obtains and Fc fusion rotein thereof can with hIL-12/IL-23 specific combination, and IL12R β 1-CHR/Fc fusion rotein has stronger avidity compared with hIL12R β 1-CHR albumen.

The 9th problem that the present invention solves is to Wistar rat tail vein injection hIL12R β 1-CHR and hIL12R β 1-CHR/Fc fusion rotein, within after injection 0.25,0.5,1,2,4,8,12,24,28,32,36,48,72,96 hour, carry out eye socket respectively and get blood, ELISA method detects drug effect concentration, its transformation period of PKsolver computed in software.

The ten problems that the present invention solves is external evoked Th1/Th17 development and cell differentiation, in stimulation T cell to while Th1/Th17 cytodifferentiation, add IL12R β 1-CHR and the IL12R β 1-CHR/Fc albumen of different concns, Th1/Th17 cytodifferentiation situation is detected by streaming technology, ELISA method detects culture supernatant Secretion of Inflammatory Factors and expresses, Q-PCR method detects the gene expression dose of Th1/Th17 cell correlation factor, thus research receptor protein is on the impact of Th1/Th17 development and cell differentiation.

The 11 problem that the present invention solves sets up multiple sclerosis animal model (EAE), normal group is set, model group, IL12R β 1-CHR group, IL12R β 1-CHR/Fc group and S-Neoral positive drug group, study and compare IL12R β 1-CHR albumen and what advantage IL12R β 1-CHR/Fc fusion rotein has to the therapeutic action of relative disease and compared to positive drug.

The 12 problem that the present invention solves sets up rheumatoid arthritis animal model (CIA), normal group is set, model group, IL12R β 1-CHR group, IL12R β 1-CHR/Fc group and S-Neoral positive drug group, study and compare IL12R β 1-CHR albumen and what advantage IL12R β 1-CHR/Fc albumen has to the therapeutic action of relative disease and compared to positive drug.

The 13 problem that the present invention solves sets up the systemic lupus erythematous (SLE) that chronic graft versus host disease (cGVHD) induces, normal group, model group and IL12R β 1-CHR/Fc group are set, study and compare IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc albumen to the therapeutic action of relative disease.

The tenth four problems that the present invention solves is the ulcerative colitis mouse model setting up DSS induction, normal group, model group, IL12R β 1-CHR group and IL12R β 1-CHR/Fc group are set, study and compare IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc albumen to the therapeutic action of relative disease.

The 15 problem that the present invention solves sets up the acute liver damage animal model of ConA induction, normal group, model group, IL12R β 1-CHR group and IL12R β 1-CHR/Fc group are set, study and compare IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc albumen to the therapeutic action of relative disease.

The 16 problem that the present invention solves sets up psoriasiform inflammatory model, normal group, model group, IL12R β 1-CHR group and IL12R β 1-CHR/Fc group are set, study and compare IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc albumen to the therapeutic action of relative disease.

The 17 problem that the present invention solves sets up mouse original position liver cancer model, normal group, model group, IL12R β 1-CHR group and IL12R β 1-CHR/Fc group are set, study and compare IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc albumen to the therapeutic action of relative disease.

The tenth Eight problems that the present invention solves is in experimentation on animals, find that IL12R β 1-CHR albumen is better with the security compared with traditional immunization inhibitor of IL12R β 1-CHR/Fc albumen, obvious bad toxic side effects is not had to indexs such as laboratory animal body weight, hemoglobin levels, to clinical treatment relative disease, there is good advantage.

Accompanying drawing explanation

Fig. 1 is IL12R β 1-CHR gene PCR product 1% nucleic acid electrophoresis figure.

M:150bp DNA Ladder Marker; 1:PCR product (650bp)

Fig. 2 is the double digestion qualification figure of PGEX-IL12R β 1-CHR.

M：250bp DNA Ladder Marker；1：PGEX-IL12Rβ1-CHR/BamH I+Xho I(4900bp，650bp)

Fig. 3 is the SDS-PAGE analysis chart of the GST-IL12R β 1-CHR fusion rotein of expressing.

M: standard molecular weight albumen Marker; 1: e. coli bl21 (DE3) empty bacterium; 2:PGEX-IL12R β 1-CHR IPTG does not induce full bacterium; Full bacterium after 3-5:PGEX-IL12R β 1-CHRIPTG induces

Fig. 4 is the SDS-PAGE analysis chart of the GST-IL12R β 1-CHR fusion rotein of purifying.

M: standard molecular weight albumen Marker; 1: the GST-IL12R β 1-CHR after purifying

Fig. 5 is the Western Blot analysis chart of GST-IL12R β 1-CHR fusion rotein.

M: pre-dyed albumen Marker; 1:PGEX-4T-2 empty plasmid; 2,3: the GST-IL12R β 1-CHR after purifying

Fig. 6 is the SDS-PAGE analysis chart not containing the restructuring IL12R β 1-CHR albumen of label after enzyme cuts purifying.

M: standard molecular weight albumen Marker; 1: enzyme does not contain the restructuring IL12R β 1-CHR albumen of label after cutting purifying

Fig. 7 is IL12R β 1-CHR/Fc recombination PCR primer 1% nucleic acid electrophoresis figure.

M:250bp DNA Ladder Marker; 1:IL12R β 1-CHR/Fc sequence overlap PCR primer (1400bp); 2: containing IL12R β 1-CHR sequence PCR primer (700bp) of signal peptide; 3: the Fc sequence PCR primer (700bp) of recombination

Fig. 8 is the double digestion qualification nucleic acid electrophoresis figure of pcDNA3.1 (+)-IL12R β 1-CHR/Fc.

M:250bp DNA Ladder Marker; 1:pcDNA3.1 (+)-IL12R β 1-CHR/Fc/Hind III+Xho I (5400bp, 1400bp); 2: pcDNA3.1 (+)-IL12R β 1-CHR/Fc that non-enzyme is cut

Fig. 9 is the nucleic acid electrophoresis detection figure of linearization plasmid.

M:DNA Marker; 1,2:pcDNA3.1 (+)-IL12R β 1-CHR/Fc plasmid is through PvuI single endonuclease digestion

Figure 10 is stable cell line Genomic PCR nucleic acid electrophoresis qualification figure.

M:DNA Marker; 1,2: two strain stable cell line Genomic PCR products of screening

Figure 11 is the SDS-PAGE electrophorogram of IL12R β 1-CHR/Fc fusion rotein.

1:IL12R β 1-CHR/Fc fusion rotein monomeric form (reduction); 2:IL12R β 1-CHR/Fc fusion rotein dimeric forms (non-reduced)

Figure 12 is the Western Blot analysis chart of IL12R β 1-CHR/Fc fusion rotein.

Figure 13 is the result that Salmonella detects the avidity of IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc fusion rotein and IL-12/IL-23.

Figure 14 is IL12R β 1-CHR albumen and the blood concentration-time graphic representation of IL12R β 1-CHR/Fc fusion rotein in rat body.

Figure 15 is IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc fusion rotein vitro inhibition mouse inflammatory factor IFN-γ, the ELISA detected result of IL-17A, IL-22 secreting, expressing.

Figure 16 is the flow cytometer showed figure that IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc fusion rotein vitro inhibition mouse Th1/Th17 break up.

Figure 17 is the result figure of the genetic expression of IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc fusion rotein vitro inhibition mouse Th1/Th17 cell transcription factor T-bet and ROR γ t.

Figure 18 is the flow cytometer showed figure that IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc fusion rotein vitro inhibition people Th1/Th17 break up.

Figure 19 is L12R β 1-CHR albumen and IL12R β 1-CHR/Fc fusion rotein vitro inhibition people IFN-γ, the ELISA detected result of IL-17A secreting, expressing.

Figure 20 is the result figure of the genetic expression of IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc fusion rotein vitro inhibition people Th1/Th17 cell transcription factor T-bet and ROR γ r.

Figure 21 is the clinical score figure of IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc fusion rotein treatment multiple sclerosis mouse EAE model.

Figure 22 is EAE model group, the HE pathological section figure of IL12R β 1-CHR administration group and IL12R β 1-CHR/Fc administration group mouse brain and spinal cord.

Figure 23 is the ELISA detected result that IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc fusion rotein suppress that in EAE mice serum, inflammatory factor IFN-γ and IL-17A secretes.

Figure 24 is the flow cytometer showed figure that IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc fusion rotein suppress Th1/Th17 cytodifferentiation in EAE mouse boosting cell.

Figure 25 is the clinical score figure of IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc fusion rotein treatment rheumatoid arthritis in rats CIA model.

Figure 26 is normal group, CIA model group, the X ray image check result of IL12R β 1-CHR administration group and IL12R β 1-CHR/Fc administration group rat.

Figure 27 is normal group, CIA model group, the ankle joint HE pathological section figure of IL12R β 1-CHR administration group and IL12R β 1-CHR/Fc administration group rat.

Figure 28 is the ELISA detected result that IL12R β 1-CHR and IL12R β 1-CHR/Fc fusion rotein suppress the Cytokine Expression Levels such as TNF-α, IFN-γ and IL-17A in CIA rat blood serum.

Figure 29 is that the anti-ss-DNA anti-body contg that mouse different time points respectively organized by SLE model compares.

Figure 30 is the immunofluorescence figure that mouse kidney respectively organized by SLE model.

Figure 31 is HE and the PAS coloration result that mouse kidney pathological section respectively organized by SLE model.

Figure 32 is the ELISA detection figure that IL12R β 1-CHR and IL12R β 1-CHR/Fc fusion rotein suppress IFN-γ and IL-17A secreting, expressing in SLE mice serum.

Figure 33 is the DAI scoring of the ulcerative colitis mouse model of IL12R β 1-CHR and IL12R β 1-CHR/Fc fusion rotein treatment DSS induction.

Figure 34 is Ulcerative Colitis Model group, the pathological section of IL12R β 1-CHR administration group and IL12R β 1-CHR/Fc administration group mouse Colon tissue.

Figure 35 is the ELISA detection figure that IL12R β 1-CHR and IL12R β 1-CHR/Fc fusion rotein suppress that in ulcerative colitis mouse Colon tissue, inflammatory factor IFN-γ and IL-17A expresses.

Figure 36 is the result figure of acute hepatic injury model each experimental mice serum transaminase (ALT) level.

Figure 37 is normal group, the HE pathological section figure of acute hepatic injury model group and IL12R β 1-CHR/Fc administration group liver organization.

Figure 38 is the ELISA detection figure that IL12R β 1-CHR and IL12R β 1-CHR/Fc fusion rotein suppress that in acute hepatic injury model mice serum, inflammatory factor TNF-α and IL-6 expresses.

Figure 39 is skin HE colored graph after each experimental mice of Psoriasis-like Model is put to death for 14 days.

Figure 40 is the ELISA detection figure that IL12R β 1-CHR and IL12R β 1-CHR/Fc fusion rotein suppress that in Psoriasis-like Model mice serum, inflammatory factor IL-17A, IL-22 and IFN-γ expresses.

Figure 41 is that after each experimental mice of primary liver cancer model is put to death for 14 days, liver neoplasm organizes HE colored graph.

Figure 42 is the Q-PCR detection figure that receptor protein suppresses IL-17A and IFN-γ mrna expression level in primary liver cancer model mouse liver tissue.

Embodiment

Below in conjunction with embodiment; further elaboration the present invention; be understood that; these embodiments are only for illustration of the present invention; but be not used for limiting the scope of the invention; under concept thereof of the present invention to the base same sense mutation of IL12R β 1-CHR albumen of the present invention and IL12R β 1-CHR/Fc fusion rotein, Fc replaces, carrier is replaced, Host Strains and express cell changes, purification process changes, to the structure of the animal model of associated autoimmune disease and the increase of indication, all belongs to the scope of protection of present invention.

The structure of embodiment 1 pGEX-IL12R β 1-CHR prokaryotic expression carrier

1.PCR amplification IL12R β 1-CHR gene

From Normal human peripheral's blood leukocytes, extract total serum IgE, reverse transcription synthesis cDNA first chain, then be template amplification IL12R β 1-CHR sequence with it.The primer sequence is as follows:

Upstream primer: F:5 ' CGGGATCC TGCAGAACCAGTGAGTGCT 3 ' (BamH I)

Downstream primer: R:5 ' CCGCTCGAGTTAAAAATACAAAATTAGCCGGG 3 ' (Xho 1)

PCR reaction conditions is as follows:

The first step: 98 DEG C of denaturation 30s, 1 circulation;

Second step: 98 DEG C of sex change 30s, 55 DEG C of annealing 15s, 72 DEG C extend 90s, totally 30 circulations;

3rd step: 72 DEG C extend 10min, 1 circulation.

PCR primer is identified through 1% nucleic acid electrophoresis, the object band (Fig. 1) of visible about 650bp.

The structure of 2.pGEX-IL12R β 1-CHR prokaryotic expression carrier

The IL12R β 1-CHR gene fragment that rubber tapping is reclaimed and PGEX-4T-2 carrier carry out double digestion respectively, then are spent the night by the 16 DEG C of connections of T4 ligase enzyme.The connexon transformation of E. coli E.coli DH5 α competent cell obtained, converted product coats the LB culture medium flat plate containing 100 μ g/mL Amp, 16h cultivated by 37 DEG C of incubators, picking list bacterium colony screening positive clone, double digestion and order-checking qualification obtain and build the correct expression vector (Fig. 2) containing goal gene.

The expression of embodiment 2 pGEX-IL12R β 1-CHR prokaryotic expression carrier and separation and purification

The abduction delivering of 1.pGEX-IL12R β 1-CHR prokaryotic expression carrier

By pGEX-IL12R β 1-CHR transformation of E. coli E.coli BL21 (DE3), converted product coats the LB culture medium flat plate containing 100 μ g/mLAmp, 16h cultivated by 37 DEG C of incubators, picking list bacterium colony is to 3mL containing in the LB substratum of 100 μ g/mLAmp, and in 37 DEG C of shaking tables, 220rpm rotating speed is cultured to bacterium liquid OD ₆₀₀for 0.6-0.8, add 1.0mM IPTG and continue 37 DEG C of cultivation 6h, induction target protein is expressed, and full bacterium runs SDS-PAGE electrophoresis visible 50KDa place object band (Fig. 3).

The enlarged culturing of 2.pGEX-IL12R β 1-CHR

The conservation that spreads cultivation bacterium liquid is to 1L LB substratum, and in 37 DEG C of shaking tables, 220rpm rotating speed is cultured to bacterium liquid OD ₆₀₀for 0.6-0.8, add 0.2mM IPTG 16 DEG C of shaking table overnight incubation.8500rpm, 4 DEG C of centrifugal 10min collect thalline, weigh wet bacterium heavy.Heavily add brokenly bacterium damping fluid by the 10mL/g bacterium that wets, be stirred well to thalline thoroughly resuspended.Power 400w, ultrasonic 3s, interval 3s, ice-bath ultrasonic 15min.The ultrasonic mixture obtained puts the centrifugal 20min of 12000rpm in superspeed refrigerated centrifuge, collects supernatant.Fusion rotein is present in supernatant with natural, solvable structure formation.

The separation and purification of 3.GST-IL12R β 1-CHR albumen

First with the broken bacterium damping fluid balance GST post of 10 times of column volumes, the centrifugal ultrasonic supernatant obtained is with 0.6 ~ 1.0mL/min flow velocity loading after 0.45 μm of frit, and the fusion rotein of band GST label will be adsorbed on affinity column.Note during loading keeping low temperature environment to prevent protein denaturation.Broken bacterium damping fluid balance pillar again with 10 times of column volumes after end of the sample, washes away combination foreign protein loosely.With the slow wash-out of broken bacterium damping fluid containing GSH of freshly prepared 10mM pH7.5, elutriant collected by 1.5mL EP pipe.The protein solution collected is analyzed through SDS-PAGE and Western Blotting, high purity 90% (Fig. 4, Fig. 5).

The separation and purification of 4.IL12R β 1-CHR albumen

The gst fusion protein that purifying obtains, the consumption cutting 2mg albumen by 1U enzyme adds zymoplasm, 4 DEG C of enzymes cut 48h, proteolytic cleavage product crosses GST post, the stream collected is worn liquid and is not containing the IL12R β 1-CHR albumen of GST label, SDS-PAGE electroresis appraisal visible 24KDa place target protein (Fig. 6).

The structure of embodiment 3 pcDNA3.1 (+)-IL12R β 1-CHR/Fc expression vector

1.Over-lap PCR builds IL12R β 1-CHR/Fc fusion gene

Total serum IgE is extracted, reverse transcription synthesis cDNA first chain from Normal human peripheral's blood leukocytes, then with it for template, respectively with P1, P2 and P3, P4 position upstream and downstream primer amplification contains IL12R β 1-CHR sequence and the IgG1Fc gene of signal peptide, and the primer sequence is as follows:

IL-12R β 1 upstream primer: P1:5 ' CCCAAGCTTATGGAGCCGCTGGTGA 3 ' (Hind III)

IL-12R β 1 downstream primer: P2:5 ' GGGGTTTTCAGGGGGAAC 3 '

Fc upstream primer: P3:5 ' GTTCCCCCTGAAAACCCCACAAAGGGCCCTTCTGTG 3 '

Fc downstream primer: P4:5 '-CCTCTCGAGTCACTTGCCGGGGGAC-3 ' (Xho I)

PCR reaction conditions is as follows:

The first step: 98 DEG C of denaturation 30s, 1 circulation;

3rd step: 72 DEG C extend 10min, 1 circulation.

PCR primer is identified through 1% nucleic acid electrophoresis, visible about 700bp containing signal peptide IL12R β 1-CHR sequence band and the IgG1Fc sequence band (Fig. 7) of about 700bp.

Two fragments that rubber tapping is reclaimed are that primer is spliced by the method for Over-lap PCR with P1, P4, and for improving specificity, adopt the method for Touchdown, PCR reaction conditions is as follows:

The first step: 98 DEG C of denaturation 30s, 1 circulation;

Second step: 98 DEG C of sex change 30s, 65 DEG C of annealing 15s, 72 DEG C extend 90s, and later each circulation reduces by 1 degree successively, until 55 DEG C, totally 10 circulations;

3rd step: 98 DEG C of sex change 30s, 55 DEG C of annealing 15s, 72 DEG C extend 90s, 20 circulations;

4th step: 72 DEG C extend 5min, 1 circulation.

PCR primer is identified through 1% nucleic acid electrophoresis, IL12R β 1-CHR/Fc goal gene band (Fig. 7) of visible about 1400bp.

The structure of 2.pcDNA3.1 (+)-IL-12R β 1-CHR/Fc expression vector

The IL-12R β 1-CHR/Fc gene fragment that rubber tapping is reclaimed and pcDNA3.1 (+) carrier carry out double digestion respectively, are spent the night by the 16 DEG C of connections of T4 ligase enzyme.Get full dose connexon transformation of E. coli E.coli DH5 α competence, converted product coats the LB culture medium flat plate containing 100 μ g/mL Amp, 16h cultivated by 37 DEG C of incubators, picking list bacterium colony screening positive clone, order-checking qualification and double digestion are identified and are obtained the correct carrier for expression of eukaryon (Fig. 8) of structure containing IL12R β 1-CHR/Fc goal gene.

The transient transfection of embodiment 4 IL-12R β 1-CHR/Fc fusion rotein is expressed

1. the preparation of transfection recombinant plasmid

Transfection plasmid extraction process is with reference to sky root without the large extraction reagent kit of intracellular toxin plasmid, and UV spectrophotometer measuring plasmid solution OD260/OD280, when result is 1.8 ~ 2.0 time, surveys A value (OD ₂₆₀), and be calculated as follows plasmid concentration:

Concentration (μ g/ μ L)=A value × extension rate × 50/1000

2. the preparation of cell to be transfected

24 hours before transfection, go down to posterity again to 293T cell, and when ensureing transfection, cell state is good and density reaches 70-90% as far as possible; Before transfection, first old substratum is replaced by fresh not containing the DMEM substratum of serum.

3.PEI transient transfection

According to the difference of culture apparatus, according to PEI (μ g): the transfection composite of the enough consumptions of proportional arrangement of plasmid (μ g)=2: 1 to 5: 1: plasmid and PEI are diluted in equal-volume opti-MEM respectively, again PEI diluent is added drop-wise in DNA diluent, mix gently, incubated at room 10-15min, obtains the PEI/DNA transfection composite of 1/10 times of culture volume.Evenly be added drop-wise in culture apparatus by transfection composite, shake makes it mix with fresh culture gently, is replaced by serum free medium after transfection 4-6 hour, cultivates 3-5 days, collects culture supernatant.

The screening of the stable expression cell strain of embodiment 5 IL-12R β 1-CHR/Fc fusion rotein

1. the preparation of linearization plasmid

Get 3 μ g DNA according to DNA sample concentration, add PvuI enzyme 37 DEG C of water-baths enzyme that spends the night and cut.Enzyme gets 100ng linearization plasmid electrophoresis detection after cutting, check whether completely (Fig. 9) linearization for enzyme restriction.Add the 3.0M NaOAC of 1/10 volume in the centrifuge tube of sample place, the freezing dehydrated alcohol (-20 DEG C) of 2 times of volumes, place 2 hours for-80 DEG C after mixing.The centrifugal 10min of 13200rpm, carefully sucks supernatant afterwards, dry 10min.

2. the preparation of cell to be transfected

The Chinese hamster ovary celI of logarithmic phase will be in, remove supernatant liquor, after adding 37 DEG C, appropriate trypsinase digestion 2-3min (under mirror visible cell floating and disperse), add the appropriate substratum containing serum and stop digestion, add after cell dispels in centrifuge tube, the centrifugal 5min of 1000rpm.Abandon supernatant, flick bottom centrifuge tube, cell precipitation is hiked up.Add appropriate 1 × PBS to wash, liquid volume in record centrifuge tube also does cell counting.The centrifugal 5min of 1000rpm, removes supernatant, flicks bottom centrifuge tube, and cell precipitation is hiked up.According to cell counts, add appropriate 1 × PBS, make cell density to 4x10 ⁶individual cell/mL.

3. electrotransfection

Get the 1 × PBS liquid of 50 μ L containing 2.5 μ g linearization plasmids, add 50 μ l cell suspensions, all join after mixing in the electric shock cup of precooling.2, set electroporation apparatus voltage 160V, 15ms shocks by electricity 1 time.Transfer pipet transitional cell is cultivated to containing in 3%FBS nutrient solution.

4.G418 screening mono-clonal

(1) electricity turn after cell cultures 24 as a child added G418, concentration is 400 μ g/mL, and every day, observation of cell state, if dead cell increases, then reduced the concentration of G418 in right amount.As cell proliferation is obvious, then improve G418 concentration, kill negative cells.Within every 2 days, change liquid once, resistance screening 10 days.

(2) collect and count survivaling cell, being diluted to 1 cell/200 μ L with nutrient solution.Every hole 200 μ L, adds in 96 well culture plates, selects mono-clonal after 8 days, is taken out by monoclonal cell culture fluid and detect ELISA after 10 days.According to ELISA result, select 6 best mono-clonals and be transferred to 6 orifice plates cultivations, within 5 days, sample afterwards and detect ELISA.According to ELISA result, choose two best cell strains of ELISA and be transferred to 10cm culture dish continuation enlarged culturing, get supernatant after 48 hours and detect ELISA, cell is by PCR method qualification stable cell line correct (Figure 10).Frozen stable cell line is in liquid nitrogen container.

The separation and purification of embodiment 6 Fc fusion rotein

With the binding buffer liquid of 10 times of column volumes balance ProteinA post, the centrifugal 20min of 12000rpm collects culture supernatant, after 0.45 μm of membrane filtration removal of impurities, with binding buffer liquid in after 1: 1 ratio mixed diluting one times with the flow velocity loading of 1mL/min.After loading, the binding buffer liquid of 10 times of column volumes is washed assorted.The 1M Tris that the 1.5mL EP pipe preparing to collect sample adds 0.06 ~ 0.2mL in advance in and the pH of elutriant.With the citrate buffer solution wash-out target protein of 2 ~ 5 times of column volumes, SDS-PAGE and Western Blot identifies reduction and the non-reduced form of albumen, prove that IL-12R β 1-CHR/Fc fusion rotein dimeric forms can exist (Figure 11, Figure 12) under non-reduced state.

The avidity of example item 7:IL12R β 1-CHR albumen and Fc fusion rotein and IL-12 and IL-23 detects---Salmonella

1. enzyme mark bar every hole bag is by 10ng hIL-12 or hIL-23,4 DEG C of overnight incubation.

2.PBST washs 3 times, each 10min, and every hole adds the BSA solution of 100 μ L 5%, hatches 2h for 37 DEG C.

3.PBST washs 3 times, each 10min, and every hole adds IL12R β 1-CHR albumen or the IL12R β 1-CHR/Fc fusion rotein of 1000,500,250,125,62.5,31.3,15.6nM successively, hatches 2h for 37 DEG C.

4.PBST washs 3 times, each 10min, and every hole adds the primary antibodie solution of the anti-human IL12R β 1 of 100 μ L, hatches 2h for 37 DEG C.

5.PBST washs 3 times, each 10min, and every hole adds the two anti-solution that 100 μ LHRP mark, and hatches 40min for 37 DEG C.

6.PBST washs 4 times, each 10min, and every hole adds 100 μ L TMB solution, and 37 DEG C of lucifuges place 20min, add 100 μ L2M sulfuric acid immediately, and color development stopping is reacted, and measure OD ₄₅₀value, utilizes the matched curve of Graphpad Software on Drawing, and juice calculates EC ₅₀value.Result shows, IL12R β 1-CHR albumen and Fc fusion rotein thereof all can with people IL-12 and IL-23 specific binding (Figure 13), for ensuing vivo and vitro activity research has established good experiment basis.

Embodiment 8: the pharmacokinetic of receptor protein in rat body

Get Wistar rat and be divided into 2 groups at random, often organize 6, respectively tail vein injection 2mg/kg IL12R β 1-CHR albumen and 4mg/kg IL12R β 1-CHR/Fc fusion rotein.After administration 0.25, 0.5, 1, 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 48, 72, 96, 120h equi-time point eye socket gets blood, blood sample room temperature leaves standstill 30min, carefully serum is drawn with microsyringe after the centrifugal 15min of 3000r/min, ELISA detects protein content in serum, calculate the Plasma Concentration of different time, drug application pharmacokinetic analysis software PKSolver matching c-t curve (Figure 14), calculating and obtaining IL12R β 1-CHR protein drug plasma half-life is 4h, and the IL12R β 1-CHR/Fc fusion rotein plasma drug transformation period comparatively IL12R β 1-CHR greatly improve, for 40h.

Embodiment 9: receptor protein external activity---suppress the differentiation of mouse Th1/Th17 cells in vitro

1. the preparation of mouse mixed lymphocytes suspension

Disconnected neck puts to death C57BL/6 mouse, 75% alcohol immersion sterilization 3-5min; Spleen is won, aseptic PBS washing in super clean bench; Spleen is placed on 70 μm of wetting in advance cell strainer, grinds pressure gently, filtered by cell suspension in 50mL aseptic plastic centrifuge tube with syringe nook closing member, collects spleen single cell suspension; The centrifugal 5min of 1000rpm, abandons supernatant, adds erythrocyte cracked liquid according to the amount of 1mL/ every spleen, lysis at room temperature about 4min, adds about 10 times of volume PBS and stops scission reaction; Centrifugally abandon supernatant, then with PBS washed cell 2 times, IMDM is resuspended, cultivates 8-24h in plastics plate, collects suspension cell (i.e. mixed lymphocytes), viable count, stand-by.

2. mouse CD4 ⁺the enrichment of T cell

Aseptic separating mouse spleen, prepares Single-cell suspensions, erythrocyte cracked liquid room temperature effect 3-5min, and 10 times of aseptic PBS of volume stop scission reaction; Centrifugally abandon supernatant, 10 times of aseptic PBS washed cells of volume 1 time, by every 10 ⁷cell adds 90 μ L magnetic bead sorting damping fluids, resuspended splenocyte; Add 10 μ L CD4 (L3T4) magnetic beads, mix, hatch 15min for 4 DEG C; Add 1-2mL magnetic bead sorting liquid, once, after resuspended, the centrifugal 10min of 1000rpm, abandons supernatant to cleaning cell, adds 500 μ L magnetic bead sorting liquid re-suspended cell precipitations, mixes; Take out MS magnetic bead sorting post, be placed on sorting magnetic frame, add 500 μ L magnetic bead sorting liquid profit posts; Under gravity after sorting liquid flows to end naturally, the cell suspension of hatching end is transferred in sorting post, utilizes action of gravity, treat that liquid flows to end naturally; Add 500 μ L sorting liquid in sorting post, after naturally flowing to end, twice, cleaning sorting post; Take off sorting post and be placed in aseptic 15mL centrifuge tube, adding 1-2mL sorting liquid, pushing away rapidly most liquid with sorting post inner core, collecting the CD4 flowed out ⁺cell, with nutrient solution cleaning once, stand-by.

3. the stimulated in vitro differentiation of mouse Th1/Th17 cell

With every hole 10 ⁶the density of individual cell, by mixed lymphocytes suspension or CD4 ⁺t cell suspension adds in 24 orifice plates wrapped in advance by anti-CD3 antibody, add mouse Th17 cells in vitro polarization stimulating factor IL-23 again, IL-6 and TGF-β (or Th1 cells in vitro polarization stimulating factor IL-12, anti-IL-4), and arrange blank group, Th1/Th17 induction group, IL12R β 1-CHR albumen intervention group and IL12R β 1-CHR/Fc fusion rotein intervention group (receptor protein adding different concns investigates its restraining effect), p40 antibody positive medicine group, 37 DEG C of CO ₂incubator cultivates 72 hours.

4.ELISA detects the cytokine in cell conditioned medium

Mouse lymphocyte is after stimulated in vitro 72h, the centrifugal 5min of 1000rpm, isolated cell precipitation and culture supernatant, cell precipitation is with stand-by after PBS cleaning once, cells and supernatant is used for the secreting, expressing that ELISA detects inflammatory factor IFN-γ and IL-17A, operates according to ELISA kit specification sheets.As shown in figure 15, IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc fusion rotein can suppress the secreting, expressing of Th1/Th17 specific cytokines IFN-γ and IL-17A, and Fc fusion rotein has higher suppression efficiency.

5. the level of differentiation of FCM analysis mouse Th1/Th17 cell

Mouse lymphocyte is after Th1/Th17 polarization, and add the ionomycin effect 4-6h that PMA that concentration is 10ng/mL and final concentration are 1 μM, the ratio according to 1: 1500 adds Golgistop effect 1h; Centrifugal collecting cell precipitates, and dye solution washed cell once; Centrifugally abandon supernatant, every tube cell adds the confining liquid containing anti-mouse CD16/CD32 antibody, and 30min is hatched in dark place on ice.Once, centrifugally abandon supernatant, add CD4 surface marker antibody, 30min is hatched in dark place on ice in dye solution washing.After cell fixes rupture of membranes, add IL-17A or IFN-γ born of the same parents internal labeling and resist, 30min is hatched in room temperature dark place.Last with 500 μ L dye solution re-suspended cells, upper machine testing.As shown in figure 16, IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc fusion rotein can suppress the differentiation and development of Th17 cell, and Fc fusion rotein has higher suppression efficiency.

6. quantitative fluorescent PCR analyzes the change of Th1/Th17 cell transcription factor gene expression dose

According to the operation of total RNA extraction reagent box specification sheets, extract each group of cell total rna; Again with for template, utilize reversed transcriptive enzyme, the synthesis such as random primer cDNA Article 1 chain; Be designed for the upstream and downstream primer of fluorescence quantitative PCR detection genes involved (as m β-actin, mT-bet and ROR γ t etc.).Using β-actin as reference gene, SYBR Green mark fluorescent, by △ △ Ct value method, the relative expression levels of goal gene in quantitatively each group sample, result shows that IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc fusion rotein can reduce the gene expression dose of T-bet and ROR γ t, and Fc fusion rotein has better reducing effect (Figure 17).

Embodiment 10; Receptor protein external activity---suppress the differentiation of people Th1/Fh17 cells in vitro

1. the extraction of human peripheral lymphocyte

Injection physiological saline mixes by 1: 1 with Freshman anticoagulated blood, is slowly added in the centrifuge tube containing equal-volume lymphocyte separation medium with dropper along centrifugal tube wall, keeps interface clear, the centrifugal 20 ~ 30min of 2000rpm.Centrifugal liquid in pipe is divided into three layers, is respectively plasma layer from top to bottom, lymphocyte separation medium, and red corpuscle and granulocyte precipitation, have an obvious oyster white cloud herring bone at liquid level intersection at the middle and upper levels, and careful this place's cell of absorption, is transferred in another new centrifuge tube.Add injection physiological saline according to 1: 10 ratio (V/V), after fully mixing, horizontal centrifugal 1000rpm, 10 minutes, abandoning supernatant, cell precipitation in kind washed 2 times, and cell counting is stand-by.

2. the stimulated in vitro differentiation of people Th1/Th17 cell

With every hole 10 ⁶the density of individual cell, monocyte separation obtained earnestly liquid adds in 24 orifice plates wrapped in advance by amCD3 antibody, add people Th17 cells in vitro polarization stimulating factor IL-1 β, IL-23, IL-6 and TGF-β (or Th1 cells in vitro polarization stimulating factor IL-12 again, anti-IL-4), and arrange blank group, Th1/Th17 induction group, IL12R β 1-CHR albumen intervention group and IL12R β 1-CHR/Fc fusion rotein intervention group (receptor protein adding different concns investigates its restraining effect), 37 DEG C of CO ₂incubator cultivates 96 hours.

3.ELISA method, fluidic cell, Q-PCR method detect to analyze and find that IL12R β 1-CHR albumen and IL12R β 1-CHR/Fc fusion rotein can suppress differentiation and development and the people IFN-γ of people Th1/Th17 cell equally, the secreting, expressing of IL-17A cytokine, and Fc fusion rotein has higher suppression efficiency (Figure 18, Figure 19, Figure 20).

Embodiment 11: receptor protein activity in vivo---improve multiple sclerosis animal model EAE symptom

The foundation of 1.EAE model

6-8 week C57BL/6 female mice is divided into model group, normal group, IL12R β 1-CHR protein groups, IL12R β 1-CHR/Fc fusion rotein group and CyA positive drug group at random.The TB equal-volume low temperature mixing of the mMOG dilute aseptic PBS and complete Freund's adjuvant dilution is to being water-in-oil sample.Immunity mouse web portion subcutaneous injection 200 same day every μ L emulsification antigen, adopts 2 injections, often 100 μ L.The simultaneously immunity same day (the 0th day) and the 2nd day, every mouse orbit injects 200 μ L PTX diluents (2.5 μ g/mL).Starting to fall ill, same day tail vein gives IL12R β 1-CHR or IL12R β 1-CHR/Fc or ddH ₂o, or gavage gives CSA, and daily, successive administration 10 days, observes the change of clinical score.

2. model evaluation

Before stopping to experiment from immune 0th day, every day 2, people adopted double-blind method, measured the body weight of mouse at one time, and observed the mental status, hair, diet and behavioral aspect etc., by Benson standards of grading, carried out clinical assessment to EAE severity:

0 point: normal mouse, gloss, four limbs and afterbody are strong, without EAE Clinical signs;

1 point: afterbody near-end tension force disappears, visible minor gait is clumsy;

2 points: side hind limb weakness, crawl to move ahead, passive stand up after can recover;

3 points: two hind limb weakness, passive standing up can not be recovered, but can move after giving stimulation;

4 points: two hind limb paralysis, action, diet difficulty, companion's urinary incontinence;

5 points: moribund condition or death.

(1) IL12R β 1-CHR administration group and IL12R β 1-CHR/Fc administration group treat EAE successful, all be better than positive administration group, wherein 12R β 1-CHR/Fc administration group treatment EAE effect is more remarkable, model group mean clinical scores is up to 3.5 points, positive drug is about 2.5, and IL12R β 1-CHR administration group is up to 2 points, IL12R β 1-CHR/Fc administration group, only up to 1.5 points, has significant difference (Figure 21) with model group.

(2) monitoring of each experimental group body weight is found simultaneously, positive drug group just occurred that body weight obviously declined giving medicine the same day, continue to after stopping administration, MAX DES reaches 18%, and obvious weight loss does not appear in the mouse of administration receptor protein always; And that studies discovery positive drug gives the level seriously reducing erythrocytic quantity and oxyphorase, and the administration group mouse blood index of receptor protein does not change, prompting receptor protein, than these nonspecific immunosuppressor, has better security while may playing more excellent curative effect clinically.

3. pathological index detects:

1) separating mouse brain and spinal cord are also soaked in formalin immediately and fix 24-48 hour, make paraffin section, and hematoxylin-eosin (HE) dyes, and analyze the damage of EAE central nervous system of mice demyelination and inflammatory cell infiltration situation.Compared with model group, the treatment of IL12R β 1-CHR and IL12R β 1-CHR/Fc can alleviate brain and myeloid tissue's inflammatory cell infiltration and spinal cord demyelination symptom (Figure 22).

2) in immune sequela peak period, mouse orbit gets blood, centrifugation serum, ELISA detects the expression amount of the cytokines such as IFN-γ and IL-17A, and the expression level of IL12R β 1-CHR and the IL12R β 1-CHR/Fc administration group serum inflammatory factor comparatively model group significantly reduces (Figure 23).

3) flow cytometer detection Th1/Th17 cytodifferentiation: at onset peak period separating mouse spleen, magnetic bead sorting CD4 ⁺cell, add the ionomycin effect 4-6h that PMA that concentration is 10ng/mL and final concentration are 1 μm, ratio according to 1: 1500 adds Golgistop effect 1h, fluorescent dye mark in the outer born of the same parents of born of the same parents, the differentiation rate of flow cytometer detection Th1/Th17, compared to model group, IL12R β 1-CHR and IL12R β 1-CHR/Fc gives the differentiation (Figure 24) that inhibit Th1/Th17 cell greatly.

Embodiment 11: receptor protein activity in vivo---improve rheumatoid arthritis animal model CIA symptom

The foundation of 1.CIA model

6-8 Wistar rat in age in week is divided into model group, Normal group, IL12R β 1-CHR, IL12R β 1-CHR/Fc and positive drug CsA group at random.The chicken II Collagen Type VI peptide of 100mM acetic acid and Freund's complete adjuvant equal-volume low temperature mix prepares immunizing antigen, the 0th day root of the tail portion intradermal injection 400 μ g antigen, and the method induction Wistar rat of 7 days same dosage booster immunizations afterwards produces sacroiliitis.IL12R β 1-CHR and IL12R β 1-CHR/Fc group give IL12R β 1-CHR and IL12R β 1-CHR/Fc albumen at immunity the 0th day tail vein, and positive drug group gavage gives CsA.

2. model evaluation

Before stopping to experiment from immune 0th day, every day 2, people adopted double-blind method, measure rat body weight at one time, and observe the mental status, hair, diet and Behavioral change etc., by arthritis index (arthritis index, AI) standards of grading, clinical assessment is carried out to CIA severity:

0 point: normal, without sacroiliitis;

1 point: have punctation or mild redness (ankle obviously red and swollen or redness only limits to single toes)

2 points: the swelling of joint part moderate;

3 points: serious swelling (whole sufficient pawl is red and swollen, comprises toes severe red and swollen);

4 points: severe swelling and can not bearing a heavy burden (redness involves whole limbs, comprises multiarticulate inflammation);

(1) AI of every rat is hind leg AI sum, is 8 points to the maximum.Carry out AI scoring to experimental rat every three days after initial immunity, data are as the highest in Figure 25, IL12R β 1-CHR group average A I marks is no more than 4 points, and IL12R β 1-CHR/Fc group average A I scoring is the highest is no more than 2 points, is significant difference with CIA model group.

(2) monitor each experimental group body weight every day to find, just there is weight loss giving medicine in positive drug group the same day simultaneously, MAX DES reaches 22%, and obvious weight loss does not all appear in the mouse of administration receptor protein.

3. pathological index detects

1) imaging examination: X-ray examination is carried out to experimental rat hind leg ankle joint, observes and compare the change in ankle joint iconography for after initial immunity, initial immunity 46 days.Normal group X-ray display cartilaginous tissue is without swelling, sclerotin is without destruction, toes Minor articulus gap is clear, and the changes such as the swelling of ankle joint surrounding soft tissue and bone erosion then appear in model group, IL12R β 1-CHR and IL12R β 1-CHR/Fc administration group pathology situation comparatively light (Figure 26).

2) H & E dyeing pathological section: put to death rat in after initial immunity 46 days, be separated hind leg ankle joint, formalin is fixed, and carries out decalcification process, and paraffin embedding ankle joint is cut into slices, H & E staining analysis finds the serious hyperplasia of model group rats synovial membrane, massive inflammatory cells infiltrated, forms pannus in joint cavity, discharges multiple inflammatory mediator and proteolytic ferment etc., destroy cartilage surface, bone involvement is serious; IL12R β 1-CHR administration group synovial hyperplasia is not obvious, and inflammatory cell infiltration degree is low, and cartilage surface is smooth, has no destruction, and bone involvement degree is low; IL12R β 1-CHR/Fc administration group almost loses inflammatory cell infiltration, and cartilage surface is smooth, has no destruction, bone involvement degree low (Figure 27).

3) ELISA method detects the expression level of relevant cell factor in rat blood serum: rat onset peak period eye socket gets blood, separation of serum, ELISA method to detect in serum the Cytokine Expression Levels such as TNF-α, IFN-γ, IL-17A, and the secreting, expressing amount of administration group inflammatory factor comparatively model group all has remarkable reduction (Figure 28).

Embodiment 12: receptor protein activity in vivo---improve systemic lupus erythematous (SLE) the model symptom that chronic graft versus host disease (cGVHD) is induced

The foundation of 1.SLE model:

8-10 week age (DAB/2 × C57BL/6J) F1 generation hybridize mice is divided into model group, CsA group at random, and IL12R β 1-CHR/Fc prevents administration group, IL12R β 1-CHR/Fc therapeutic administratp group and Normal group.Disconnected neck put to death 8-10 DBA in age in week 2 mouse, separating Morr. cell makes single cell suspension, and ammonium chloride lysate removes red corpuscle, and physiological saline re-suspended cell precipitates, and cell counting is stand-by; Every BDF1 mouse tail vein injection 5x10 ⁷individual cell, the physiological saline of Normal group injection equivalent.IIL12R β 1-CHR and IL12R β 1-CHR/Fc administration group give corresponding protein at the 2nd week tail vein.

2. pathological hallmarks detects and model evaluation

1) blood specimen: respectively organize weekly mouse orbit and get blood, indirect elisa method detects the content of anti-ssDNA antibody in serum.After 2 weeks, the content of the anti-ssDNA antibody of model group raises rapidly, and the content elevation amplitude of the anti-ssDNA antibody of IL12R β 1-CHR administration group comparatively model group significantly reduce, at the 3rd week, IL12R β 1-CHR/Fc administration group just occurs that anti-ssDNA antibody raises, and administration group anti-body contg peak value comparatively model group all have obvious reduction (Figure 29).

2) nephridial tissue sample: disconnected neck put to death each group of mouse in 10 weeks, take out Bilateral Renal, a part makes frozen section, direct IF staining, marks with FITC-goat anti-mouse IgG, uses laser scanning fluorescence microscopic examination immune deposits, as shown in figure 30, model group fills the air granular fluorescent substance deposition as seen, and IL12R β 1-CHR administration group phosphor deposit obviously reduces, and IL12R β 1-CHR/Fc administration group is visible few fluorescent substance deposition only; Paraffin section is prepared after another part 10% formaldehyde fixes 48h, the dyeing such as HE, PAS, observe under ordinary optical microscope, as shown in figure 31, the visible glomerular volume of model group renal tissue obviously increases, severe mesangial matrix * with cellularity crescent, a large amount of inflammatory cell infiltration of interstitial, there is renal glomerulus local hardening, a large amount of immune complex deposit; And IL12R β 1-CHR administration group renal tissue pathology there is a change for the better, rarely seen slight mesangial matrix *, there is a small amount of neutrophil leucocyte in mesangial region, visible a small amount of immune complex deposit; IL12R β 1-CHR/Fc administration group renal tissue pathology improving situation, there is a small amount of neutrophil leucocyte in mesangial region, without immune complex deposit.

3) Th17 cell correlation factor is expressed and is measured: ELISA to detect in serum the Cytokine Expression Levels such as IFN-γ and IL-17A, and administration group Secretion of Inflammatory Factors expression amount comparatively model group significantly reduces (Figure 32)

Embodiment 13: receptor protein activity in vivo---improve Animal Model of Ulcerative Colitis symptom

1. the foundation of acute DSS enteritis animal model

6-8 male C 57 BL mouse in age in week is divided into model group, IL12R β 1-CHR administration group, IL12R β 1-CHR/Fc administration group and Normal group at random.Except control group, other groups all replace common drinking-water freely to drink with 5%DSS solution, and Ganderwa is 7 days, and after 7 days, all experiment mices all continue freely to drink distilled water.IL12R β 1-CHR administration group and IL12R β 1-CHR/Fc administration group abdominal injection every day fusion rotein 1 time, successive administration 7 days.

2. model evaluation

In modeling process, every day observes the generalized case such as mouse diet, activity, monitoring weight, observe ight soil proterties and with or without occulting blood, observe mouse-as situation, primary part observation body weight change, stool and the situation of having blood in stool record, colitis severity is assessed by disease activity index (disease activity index, DAI).DAI scoring comprises 3 aspects: stool, stool blood, weight decline.Integration 0 ~ 4 point.0 point: stool is normal, without stool blood, declines without weight; 1 point: stool is normal, without stool blood, weight declines 1% ~ 5%; 2 points: defecate loose, stool blood is positive, weight decline > 5% ~ 10%; 3 points; Defecate loose, stool blood is positive, weight decline > 10% ~ 15%; 4 points: just rare, naked eyes bloody stool, weight decline > 15% (normally defecates: be shaped stool; Loose stool: do not attach to the pasty state of anus, semiformed stool; Just rare: the just shapeless of anus can be attached to).

Namely there is seizure of disease in the 2nd day after modeling in model group and protein medicine-feeding group, model group inflammation in whole experimentation continues, and is inclined to without obvious self-healing; And relative to model group, IL12R β 1-CHR administration group and IL12R β 1-CHR/Fc fusion rotein administration group start to occur that DAI increases sluggish trend respectively after the treatment on the the 5th and the 4th day.After modeling the 7th day, compare with model group, administration group DAI is starkly lower than model group (P < 0.05), and prompting IL12R β 1-CHR and IL12R β 1-CHR/Fc fusion rotein can improve colitis in mice degree of inflammation (Figure 33).

3. pathological index detects

1) H & E dyes pathological section: mouse is put to death in modeling the 7th day, and aseptic mouse Colon of getting organizes about 1cm, is placed in 10% formalin and fixes, and through dehydration, embedding, section, HE dyeing, examines under a microscope colon's pathological change.Model group mouse Colon organizes visible multifocal fester, and most of body of gland is destroyed, and body of gland normal configuration is lost, arrangement disorder, and lumen of gland disappears, and visible intensive inflammatory cell infiltration; Fusion rotein administration group mouse Colon body of gland destroys comparatively light, and structure recovers normal substantially, and the inflammatory cell infiltration degree of depth comparatively model group is slight, and inflammatory cell quantity also significantly reduces, and degree of inflammation obviously alleviates (Figure 34).

2) each experimental mice gets 1cm colon, tissue homogenate is made under condition of ice bath, get supernatant liquor after centrifugal, measure IFN-γ and IL-17A concentration by ELISA method, administration group Secretion of Inflammatory Factors expression amount comparatively model group significantly reduces (Figure 35).

Embodiment 14: receptor protein activity in vivo---improve the acute liver damage animal model symptom of ConA induction

1. the foundation of liver injury model

6-8 female BAl BIc/c mouse in age in week is divided into model group, IL12R β 1-CHR administration group, IL12R β 1-CHR/Fc administration group and Normal group at random.Stroke-physiological saline solution dilution ConA, with 20mg/kg dosage Tail Vein injection Mouse guidance model, control group injection equal-volume PBS.Before the modeling of fusion rotein administration group, tail vein gives IL12R β 1-CHR and IL12R β 1-CHR/Fc fusion rotein.12 h before harvest serum and liver tissue sample are used for transaminase ALT and measure and pathologic group fractional analysis.

2. pathological index detects

1) serum aminotransferase determination: 12h after ConA process, experiment mice eye socket gets blood, centrifuging and taking supernatant, respectively organizes ALT level with gpt (ALT) kit measurement.Compared with normal group, model group Serum ALT levels significantly raises, and causes serious damage after prompting Con A injection to mouse liver cell.The serum aminotransferase levels at commencement of IL12R β 1-CHR and IL12R β 1-CHR/Fc fusion rotein administration group mouse comparatively model group has significance to reduce, and prompting receptor protein has therapeutic action (Figure 36) to hepatocellular injury.

2) pathology of hepar analysis: each experimental mice liver organization of aseptic separation, fixes through 10% formaldehyde, paraffin embedding, and after section, hepar damnification situation is observed in HE dyeing.Model group mouse liver cell swelling and degeneration is obvious, and the even large sheet of visible loose spotty necrosis is downright bad, massive inflammatory cells infiltrated around portal area and necrotic tissue.And administration group pathological section swelling of liver cell and downright bad in be all improved effect (Figure 37).

3) serum cytokines detects: ELISA kit detects the important hepatitis inflammatory parameters of different group blood supernatant TNF-α and IL-6 two.In the serum of protein medicine-feeding group mouse, TNF-α and IL-6 level all have significance to reduce (Figure 38) compared with model group.

Embodiment 15: receptor protein activity in vivo---improve psoriasiform inflammatory model symptom

1. the foundation of psoriasiform inflammatory model

Normal group, model group is divided at random, IL12R β 1-CHR administration group and IL12R β 1-CHR/Fc fusion rotein administration group by female BAl BIc/c mouse in 5 week age.Mouse back shaves hair, and area is about 3cm × 5cm, and except control group gives external-use substrate emulsifiable paste, all the other each group all gives imiquimod cream, once a day, continuous 14 days, singly refers to light pressing massage about 60 times.Administration group is timing every day abdominal injection IL12R β 1-CHR or IL12R β 1-CHR/Fc fusion rotein from the 1st day.

2. model evaluation

1) PASI scoring is corrected: observe local skin change every day, photographic recording, the correction PASI calculating psoriasiform inflammation marks.Because area of getting involved is relatively fixing, correct PASI according to erythema, the scales of skin that peel off and pachyderma degree, each index is as follows to reserved portion, and three adds up to last PASI total score.

Score	Grading	Severity
			0	1	Asymptomatic
1	2	Slightly (spot color is light red; Chaff shape furfur)
			2	3	(spot is red for moderate; The thin covering of the sheet scales of skin that peel off)
3	4	(spot color is scarlet or dark red for severe; The scales of skin that peel off is thick, and part covers)
			4	5	It is very serious that (spot color is purplish red; Thick bits, skin damages holostrome and covers)

From PASI grade trend figure, normal group mouse back skin has no obvious change in whole medication process, and model group mouse back skin is at external application imiquimod cream after 3 days, gradually there is red macula, patch and the scales of skin that peel off, 8th day the most obvious, skin of back severe erythema, the covering thick-layer silvery white scales of skin that peel off, change in typical psoriasiform.IL12R β 1-CHR administration group and IL12R β 1-CHR/Fc administration group, start gradually to occur slight erythema and the thin layer scales of skin that peel off respectively at the 9th day and the 11st day, but after this erythema and scales of skin that peel off degree naked eyes without considerable change.

2) HE pathological section: got mouse back skin at the 7th day and be H & E and dye, under mirror, control group mice skin histology is normal, and keratinocyte only has 2 ~ 3 layers; Model group mouse skin damages to show as and obviously thickens (hyperkeratosis), keratinocyte core residual (parakeratosis), acanthosis and corium inflammatory cell infiltration; Administration group has slight hyperkeratosis, acanthosis and inflammatory infiltration, but comparatively psoriatic group obviously alleviates (Figure 39).

3. pathological index detects

ELISA method detects the content of cytokine IL-17A, IL-22 and IFN-γ in each group of mice serum, research show administration group cytokine content comparatively model group significantly decline (Figure 40).

Embodiment 16: acceptor activity in vivo---improve mouse original position liver cancer model symptom

1. the foundation of mouse original position liver cancer model

6-8 female BAl BIc/c mouse in age in week, is divided into Normal group, liver cancer model group, IL12R β 1-CHR administration group and IL12R β 1-CHR/Fc administration group at random.Get the fresh H22 cell strain that mouse peritoneal goes down to posterity, after conventional washing, physiological saline adjusts cell concn to be 1 × 10 ⁶individual cell/mL.After mouse anesthesia, under opening abdomen direct-view, 0.1mL cancer cell suspension is injected liver.Normal group injection Isodose physiological saline.After administration group inoculation H22 liver cancer cell, except abdominal injection IL12R β 1-CHR or IL12R β 1-CHR/Fc fusion rotein every day, continuous 13 days.

2. pathological index detects

1) HE pathological section: put to death mouse in after modeling the 14th day, get liver neoplasm tissue and fix, conventional H E dyes, and carries out Tissue pathological diagnosis.Model group can see larger tumour cancer stove, and cancer cells fills the air infiltration, and without obvious border, closely, interstitial is few in arrangement, visible a small amount of tumor giant cell and necrosis region; The reduction compared with model group of administration group tumour cancer stove, and clearer with normal liver tissue border (Figure 41).

2) Trizol method extracts each experimental group liver organization total serum IgE, and reverse transcription synthesis cDNA, Q-PCR detect the expression level of IL-17A and IFN-γ mRNA.Administration group liver organization IL-17A and IFN-γ mrna expression level are all starkly lower than model group (Figure 42).

Claims

1. a people IL12R β1receptor extracellular region cytokine is in conjunction with homologous region albumen, i.e. people IL12R β 1-CHR albumen, it is characterized in that, described IL12R β 1-CHR is the critical area that IL-12R β1receptor extracellular region is combined with people 1L-12/IL-23, there is as shown in SEQ ID.1 gene order and aminoacid sequence as shown in SEQ ID.2, and according to codon degenerate, same acid sequence of encoding, but the gene order that Nucleotide is replaced.

2. a people IL12R β 1-CHR/Fc fusion rotein, it is characterized in that, described IL12R β 1-CHR is except having as shown in SEQ ID.1 gene order and aminoacid sequence as shown in SEQ ID.2, also there is as shown in SEQ ID.3 gene order and the N end signal peptide sequence of one section of 69 nucleotide coding of aminoacid sequence as shown in SEQ ID.4, and according to codon degenerate, to encode same acid sequence, but the gene order that Nucleotide is replaced.

3. fusion rotein according to claim 2, it is characterized in that, described Fc fragment comprises hinge area, CH2 district and CH3 district, described Fc fragment is selected from the immunoglobulin IgG of human or animal, IgM, IgD, IgA or their hypotype, described Fc fragment be natural type Fc or to acceptor and/or the complement saltant type Fc in conjunction with relevant amino acid change.

4. fusion rotein according to claim 2, is characterized in that, described Fc fragment is preferable over and derives from people 1gG1, including but not limited to SEQ ID.5 and SEQ ID.6; Simultaneously including but not limited to aminoacid sequence SEQ ID7 corresponding with it and SEQ ID8.

5. fusion rotein according to claim 2, is characterized in that, for directly to merge or to be merged by catenation sequence between described IL12R β 1-CHR and Fc sequence.

6. fusion rotein according to claim 2, is characterized in that, described antigen-4 fusion protein gene sequence codon is optimized, obtain two kinds can inside Chinese hamster ovary celI the gene order of high expression, be respectively SEQ ID 9 and SEQ ID10.

7. comprise a carrier for any one protein coding DNA sequence in claim 1-6, it is characterized in that, described is eukaryotic vector and prokaryotic vector.

8. carrier according to claim 7, is characterized in that, described eukaryotic vector is mammalian cell expression vector.

9. comprise a host cell for the carrier of any one protein coding DNA sequence in claim 1-6, it is characterized in that, described host cell is selected from eukaryotic cell or prokaryotic cell prokaryocyte.

10. fusion rotein according to claim 9, is characterized in that, described eukaryotic cell is Chinese hamster ovary celI, and described prokaryotic cell prokaryocyte is Bacillus coli cells.

The autoimmune disorder purposes that protein for treatment in 11. claim 1-6 described in any one is feature with Th1/Th17 dysfunction and IL-12/IL-23 high expression level, include but not limited to rheumatoid arthritis, Ke Luoshi is sick, multiple sclerosis and systemic lupus erythematous etc.

Albumen in 12. claim 1-6 described in any one improves multiple sclerosis animal model EAE disease symptoms.

Albumen in 13. claim 1-6 described in any one improves rheumatoid arthritis animal model CIA disease symptoms.

Albumen in 14. claim 1-6 described in any one improves systemic lupus erythematous animal model disease symptoms.

Albumen in 15. claim 1-6 described in any one improves enteritis animal model disease symptoms.

Albumen in 16. claim 1-6 described in any one improves hepatitis animal disease symptoms.

Albumen in 17. claim 1-6 described in any one improves psoriasiform inflammatory model disease symptoms.

Albumen in 18. claim 1-6 described in any one improves mouse original position liver cancer model disease symptoms.

Albumen in 19. claim 1-6 described in any one improves the application of the autoimmune disorder animal model disease symptoms that other are feature with Th1/Th17 dysfunction and IL-12/IL-23 high expression level.

20. pharmaceutical composition and the application thereof comprising the albumen in claim 1-6 described in any one.