CN101434656B

CN101434656B - Preparation and use of OPG-HSP70 fusion protein

Info

Publication number: CN101434656B
Application number: CN2007101879321A
Authority: CN
Inventors: 王炜; 赵文明; 马静; 李慎涛
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2007-11-16
Filing date: 2007-11-16
Publication date: 2011-12-14
Anticipated expiration: 2027-11-16
Also published as: CN101434656A

Abstract

The invention relates to 'a preparation method of OPG-HSP70 fusion protein and an application thereof', pertaining to the new technology field of biological medicaments. The preparation method provides an OPG-HSP70 fusion protein (osteoprotegerin-heat shock protein 70) medicament. According to the most significant pathological features of the rheumatoid arthritis (RA) i.e. the arthrosynovitis with cartilage and bone destruction, the medicament uses the osteoprotegerin as the decoy receptor to combine with the receptor activator of nuclear factor-kB ligand (RANKL) of the nuclear factor kB expressed by osteoblast and other cells, and to interrupt the combination between the RANKL and the RANK expressed by the osteoclast so as to inhibit the bone resorption involved by the osteoclast; and the medicament uses the protective polypeptide segments of the heat shock protein (HSP) to inhibit the inflammatory arthritis. The method contains the DNA sequence which encodes the fusion protein, the method for generating the fusing protein by the recombinant technology, the biological activity of the fusion protein and the like.

Description

The Preparation method and use of OPG-HSP70 fusion rotein

Technical field

The present invention relates to use escherichia coli expression to prepare the method for reorganization osteoprotegerin-heat shock protein 70 (OPG-HSP70) fusion rotein, contain the pharmaceutical preparation of the reorganization osteoprotegerin-heat shock protein 70 (OPG-HSP70) for preparing by this method and the purposes in the medicine of this pharmaceutical preparation in preparation prevention or treatment rheumatoid arthritis.

Technical background

(Rheumatoid arthritis is that a kind of T is cell-mediated RA) to rheumatoid arthritis, is principal character with the synovial membrane inflammation, and the while is with the autoimmune disorder of joint cartilage and bone injury.RA is 0.32%～0.38% in the morbidity of China, and the disability rate height, and the loss that RA causes for labor force, social economy is huge.And RA is a kind of refractory disease, though existing treatment means is varied, all not satisfactory.The present invention is directed to the key pathology problem of bone injury and these two RA that are associated of chronic inflammatory reaction in the RA morbidity, development OPG-HSP70 fusion rotein is for a kind of new thinking has been opened up in the treatment of RA.

In recent years, (osteoclast, research OC) had not only caused the theoretical investigation in immunomodulatory, the bone metabolism disease, also provided new foundation for clinical diagnosis and treatment bone metabolic disease for osteoclast.To the research that OC carries out from the Celluar and Molecular Biology aspect etc., frontier (N Engl J Med.2005,353 (9): 872-75) of the medicament research and development of various bone photo related disorders have been opened up.Because OC forms the arthritic neccessary composition of aggressiveness, so it can be used as a target spot of RA treatment.

It is multiple metabolic osteopathy that the differentiation of OC and/or the bone that changes of function caused are built unbalance again, as the pathologic basis of formation such as osteoporosis, osteosclerosis and osteitis deformans (Paget osteopathy).Verified at present multiple hormone and local cytokine such as activity of vitamin d3, glucocorticosteroid, parathyroid hormone (PTH), prostaglandin(PG) (PGE2), IL-6, IL-11, IL-12 etc. can not only regulate OC and generate, the function that can also regulate OC.These factors mainly act on skeletonization/stroma cell; by regulating osteoprotegerin (osteoprotegerin; OPG) and NF-κ B receptor activation factor part (receptor activatorofNF-κ B ligand; RANKL) expression; regulation and control OPG, RANKL and the NF-κ B receptor activation factor (receptoractivator ofNF-κ B; RANK) ratio between, mediation OC generates and keeps its function.Therefore OPG, RANKL and RANK are the final regulatory factors of OC.3 kinds of molecules mutual relationship and the illustrating of the mechanism of action in OC differentiation and maturation process is for the control of multiple metabolic osteopathy provides theoretical foundation (Curr Opin Rheumatol.2003,15 (3): 280-7).

OPG was found in 1997, and it belongs to Tumor Necrosis Factor Receptors (TNFR) superfamily member, and two kinds of forms of monomer and the homodimer that is connected by disulfide linkage are arranged, and molecular weight is respectively 60 kD and 120 kD.Monomer is present in the scleroblast, and dimer can be secreted in the matrix, is a kind of secretor type glycoprotein.OPG is conservative at the evolution camber, and the homology of rat and mouse OPG is 94%, and the homology of mouse and people OPG is 89%.OPG contains 401 amino-acid residues at first, contains 380 amino acid whose mature proteins because of removing the signal peptide that 21 amino-acid residues are formed, finally becoming in building-up process.Its N end has 4 functional domains that are rich in halfcystine, and (D1～D4) combine with its part, C end and known protein do not have homology.Many histoorgans can be expressed OPG mRNA, and as lung, heart, kidney, liver, stomach and intestine, brain, backbone, Tiroidina etc., it mainly acts on is to suppress bone resorption (Cell.1997,89 (2): 309-19).Experiment shows, the C end parts (to 194aa) of removing OPG does not influence the OPG activity, the OPGN terminal sequence that therefore comprises TNFR sample functional zone for suppress the OC differentiation and maturation be essential also be enough.

Behind a year of discovery OPG, the same research group of the U.S. has found the part OPGL (Cell.1998 of OPG again, 93 (2): 165-76), be RANKL, belong to the TNF superfamily member, there are two kinds of forms of film mating type and solubility, mainly express, can promote the OC differentiation, stimulate its activation and improve the OC survival rate by scleroblast and marrow stromal cell.When these effects come across RANKL and combine with its acceptor RANK, and show as absorption at last to bone.RANK is expressed in the precursor cell surface of osteoclast, combines with the RANKL on skeletonization/stroma cell surface, can start differentiation and the maturation of OC.OPG is as the bait acceptor (decoy receptor) of RANK, mode with competition, high-affinity ground combines with the RANKL specificity, stop RANKL to combine with RANK on the OC, the differentiation of negative regulation OC and activation also promote the osteoclast apoptosis, thereby the inhibition bone resorption plays key effect in bone metabolism is regulated.Experimental study shows, serious osteoporosis appears in the OPG deficient mice, and with low bmd and multiple fracture and serious arteriosclerosis, this be since RANKL and RANK in conjunction with increase, thereby cause due to the formation of OC.Can improve the bone amount of its shin bone and femur to normal mouse reorganization OPG, and can compensate after the oophorectomize because oestrogenic hormon is lost the bone loss that causes.Obtain good effect with OPG treatment teenager Pegat osteopathy, and do not had tangible side reaction that (N Engl J Med.2005,353 (9): 918-23) take place.In the American Endocrine Society that held in 2005 (ENDO) annual meeting, reported that the OPG with reorganization is applied to 40～70 years old women, can reduce the again absorption of human body bone.These clinical studyes further illustrate OPG and have a good application prospect aspect the osteoclasia preventing.

The destruction of joint of RA comprises the erosion of synovial membrane surface, joint cartilage and subchondral bone.Recent research finds that OC is the crucial composition of RA destruction of joint; at lesions position a large amount of ripe OC and OC precursor are arranged; active in addition lymphocyte, scavenger cell, scleroblast and other cell expressing various kinds of cell factors; act on each links such as OC generation, differentiation, activation; make OC hyper-proliferative or Showed Very Brisk; broken the balance of bone metabolism, the destruction of bone occupies advantage.Think that at present RANKL that the activated T lymphocyte is expressed is the crucial regulatory factor among the RA, can be among the RA immunity system and bone metabolism provides and gets in touch (Arthritis Rheum.2006,54 (6): 1772-7).A large amount of activated T lymphocytes that RA arthropathy position occurs can directly pass through the RANKL of film mating type and the generation that solubility RANKL starts osteoclast, make joint part form more OC precursor and ripe OC, and cause bone loss.The cytokine in multiple T cell source may be disturbed the RANK signal path, thereby influences the generation of OC, as IL-12, IL-18, IL-4 etc.Wherein IL-4 is as the differentiation of anti-inflammatory cytokines by STAT-6 dependent mechanism inhibition OC, and current research shows that IL-4 is by suppressing NF-κ 3 and Ca ²⁺Signal path directly acts on OC and suppresses absorption again (J Immunol.2005,175 (2): 917-25) of bone.In adjuvant type sacroiliitis animal model, inflammation activity position can detect the expression of RANKL, and consistent with increase, the activation of OC, thereby causes serious B﹠J to destroy.Give OPG and can prevent osteoarticular destruction, but to the no improvement effect of immune inflammation reaction (Nature.1999,402 (6759): 304-9).(collagen-induced arthritis, CIA) OPG also can reduce or stop osteoclasia in the model, but synovial membrane inflammation is not acted on (Am J Pathol.2002,161 (4): 1419-27) in collagen-induced sacroiliitis.Show that thus OPG can not solve the chronic inflammatory diseases problem of RA, inflammation is still suitable stubborn problem.Animal experiment finds, the T cell plays an important role in the developing of RA inflammation, research prompting in recent years, and (heat shockprotein HSP) can be by regulating the Inflammatory response that the T cell function suppresses RA for heat shock protein(HSP).

Heat shock protein(HSP) belongs to molecular chaperone protein, and not only some protein molecule conformations and stability play the modulability effect in the pair cell, go back pair cell stress, physiological processs such as metabolism, propagation and apoptosis have important regulation.According to the size of monomer molecule amount, heat shock protein(HSP) can divide 6 family: HSP10, HSP40, HSP60, HSP70, HSP90 and HSP100.Wherein HSP70 is as immunodominant antigen, and conservative at the evolution camber, the HSP of mammiferous HSP and microorganism has very high homology, so also be easy to cause immunological cross-reaction.Along with to the particularly further investigation of regulatory T cells of immune regulation mechanism, the immunoregulation effect of HSP also is familiar with by people gradually.It is that other bacterium conservative antigens are not available that HSP induces the ability of regulatory T cells, and it may be relevant with some hsp receptor that participates in the inherent immunity responsive cell, and these acceptors are to connect inherent immunity to reply bridge with adaptive immune response.The HSP of Mammals and bacterium can direct activation APC (scavenger cell and dendritic cell), this effect may realize (Tissue Antigens.2004,64 (4): 442-51) by being attached to cell surface receptor such as CD14, CD40, TLRs, CD36, CD91 and LOX1 etc.

HSP70 can be discerned and produce corresponding immune response by body as immunodominant antigen.Analyze the epi-position of HSP70 and find, have only those can induce the epi-position of self HSP cross reactivity T cell that provide protection is just arranged.Because HSP70 evolution conservative section produces cross reaction between microorganism and Mammals, the cytokine that the autoantigen cross reactivity T lymphocyte that these conservative peptide sections produce can be secreted inflammation-inhibiting again, as IL-4, IL-10 and TGF-β, thereby activated immune regulation mechanism, directly suppress the proinflammatory effector T cell, perhaps regulate APC, stop the process of inflammatory diseases.And the protective effect of HSP peptide section and pathogenic effect are irrelevant.

Studies show that the effect that better prevention CIA is arranged with tubercule bacillus HSP70 peptide section immune animal, arthritis index obviously descends, pathological change is light, SC factor IL-4 level raises, and inflammatory cytokine IFN-γ level and anti-C II antibody horizontal reduce, significant difference (Chinese Journal of Immunology .2006,22 (12): 1092-5) have been compared with positive controls.

In view of synovitis and bone injury are two pathological characteristicses the most significant of RA, we will encode the dna fragmentation of OPG and HSP70 functional fragment connect (remove both with treatment RA irrelevant or to the deleterious peptide section of body, and help the preparation of preparation), insert prokaryotic expression carrier pET28a.On this basis, recombinant protein is carried out prokaryotic expression, and recombinant protein is carried out purifying, adopt SDS-PAGE, Western-blot method etc. that fusion rotein is carried out physics and chemistry and identify by methods such as molecular sieve or ion exchange chromatographies.Secondly, by the inside and outside biological activity test fusion rotein is further analyzed.This system expressing quantity height, controllability is strong, and production cost is relatively low, and has good biologic activity, easy realization of large-scale production.

Summary of the invention

One object of the present invention is to provide the production method for preparing the OPG fusion rotein with escherichia coli expression OPG-HSP70 fusion rotein and separation and purification.

The present invention also provides a kind of OPG-HSP70 fusion rotein, and is used for preventing and treating the purposes of the medicine of rheumatoid arthritis in preparation.

Another object of the present invention provides the pharmaceutical preparation that contains OPG-HSP70 fusion rotein of the present invention and pharmaceutically acceptable carrier.

OPG fusion rotein of the present invention refers to that OPG or its active variant, fragment and HSP or its variant, fragment merge the albumen that forms.

The invention provides the nucleotide sequence of a kind of OPG of coding and HSP70 fusion rotein.But in certain embodiments, can suddenly change to the nucleic acid codon according to actual needs, select intestinal bacteria preference codon for use, but the aminoacid sequence of coding OPG-HSP70 fusion rotein is constant.

OPG-HSP70 fusion rotein of the present invention, it is the R1-L-R2 form, and wherein R1 is OPG albumen or its variant or fragment, and R2 is HSP albumen or its variant or fragment, and L is a joint albumen.

Described OPG albumen, or its variant, fragment are selected from:

(a) protein sequence shown in the SEQIDNo.1;

(b) aminoacid sequence 22-X, wherein X is the arbitrary amino acid residue that comprises position 185-401 shown in the SEQIDNo.1.

Described HSP70 albumen or its variant or fragment are selected from:

(a) aminoacid sequence shown in the SEQIDNo.2;

(b) aminoacid sequence ITDAVITTPAYFNDA.

Described joint is selected from:

(a) ala-(ala) n-ala, n can be 1～4 a integer;

(b) gly-(gly) n-gly, n can be 1～4 a integer;

(c)gly-pro-gly；

(d)gly-gly-pro-gly-gly；

(e) ser-gly-(gly) n-gly, n can be 1～4 a integer;

(f)val；

(g)tyr-val；

(h) any combination of inferior part (a)-(g).

The intestinal bacteria of indication comprise e. coli bl21 host cells such as (DE3) among the present invention.Expression vector used in the present invention can be selected various known prokaryotic expression carriers for use, and recombinant expression vector is converted into host cell, utilizes appropriate condition screening recon, abduction delivering OPG-HSP70 fusion rotein.

In a preferred embodiment of the present invention, comprise in the step of appropriate condition bottom fermentation culturing engineering bacterium:

Enzyme is cut correct recombinant clone plasmid transformation escherichia coli BL21 (DE3) competent cell of evaluation, select single colony inoculation in 5mL 2 * YT substratum (containing the 25mg/L kantlex), 37 ℃ of shaken overnight.After the switching 1: 50 next day, cultivate 2h for 37 ℃, it is 0.1mmol/L that adding IPTG makes it final concentration, centrifugal collection thalline behind 30 ℃ of continuation shaking culture 5h.

In a preferred embodiment of the present invention, purifies and separates and renaturation go out the OPG-HSP70 fusion rotein and comprise following main points from tunning:

The positive colony bacterial strain is in 0.1mmol/L IPTG, behind 30 ℃ of inducing culture 5h, 4 ℃, the centrifugal 15min of 6000r/min, collect thalline,, add the Buffer A[1M Tris-HCl (pH8.0) of 10 times of volumes by every gram wet thallus with 20mM Tris-HCL (pH=7.9) the damping fluid washing thalline of precooling, 5M NaCl, 0.5M EDTA (pH8.0), 0.5%Triton100,1MDTT] resuspended, add N,O-Diacetylmuramidase to 1mg/ml, add PMSF to 1mM simultaneously, place 30min on ice after, ultrasonication thalline 10min intermittently, centrifugal collection inclusion body precipitation, respectively wash twice with 2M urea inclusion body washings and BufferA respectively again, then with the urea-denatured liquid dissolving of 8M inclusion body, 4 ℃, the centrifugal 45min of 12000r/min gets supernatant.Supernatant is the sex change liquid that contains the purpose recombinant protein again through the filtering with microporous membrane of 0.22um.The recombinant protein sex change liquid of great expression is passed through the method simmer down to 6mg/mL of ultrafiltration and concentration.Prepare renaturation solution (4M urea by progressively the successively decrease method of (4M-3M-2M-1M-0.5M-0M) of urea concentration, the 1mM reduced glutathion, 0.1mM Sleep-promoting factor B, 20% glycerine, 5% glucose, PBS, pH7.2), spissated protein denaturation liquid 5mL is placed in the dialysis tubing, and dialysis tubing places the renaturation solution of large volume slowly to stir dialysis for 4 ℃.Whether separate out and what of the amount of separating out are determined best dialysis conditions according to albumen in the dialysis procedure, changed the dialyzate that urea concentration reduces gradually in about four hours, protein denaturation liquid is carried out dilution refolding.Albumen after the renaturation is dissolved in the PBS solution of pH7.2 at last.

Prove conclusively, the OPG-Fc fusion rotein is safely and effectively in clinical application, and therefore, the OPG-HSP70 fusion rotein can be used for drug use.

The OPG-HSP70 fusion rotein can be used for diseases such as prevention and treatment rheumatoid arthritis, suppresses the osteoclasia effect and brings into play the inflammation-inhibiting effect.

Description of drawings

Fig. 1 shows construction of recombinant plasmid synoptic diagram of the present invention

Fig. 2 shows the synoptic diagram of expression vector of the present invention

Fig. 3 OPG/OPG-HSP70PCR product agarose gel electrophoretogram

Fig. 4 OPG-HSP70 fusion rotein SDS-PAGE electrophoretogram

The SDS-PAGE electrophoretogram of Fig. 5 OPG-HSP70 inclusion body washing process

Fig. 6 Western-blotting analyzes the OPG-HSP70 fusion rotein

The SDS-PAGE electrophoretogram of OPG-HSP70 fusion rotein after Fig. 7 renaturation

Fig. 8 OPG-HSP70 fusion rotein suppresses the differentiation of osteoclast

The restraining effect that Fig. 9 OPG-HSP70 fusion rotein produces inflammatory cytokine

Figure 10 OPG-HSP70 fusion rotein is to the scorching effect of pressing down of inflammatory model mouse

Embodiment

Embodiment 1OPG Cloning of Entire Gene

Sequence (GenBank NM_002546) according to the people OPG coding region cDNA of bibliographical information, design 2 Oligonucleolide primers at OPG coding region cDNA, it is early stage that human osteosarcoma cell line MG63 is cultured to logarithmic growth, add rhBMP-2 (final concentration reaches 100ug/L in substratum), continue to be cultured to the logarithmic growth after date, cell counting, centrifugal collecting cell.Extract cell total rna according to Trizol Reagent total RNA extraction reagent specification sheets.According to the MMLV first chain cDNA synthetic agent box specification sheets operation steps, be synthetic cDNA first chain of primer with oligomerization (dT).Getting 8uL reverse transcription product is template, under the effect of TaqDNA polysaccharase, uses P1 respectively, and the P2 primer carries out PCR, and reaction conditions is: behind 95 ℃ of sex change 5min, circulate 35 times by following parameter: 94 ℃ of sex change 45s, and 52 ℃ of annealing 30s, 68 ℃ are extended 1min.PCR product behind the purifying and plasmid pGEM-T-Easy are under the effect of T4DNA ligase enzyme, 4 ℃ of connections are spent the night, connect product Transformed E .coli DH5a competent cell, select single colony inoculation in the 5ml LB substratum test tube of (containing the 100mg/L penbritin), 37 ℃ of shaken overnight are collected thalline, and alkaline lysis extracts plasmid in a small amount, cut evaluation through the EcoRI enzyme, construction recombination plasmid pGEM-T-Easy-OPG.Getting enzyme cuts and identifies that correct recombinant clone carries out sequencing.

The structure of embodiment 2OPG-HSP70 fusion protein prokaryotic expression carrier

With the recombinant plasmid pGEMT-Easy-OPG that checks order correct is template, and P3 and P4 are that primer carries out the pcr amplification first time, and the PCR product with this is a template then, and P3 and P5 are that primer carries out the pcr amplification second time, obtain the OPG-HSP70 fusion gene.The purified back of PCR product utilizes Nco I and Xho I restriction enzyme to carry out the double digestion reaction with prokaryotic expression carrier pET-28a simultaneously.Carrier after enzyme is cut is purified with target DNA, be connected after, in the Transformed E .Coli DH5a competent cell, select single colony inoculation in the 5mL LB substratum Erlenmeyer flask of (containing the 25mg/L kantlex), 37 ℃ of shaken overnight.Collect thalline, extract plasmid in a small amount, identify through Nco I and Xho I double digestion again.Getting enzyme cuts and identifies that correct recombinant clone carries out sequencing.

The abduction delivering of embodiment 3 OPG-HSP70 fusion roteins in intestinal bacteria

Enzyme is cut correct recombinant clone plasmid transformation escherichia coli E.coli BL21 (DE3) competent cell of evaluation, select single colony inoculation in 5mL 2 * YT substratum (containing the 25mg/L kantlex), 37 ℃ of shaken overnight.After the switching 1: 50 next day, cultivate 2h for 37 ℃, it is 0.1mmol/L that adding IPTG makes it final concentration, centrifugal collection thalline behind 30 ℃ of continuation shaking culture 5h.

Purifying and the renaturation of embodiment 4 OPG-HSP70 fusion roteins in intestinal bacteria

Embodiment 5 activity experiments: the OPG-HSP70 fusion rotein suppresses the differentiation of osteoclast

After frozen former generation bone marrow cells in mice recovery, cultivate under 37 ℃, 5%CO2 saturated humidity condition.Add then macrophage colony stimulating factor M-CSF to final concentration be 25ng/mL, under 37 ℃, 5%CO2 saturated humidity condition, cultivate 24h.Next day, the collecting cell suspension was the non-tack myelomonocyte of M-CSF dependency.1000r/min, the centrifugal 5min of normal temperature abandons supernatant, and PBS washes twice.With 1640 substratum (containing the M-CSF of 25ng/mL and the RANKL of 40ng/mL) re-suspended cell, counting, and the adjustment cell concn is 1 * 10 ⁶/ mL.A slide is placed in each hole respectively in 24 orifice plates, and each hole adds nutrient solution 1,640 200 μ L, sucking-off nutrient solution behind 37 ℃ of incubation 2h respectively.Be divided into experimental group and control group according to the difference that adds OPG-HSP70 fusion rotein or PBS, 10 every group multiple holes.It is 10 μ g/mL to final concentration that experimental group adds the OPG-HSP70 fusion rotein, and control group adds same PBS liquid.Respectively add the resuspended bone marrow cell suspension 1mL of M-CSF and RANKL then in each hole respectively, cultivate under 37 ℃, 5%CO2 saturated humidity condition.For the first time change liquid after three days, changed liquid once in after this per two days, change liquid 50% at every turn, when being cultured to the 7th day, discard substratum, PBS washes fixedly 15min of cell twice, 4% (volume fraction) formaldehyde solution, and then washes cell twice, seasoning in the air with PBS.According to the capable TRAP dyeing of Sigma specification sheets, count the positive osteoclast of a plurality of nuclear TRAP down in opticmicroscope.2 above nucleus, dye the osteoclast that is for redness.

Embodiment 6 activity experiments: the OPG-HSP70 fusion rotein is to the scorching effect of pressing down of inflammatory model mouse

The mouse part skin depilation, the about 3cm * 3cm of scope, 40 μ L evenly smear sensitization with 1% dinitrofluorobenzene (DNFB) solution.Fixedly mouse 5sec makes the skin surface solvent evaporates.Give experimental mice abdominal injection OPG-HSP70 fusion rotein simultaneously, 0.2mg//time; Control group is intraperitoneal injection of saline simultaneously.The next day, inject, and injects altogether three times.Smearing DNFB sensitization next day again strengthens once.After the sensitization the 5th day, evenly be applied in mouse right ear (two sides) with 1%DNFB solution 10 μ L and attack.Behind 24 h, disconnected neck is put to death mouse, is the swelling degree with the difference of vernier caliper measurement left and right sides ear thickness.Adopt one-way analysis of variance to compare the difference of each experimental group and control group.The difference of left and right sides ear thickness is represented the degree of DTH.

In the mouse invasion peak, promptly attack back 24h, disconnected neck is put to death mouse, soaks about 2min filling in the large beaker of alcohol, then mouse is transferred in the Bechtop.Get a sterile petri dish, add 1640 cell culture fluids of 8mL within it, 200 order cells sieve is immersed wherein, isolating spleen is put on the cell sieve, evenly grind spleen to there not being the macroscopic fragment of organizing with piston.Draw the undersized 8mL splenocyte suspension of cell in aseptic 15mL centrifuge tube with sharp suction pipe, blow and beat mixing gently.Draw splenocyte suspension with sharp suction pipe and be added drop-wise in the 15mL centrifuge tube that fills the 4mL lymphocyte separation medium, make splenocyte suspension be overlapped in the separation liquid level.The centrifugal 20min of horizontal centrifuge 2000rpm.The most mononuclearcells in centrifugal back are suspended in blood plasma and parting liquid interface, are the tunica albuginea shape.Sharp suction pipe gently is inserted to the tunica albuginea layer,, moves in another aseptic 15mL centrifuge tube along test tube wall edge sucking-off mononuclearcell.Add 5mL 1640 cell culture fluids, abundant mixing, abandon supernatant behind the centrifugal 15min of 1500rpm, sedimentation cell is resuspended in 5mL 1640 cell culture fluids once more, abandon supernatant behind the centrifugal 15min of 1500rpm, the exhaustion raffinate adds the perfect medium 50 μ L that do not contain cell stimulatory agents then in pipe, behind the piping and druming mixing it is transferred in the 1.5mL centrifuge tube.Take out above-mentioned stoste 4 μ L and in the centrifuge tube that fills 400 μ L physiological saline, (be about to 100 times of cell stoste dilutions), fully get 10 μ L behind the mixing, add from the side and built on the tally of cover glass, get 4 the big lattice in four angles and count.After counting finishes, calculate the concentration of cell stoste, formula is total cellular score/4 * 10 of cell original liquid concentration=4 a big lattice ⁴* extension rate (100), with the perfect medium dilution that contains cell stimulatory agents, the final concentration that makes cell is 3 * 10 ⁶/ mL.

Get 96 orifice plates (MAIPAN4510 plate) of pvdf membrane bag quilt, be used for detecting IFN-γ.Thing to be checked is divided into experimental group and 2 groups of control group.Diplopore detects, and averages at last.Every hole adds 100 μ l, the 70% ethanol pvdf membrane of prewetting, and covers the plate lid, incubated at room 5min.Ethanol in sucking-off or the ejection hole washes twice with PBS solution immediately then, pats dry on thieving paper.Every hole adds the coated antibody of 50 μ L with 5mL PBS dilution, adds aseptic PBS to 100 μ L, covers the plate lid, and 4 ℃ of bags are spent the night.Discard liquid in the hole, with lavation buffer solution PBST washing, throw away liquid after leaving standstill 15～30s, repeated washing 5 times is at aseptic thieving paper arsis dry plate.Every then hole adds 1 * confining liquid R (with 10 times of PBS solution dilutions) of 200 μ L, covers the plate lid, hatches 1h (or incubated at room 2h) for 37 ℃.Discard confining liquid in the hole, every hole adds 100 μ L cell suspensions, and (cell concn is 3 * 10 ⁶/ mL, and contain cell stimulatory agents), cover the plate lid, at 37 ℃, 5%CO ₂, 100% humidity environment under hatch 20～24h.Throw away the cell in the reacting hole, add rapidly the PBS solution 250 μ L under the room temperature, firmly throw away then in every hole, repeats 3 times after, wash 5 times with PBST.Every then hole adds 100 μ L biotinylations and detects antibody diluent, seals Sptting plate with the shrouding film, hatches 1h in 37 ℃.Discard liquid in the hole, the both sides of usefulness PBST flushing membrane 5 times.Every then hole adds 100 μ L Streptomycin sulphates-HRP avidin diluent, seals Sptting plate with the shrouding film, hatches 1h in 37 ℃.Discard liquid in the hole, the both sides of usefulness PBST flushing membrane 5 times.Every then hole adds the AEC substrate solution that 100 μ L thaw immediately, covers the plate lid, room temperature lucifuge reaction 15～30min; After spot forms clearly, discard liquid in the hole and with the both sides of the abundant flushing membrane of DDW with termination reaction.Dry PVDF plate under the room temperature is with immunodotting image analyzer counting.Statistical procedures Eli-spot result: adopt the one-way analysis of variance method in the SPSS statistical software 12.0 to carry out the respectively data analysis between the group.

Sequence table

＜110〉the quiet Li Shen great waves of Wang Wei Zhao Wenming horse

＜120〉Preparation method and use of OPG-HSP70 fusion rotein

<160>9

<170>PatentIn?version?3.3

<210>1

<211>401

<212>PRT

<213>Artificial

<220>

＜223〉OPG albumen

<220>

<221>MISC_FEATURE

<222>(1)..(401)

<400>1

Met?Asn?Lys?Leu?Leu?Cys?Cys?Ala?Leu?Val?Phe?Leu?Asp?Ile?Ser?Ile

1 5 10 15

Lys?Trp?Thr?Thr?Gln?Glu?Thr?Phe?Pro?Pro?Lys?Tyr?Leu?His?Tyr?Asp

20 25 30

Glu?Glu?Thr?Ser?His?Gln?Leu?Leu?Cys?Asp?Lys?Cys?Pro?Pro?Gly?Thr

35 40 45

Tyr?Leu?Lys?Gln?His?Cys?Thr?Ala?Lys?Trp?Lys?Thr?Val?Cys?Ala?Pro

50 55 60

Cys?Pro?Asp?His?Tyr?Tyr?Thr?Asp?Ser?Trp?His?Thr?Ser?Asp?Glu?Cys

65 70 75 80

Leu?Tyr?Cys?Ser?Pro?Val?Cys?Lys?Glu?Leu?Gln?Tyr?Val?Lys?Gln?Glu

85 90 95

Cys?Asn?Arg?Thr?His?Asn?Arg?Val?Cys?Glu?Cys?Lys?Glu?Gly?Arg?Tyr

100 105 110

Leu?Glu?Ile?Glu?Phe?Cys?Leu?Lys?His?Arg?Ser?Cys?Pro?Pro?Gly?Phe

115 120 125

Gly?Val?Val?Gln?Ala?Gly?Thr?Pro?Glu?Arg?Asn?Thr?Val?Cys?Lys?Arg

130 135 140

Cys?Pro?Asp?Gly?Phe?Phe?Ser?Asn?Glu?Thr?Ser?Ser?Lys?Ala?Pro?Cys

145 150 155 160

Arg?Lys?His?Thr?Asn?Cys?Ser?Val?Phe?Gly?Leu?Leu?Leu?Thr?Gln?Lys

165 170 175

Gly?Asn?Ala?Thr?His?Asp?Asn?Ile?Cys?Ser?Gly?Asn?Ser?Glu?Ser?Thr

180 185 190

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

195 200 205

Phe?Ala?Val?Pro?Thr?Lys?Phe?Thr?Pro?Asn?Trp?Leu?Ser?Val?Leu?Val

210 215 220

Asp?Asn?Leu?Pro?Gly?Thr?Lys?Val?Asn?Ala?Glu?Ser?Val?Glu?Arg?Ile

225 230 235 240

Lys?Arg?Gln?His?Ser?Ser?Gln?Glu?Gln?Thr?Phe?Gln?Leu?Leu?Lys?Leu

245 250 255

Trp?Lys?His?Gln?Asn?Lys?Ala?Gln?Asp?Ile?Val?Lys?Lys?Ile?Ile?Gln

260 265 270

Asp?Ile?Asp?Leu?Cys?Glu?Asn?Ser?Val?Gln?Arg?His?Ile?Gly?His?Ala

275 280 285

Asn?Leu?Thr?Phe?Glu?Gln?Leu?Arg?Ser?Leu?Met?Glu?Ser?Leu?Pro?Gly

290 295 300

Lys?Lys?Val?Gly?Ala?Glu?Asp?Ile?Glu?Lys?Thr?Ile?Lys?Ala?Cys?Lys

305 310 315 320

Pro?Ser?Asp?Gln?Ile?Leu?Lys?Leu?Leu?Ser?Leu?Trp?Arg?Ile?Lys?Asn

325 330 335

Gly?Asp?Gln?Asp?Thr?Leu?Lys?Gly?Leu?Met?His?Ala?Leu?Lys?His?Ser

340 345 350

Lys?Thr?Tyr?His?Phe?Pro?Lys?Thr?Val?Thr?Gln?Ser?Leu?Lys?Lys?Thr

355 360 365

Ile?Arg?Phe?Leu?His?Ser?Phe?Thr?Met?Tyr?Lys?Leu?Tyr?Gln?Lys?Leu

370 375 380

Phe?Leu?Glu?Met?Ile?Gly?Asn?Gln?Val?Gln?Ser?Val?Lys?Ile?Ser?Cys

385 390 395 400

Leu

<210>2

<211>625

<212>PRT

<213>Artificial

<220>

＜223〉HSP albumen

<220>

<221>MISC_FEATURE

<222>(1)..(625)

<400>2

Met?Ala?Arg?Ala?Val?Gly?Ile?Asp?Leu?Gly?Thr?Thr?Asn?Ser?Val?Val

1 5 l0 15

Ser?Val?Leu?Glu?Gly?Gly?Asp?Pro?Val?Val?Val?Ala?Asn?Ser?Glu?Gly

20 25 30

Ser?Arg?Thr?Thr?Pro?Ser?Ile?Val?Ala?Phe?Ala?Arg?Asn?Gly?Glu?Val

35 40 45

Leu?Val?Gly?Gln?Pro?Ala?Lys?Asn?Gln?Ala?Val?Thr?Asn?Val?Asp?Arg

50 55 60

Thr?Val?Arg?Ser?Val?Lys?Arg?His?Met?Gly?Ser?Asp?Trp?Ser?Ile?Glu

65 70 75 80

Ile?Asp?Gly?Lys?Lys?Tyr?Thr?Ala?Pro?Glu?Ile?Ser?Ala?Arg?Ile?Leu

85 90 95

Met?Lys?Leu?Lys?Arg?Asp?Ala?Glu?Ala?Tyr?Leu?Gly?Glu?Asp?Ile?Thr

100 105 110

Asp?Ala?Val?Ile?Thr?Thr?Pro?Ala?Tyr?Phe?Asn?Asp?Ala?Gln?Arg?Gln

115 120 125

Ala?Thr?Lys?Asp?Ala?Gly?Gln?Ile?Ala?Gly?Leu?Asn?Val?Leu?Arg?Ile

130 135 140

Val?Asn?Glu?Pro?Thr?Ala?Ala?Ala?Leu?Ala?Tyr?Gly?Leu?Asp?Lys?Gly

145 150 155 160

Glu?Lys?Glu?Gln?Arg?Ile?Leu?Val?Phe?Asp?Leu?Gly?Gly?Gly?Thr?Phe

165 170 175

Asp?Val?Ser?Leu?Leu?Glu?Ile?Gly?Glu?Gly?Val?Val?Glu?Val?Arg?Ala

180 185 190

Thr?Ser?Gly?Asp?Asn?His?Leu?Gly?Gly?Asp?Asp?Trp?Asp?Gln?Arg?Val

195 200 205

Val?Asp?Trp?Leu?Val?Asp?Lys?Phe?Lys?Gly?Thr?Ser?Gly?Ile?Asp?Leu

210 215 220

Thr?Lys?Asp?Lys?Met?Ala?Met?Gln?Arg?Leu?Arg?Glu?Ala?Ala?Glu?Lys

225 230 235 240

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

245 250 255

Tyr?Ile?Thr?Val?Asp?Ala?Asp?Lys?Asn?Pro?Leu?Phe?Leu?Asp?Glu?Gln

260 265 270

Leu?Thr?Arg?Ala?Glu?Phe?Gln?Arg?Ile?Thr?Gln?Asp?Leu?Leu?Asp?Arg

275 280 285

Thr?Arg?Lys?Pro?Phe?Gln?Ser?Val?Ile?Ala?Asp?Thr?Gly?Ile?Ser?Val

290 295 300

Ser?Glu?Ile?Asp?His?Val?Val?Leu?Val?Gly?Gly?Ser?Thr?Arg?Met?Pro

305 310 315 320

Ala?Val?Thr?Asp?Leu?Val?Lys?Glu?Leu?Thr?Gly?Gly?Lys?Glu?Pro?Asn

325 330 335

Lys?Gly?Val?Asn?Pro?Asp?Glu?Val?Val?Ala?Val?Gly?Ala?Ala?Leu?Gln

340 345 350

Ala?Gly?Val?Leu?Lys?Gly?Glu?Val?Lys?Asp?Val?Leu?Leu?Leu?Asp?Val

355 360 365

Thr?Pro?Leu?Ser?Leu?Gly?Ile?Glu?Thr?Lys?Gly?Gly?Val?Met?Thr?Arg

370 375 380

Leu?Ile?Glu?Arg?Asn?Thr?Thr?Ile?Pro?Thr?Lys?Arg?Ser?Glu?Thr?Phe

385 390 395 400

Thr?Thr?Ala?Asp?Asp?Asn?Gln?Pro?Ser?Val?Gln?Ile?Gln?Val?Tyr?Gln

405 410 415

Gly?Glu?Arg?Glu?Ile?Ala?Ala?His?Asn?Lys?Leu?Leu?Gly?Ser?Phe?Glu

420 425 430

Leu?Thr?Gly?Ile?Pro?Pro?Ala?Pro?Arg?Gly?Ile?Pro?Gln?Ile?Glu?Val

435 440 445

Thr?Phe?Asp?Ile?Asp?Ala?Asn?Gly?Ile?Val?His?Val?Thr?Ala?Lys?Asp

450 455 460

Lys?Gly?Thr?Gly?Lys?Glu?Asn?Thr?Ile?Arg?Ile?Gln?Glu?Gly?Ser?Gly

465 470 475 480

Leu?Ser?Lys?Glu?Asp?Ile?Asp?Arg?Met?Ile?Lys?Asp?Ala?Glu?Ala?His

485 490 495

Ala?Glu?Glu?Asp?Arg?Lys?Arg?Arg?Glu?Glu?Ala?Asp?Val?Arg?Asn?Gln

500 505 510

Ala?Glu?Thr?Leu?Val?Tyr?Gln?Thr?Glu?Lys?Phe?Val?Lys?Glu?Gln?Arg

515 520 525

Glu?Ala?Glu?Gly?Gly?Ser?Lys?Val?Pro?Glu?Asp?Thr?Leu?Asn?Lys?Val

530 535 540

Asp?Ala?Ala?Val?Ala?Glu?Ala?Lys?Ala?Ala?Leu?Gly?Gly?Ser?Asp?Ile

545 550 555 560

Ser?Ala?Ile?Lys?Ser?Ala?Met?Glu?Lys?Leu?Gly?Gln?Glu?Ser?Gln?Ala

565 570 575

Leu?Gly?Gln?Ala?Ile?Tyr?Glu?Ala?Ala?Gln?Ala?Ala?Ser?Gln?Ala?Thr

580 585 590

Gly?Ala?Ala?His?Pro?Gly?Gly?Glu?Pro?Gly?Gly?Ala?His?Pro?Gly?Ser

595 600 605

Ala?Asp?Asp?Val?Val?Asp?Ala?Glu?Val?Val?Asp?Asp?Gly?Arg?Glu?Ala

610 615 620

Lys

625

<210>3

<211>1206

<212>DNA

<213>Artificial

<220>

＜223〉the proteic encoding sequence of OPG

<220>

<221>misc_feature

<222>(1)..(1206)

<400>3

atgaacaagt?tgctgtgctg?cgcgctcgtg?tttctggaca?tctccattaa?gtggaccacc 60

caggaaacgt?ttcctccaaa?gtaccttcat?tatgacgaag?aaacctctca?tcagctgttg 120

tgtgacaaat?gtcctcctgg?tacctaccta?aaacaacact?gtacagcaaa?gtggaagacc 180

gtgtgcgccc?cttgccctga?ccactactac?acagacagct?ggcacaccag?tgacgagtgt 240

ctatactgca?gccccgtgtg?caaggagctg?cagtacgtca?agcaggagtg?caatcgcacc 300

cacaaccgcg?tgtgcgaatg?caaggaaggg?cgctaccttg?agatagagtt?ctgcttgaaa 360

cataggagct?gccctcctgg?atttggagtg?gtgcaagctg?gaaccccaga?gcgaaataca 420

gtttgcaaaa?gatgtccaga?tgggttcttc?tcaaatgaga?cgtcatctaa?agcaccctgt 480

agaaaacaca?caaattgcag?tgtctttggt?ctcctgctaa?ctcagaaagg?aaatgcaaca 540

cacgacaaca?tatgttccgg?aaacagtgaa?tcaactcaaa?aatgtggaat?agatgttacc 600

ctgtgtgagg?aggcattctt?caggtttgct?gttcctacaa?agtttacgcc?taactggctt 660

agtgtcttgg?tagacaattt?gcctggcacc?aaagtaaacg?cagagagtgt?agagaggata 720

aaacggcaac?acagctcaca?agaacagact?ttccagctgc?tgaagttatg?gaaacatcaa 780

aacaaagccc?aagatatagt?caagaagatc?atccaagata?ttgacctctg?tgaaaacagc 840

gtgcagcggc?acattggaca?tgctaacctc?accttcgagc?agcttcgtag?cttgatggaa 900

agcttaccgg?gaaagaaagt?gggagcagaa?gacattgaaa?aaacaataaa?ggcatgcaaa 960

cccagtgacc?agatcctgaa?gctgctcagt?ttgtggcgaa?taaaaaatgg?cgaccaagac 1020

accttgaagg?gcctaatgca?cgcactaaag?cactcaaaga?cgtaccactt?tcccaaaact 1080

gtcactcaga?gtctaaagaa?gaccatcagg?ttccttcaca?gcttcacaat?gtacaaattg 1140

tatcagaagt?tatttttaga?aatgataggt?aaccaggtcc?aatcagtaaa?aataagctgc 1200

ttataa 1206

<210>4

<211>1878

<212>DNA

<213>Artificial

<220>

＜223〉the proteic encoding sequence of HSP

<220>

<221>misc_feature

<222>(1)..(1878)

<400>4

atggctcgtg?cggtcgggat?cgacctcggg?accaccaact?ccgtcgtctc?ggttctggaa 60

ggtggcgacc?cggtcgtcgt?cgccaactcc?gagggctcca?ggaccacccc?gtcaattgtc 120

gcgttcgccc?gcaacggtga?ggtgctggtc?ggccagcccg?ccaagaacca?ggcagtgacc 180

aacgtcgatc?gcaccgtgcg?ctcggtcaag?cgacacatgg?gcagcgactg?gtccatagag 240

attgacggca?agaaatacac?cgcgccggag?atcagcgccc?gcattctgat?gaagctgaag 300

cgcgacgccg?aggcctacct?cggtgaggac?attaccgacg?cggttatcac?gacgcccgcc 360

tacttcaatg?acgcccagcg?tcaggccacc?aaggacgccg?gccagatcgc?cggcctcaac 420

gtgctgcgga?tcgtcaacga?gccgaccgcg?gccgcgctgg?cctacggcct?cgacaagggc 480

gagaaggagc?agcgaatcct?ggtcttcgac?ttgggtggtg?gcactttcga?cgtttccctg 540

ctggagatcg?gcgagggtgt?ggttgaggtc?cgtgccactt?cgggtgacaa?ccacctcggc 600

ggcgacgact?gggaccagcg?ggtcgtcgat?tggctggtgg?acaagttcaa?gggcaccagc 660

ggcatcgatc?tgaccaagga?caagatggcg?atgcagcggc?tgcgggaagc?cgccgagaag 720

gcaaagatcg?agctgagttc?gagtcagtcc?acctcgatca?acctgcccta?catcaccgtc 780

gacgccgaca?agaacccgtt?gttcttagac?gagcagctga?cccgcgcgga?gttccaacgg 840

atcactcagg?acctgctgga?ccgcactcgc?aagccgttcc?agtcggtgat?cgctgacacc 900

ggcatttcgg?tgtcggagat?cgatcacgtt?gtgctcgtgg?gtggttcgac?ccggatgccc 960

gcggtgaccg?atctggtcaa?ggaactcacc?ggcggcaagg?aacccaacaa?gggcgtcaac 1020

cccgatgagg?ttgtcgcggt?gggagccgct?ctgcaggccg?gcgtcctcaa?gggcgaggtg 1080

aaagacgttc?tgctgcttga?tgttaccccg?ctgagcctgg?gtatcgagac?caagggcggg 1140

gtgatgacca?ggctcatcga?gcgcaacacc?acgatcccca?ccaagcggtc?ggagactttc 1200

accaccgccg?acgacaacca?accgtcggtg?cagatccagg?tctatcaggg?ggagcgtgag 1260

atcgccgcgc?acaacaagtt?gctcgggtcc?ttcgagctga?ccggcatccc?gccggcgccg 1320

cgggggattc?cgcagatcga?ggtcactttc?gacatcgacg?ccaacggcat?tgtgcacgtc 1380

accgccaagg?acaagggcac?cggcaaggag?aacacgatcc?gaatccagga?aggctcgggc 1440

ctgtccaagg?aagacattga?ccgcatgatc?aaggacgccg?aagcgcacgc?cgaggaggat 1500

cgcaagcgtc?gcgaggaggc?cgatgttcgt?aatcaagccg?agacattggt?ctaccagacg 1560

gagaagttcg?tcaaagaaca?gcgtgaggcc?gagggtggtt?cgaaggtacc?tgaagacacg 1620

ctgaacaagg?ttgatgccgc?ggtggcggaa?gcgaaggcgg?cacttggcgg?atcggatatt 1680

tcggccatca?agtcggcgat?ggagaagctg?ggccaggagt?cgcaggctct?ggggcaagcg 1740

atctacgaag?cagctcaggc?tgcgtcacag?gccactggcg?ctgcccaccc?cggcggcgag 1800

ccgggcggtg?cccaccccgg?ctcggctgat?gacgttgtgg?acgcggaggt?ggtcgacgac 1860

ggccgggagg?ccaagtga 1878

<210>5

<211>23

<212>DNA

<213>Artificial

<220>

＜223〉primer

<220>

<221>misc_feature

<222>(1)..(23)

<400>5

atgaacaagt?tgctgtgctg?cgc 23

<210>6

<211>23

<212>DNA

<213>Artificial

<220>

＜223〉primer

<220>

<221>misc_feature

<222>(1)..(23)

<400>6

ttataagcag?cttattttta?ctg 23

<210>7

<211>27

<212>DNA

<213>Artificial

<220>

＜223〉primer

<220>

<221>misc_feature

<222>(1)..(27)

<400>7

catgccatgg?aaacgtttcc?tccaaag 27

<210>8

<211>61

<212>DNA

<213>Artificial

<220>

＜223〉primer

<220>

<221>misc_feature

<222>(1)..(61)

<400>8

cgtcgtgata?accgcgtcgg?taatgccgcc?gccgccgccg?cctttttgag?ttgattcact 60

g 61

<210>9

<211>51

<212>DNA

<213>Artificial

<220>

＜223〉primer

<220>

<221>misc_feature

<222>(1)..(51)

<400>9

ccgctcgagt?caggcgtcat?tgaagtaggc?gggcgtcgtg?ataaccgcgt?c 51

Claims

1. a method that produces the OPG-HSP70 fusion rotein is characterized in that, this method comprises:

(a) will the encode proteic nucleotide sequence of OPG, the proteic nucleotide sequence of L joint and the proteic nucleotide sequence of coding HSP is operably connected to expression vector, obtains OPG-HSP70 fusion rotein recombinant expression vector;

(b) recombinant expression vector in the step (a) is transformed into intestinal bacteria, screening obtains to express the engineering bacteria of OPG-HSP70 fusion rotein;

(c) cultivate the abduction delivering fusion rotein at the appropriate condition bottom fermentation;

(d) the described fusion rotein of purifies and separates in the cultured products from step (c); The form of wherein said fusion rotein is the R1-L-R2 form, and wherein R1 is an OPG albumen, and R2 is a HSP albumen, and L is a joint albumen;

Described OPG albumen is the polypeptide that the 22-194 amino acids shown in the SEQID No:1 is formed;

The proteic aminoacid sequence of described HSP70 is ITDAVITTPAYFNDA;

Described L joint is selected from:

(1) ala-(ala) n-ala, n is 1～4 a integer;

(2) gly-(gly) n-gly, n is 1～4 a integer;

(3)gly-pro-gly；

(4)gly-gly-pro-gly-gly；

(5) ser-gly-(gly) n-gly, n is 1～4 a integer;

(6)val；

(7)tyr-val；

(8) inferior partly any combination of (1)-(7).

2. the method for claim 1 is characterized in that, described step (c) comprises recombinant clone plasmid transformation escherichia coli BL21 (DE3) competent cell, selects single colony inoculation and contains 2 * YT substratum of 25mg/L kantlex, 37 ℃ of shaken overnight in 5mL; After the switching 1: 50 next day, cultivate 2h for 37 ℃, it is 0.1mmol/L that adding IPTG makes it final concentration, centrifugal collection thalline behind 30 ℃ of continuation shaking culture 5h.

3. the method for claim 1 is characterized in that, described step (d) comprises the dissolving of adopting inclusion body, and dilution refolding and sieve chromatography obtain target protein.

4. the fusion rotein that produces of each described method of claim 1-3 is used for the treatment of application in the medicine of rheumatoid arthritis in preparation.