CN102370979A

CN102370979A - Building method for autovaccine by aiming at human TNF(Tumor Necrosis Factor)-alpha molecule

Info

Publication number: CN102370979A
Application number: CN2011103039461A
Authority: CN
Inventors: 张英起; 万一; 薛晓畅; 王增禄; 赵宁
Original assignee: Fourth Military Medical University FMMU
Current assignee: Fourth Military Medical University FMMU
Priority date: 2011-10-10
Filing date: 2011-10-10
Publication date: 2012-03-14
Anticipated expiration: 2031-10-10
Also published as: CN102370979B

Abstract

The invention discloses a building method for autovaccine in-vivo induced by aiming at human TNF(Tumor Necrosis Factor)-alpha molecule. With a step-by-step cloning method, a fusion gene of hTNF-TT830-844, hTNF-HEL46-61 and hTNF-PADRE is built; point mutation (T439-A,C440-G) is introduced into a natural human TNF gene to optimize a mRNA (Ribonucleic Acid) secondary structure; the fusion gene is cloned into a pET22b prokaryotic expression vector, and efficient expression is achieved in the bacterial strain of escherichia coli; three T accessory cell epitope peptides are introduced between the epitope peptide structure domains of hTNF by the computer-aided analysis and is fused with the hTNF-alpha to overcome the immunological tolerance of an organism for the autologous protein, and therefore the organism generates high-level humoral immune response; the generated high-level hTNF-alpha neutralizing polyclone antibody can neutralize killing activity of the hTNF-alpha on L929 cells in vitro; the hTNF-PADRE has the strongest immunogenicity; the high-level antibody can be induced under the condition of using no immunological adjuvant; and the vaccine has favorable protection and curing action on mouse models suffering from rheumatoid arthritis induced by the II-type collagen, cachexia and the like induced by LPS (lipopolysaccharide).

Description

A kind of construction method that is directed against the autovaccine of humanTNF-'s molecule

Technical field

The invention belongs to the medical biotechnology field; Be specifically related to the expression in prokaryotic cell of gene clone, gene recombinaton, exogenous gene, the technical fields such as purification of destination protein, further relate to a kind of construction method that can induce in vivo to the protein vaccine of humanTNF-'s molecule autoantibody.

Background technology

1.TNF-the correlational study of alpha molecule and anti-TNF-α treatment

1.1TNF-alpha molecule

Discoveries such as Carswell in 1975; A kind of highly active tumor cytotoxicity factor is arranged in bacillus calmette-guerin vaccine and the bacterial endotoxin mice infected serum; Can make tumor generation hemorrhagic necrosis, but normal tissue cell is not had killing activity, with its called after tumor necrosis factor---TNF-α.HumanTNF-'s gene is positioned at chromosome No. 6, and gene length 4kb contains 4 exons, and is chain with mhc gene, and its precursor is made up of 232 aminoacid, contains 76 amino acid whose signal peptides.The mice gene is positioned at chromosome No. 17, is made up of 4 exons and 3 introns, and precursor is made up of 235 aminoacid.People and mice TNF-α precursor have 79% homology, and the adult form humanTNF-is made up of 157 aminoacid, and molecular weight is about 17KDa, than mice adult form TNF at histidine residues more than 73.The adult form TNF-α of people and mice all contains two cysteine (69,101 of people; 69,100 of Mus), can form intramolecular disulfide bond.The TNF-α of separation and purification from cell strain culture supernatant molecular weight under non-reduced condition differs in size, and molecular weight is 17KDa under reducing condition, and is consistent with theoretical molecular.This possibly be because the reason that TNF-α exists with in various degree polymer form.Confirmed at present the homotrimer that TNF-alpha active form is made up of the subunit of 17KDa.

TNF-α is a kind of multi-functional cytokine.It except can be in vivo, outside the killing tumor cell, also have the inflammatory reaction of inducing, viral infection resisting, adjusting immunity of organism, promote multiple functions such as cell proliferation and activation.TNF-α has direct inhibition and cytolysis to tumor, but different tumor line is different to the sensitivity of TNF-α.TNF-α antitumor mechanism imperfectly understands, and present result of study shows that TNF-α antineoplastic mechanism mainly contains:

(1) combines to bring into play cytotoxicity through surface receptor with tumor cell TNF-α.Present known receptor has two kinds, and TNFR I and TNFR II are transmembrane glycoprotein.TNFR I is through dead plot structure territory (death domain, DD) apoptosis of mediation tumor cell of about 80 amino acid residues of its cytoplasmic domain.

(2) through to the effect of vascular endothelial cell, induce the expression of thrombin, increase the secretion of plasminogen inhibitor, thereby promote the formation of intravascular coagulation and thrombosis, cause ischemia and the necrosis of tumor tissues.

(3) through mediation tumor effector lymphocyte's effect and the partial inflammatory reaction of induced tumor extremely, the effect that promotes to kill the tumor effector lymphocyte.

At anti-virus aspect, TNF-α can optionally kill and wound the cell of viral infection, and different viral infection is had different effects.As inhibited, but HIV had facilitation to viruses such as VSV, HSV.In addition, TNF-α is important inflammatory factor, and it can express the expression of MHC I class antigen and multiple adhesion molecule by stimulating endothelial cell on the one hand, promotes the expression of IL-1, IL-6, and stimulating endothelial cell and leukocyte discharge a series of inflammatory mediators.TNF-α can promote neutrophilic granulocyte, lymphocyte and mononuclear cell to be attached on the endotheliocyte on the other hand, thereby participates in inflammatory reaction.TNF-α can also promote T, B cell proliferation, and enhancing antibody produces; The activation mononuclear phagocyte improves its killing activity; Raise macrophage MHC II quasi-molecule, improve antigen presentation ability etc.

1.2 anti-TNF-α treatment

Discover that TNF-α continues in vivo, in multiple disease, play an important role during high-caliber expression, as: dyscrasia, insulin resistant, septic shock, inflammatory reaction, autoimmune disease etc.Development TNF-α agonist drug provides a good approach for treatment TNF-alpha levels increases relevant disease.

Traditional medicine that is used to treat the overexpression relevant disease of TNF-α in the body mainly contains: nonsteroidal anti-inflammatory agent (NSAIDS), alleviate the antirheumatic (DMARD) of disease, corticosteroid, immunosuppressant and analgesic.The new drug of coming out one after another in recent years has: methotrexate, leflunomide etc.But said medicine all has than obvious toxic and side effects.

Research at present mainly concentrates on TNF-α antibionts preparation, and it is evident in efficacy, side effect is little.

The TNF-α agonist drug of having invented has infliximab, etanercept, adalimumab, CDP571 and CDP870.The first three medicine is through the FDA approval.Etanercept is the fusion rotein of gene recombinaton sTNFR75 and human IgG Fc section, can suppress TNF-α and TNF-β simultaneously, and be used to treat teenager RA and psoitis by the FDA approval in November, 1998.Infliximab is people's Mus embedding of TNF-α neutrality and antibody, is used for treating RA by FDA approval in 1999, after be used for treating the Crohn disease again.Adalimumab is the TNF-α neutrality IgG1 monoclonal antibody of full-length human, and it is sick to can be used for treating RA and Crohn.Clinical research in recent years shows that anti-TNF-α has obtained curative effect preferably at clinical treatment RA.The RA of Americanism damp disease association issue in 2002 treats up-to-date guide and points out, early stage RA patient is effective to etanercept with the patient who uses DMARDS to fail to respond to any medical treatment.Use the out of contior RA patient who carries out sexual activity property of capacity methotrexate, etanercept and infliximab unite and use effectively, recommend at present separately use infliximab or with the methotrexate therapeutic alliance.

The treatment of TNF-α agonist drug also can be applicable to the treatment that other TNF-alpha levels increases relevant disease except can be used for RA, as, cachexia, crohn, ankylosing spondylitis, psoriatic arthritis etc.

Though TNF-alpha monoclonal antibodies and soluble recepter have curative effect preferably clinically,, these medicines have dosage big, need prolonged and repeated use, be prone to cause the defective of allergy.In addition, expensive expenses for medicine also is the reason that hinders this type of medicine extensive use.According to statistics; Every patient's average year cost of accepting the etanercept treatment is 1.5 ten thousand dollars clinically; Patient's average year cost of accepting Infliximab or Adalimumab treatment is 90,000 dollars, even so expensive expenses for medicine also is difficult to bear in developed country.

2. therapeutic vaccine

2.1 progress

In recent years, in the process of the acute and chronic disease of treatment, demonstrated good effect to the proteic monoclonal antibody of mankind itself.But; The costliness of cost and the inconvenience of using have limited the extensive use of monoclonal antibody; Therefore; Turn to and seek to the proteic active immunity vaccine of mankind itself by accepting this antibody protein passively,, become the developing direction of protein drug promptly with the alternative passive immunity of the therapeutic modality of active immunity.At present, the research of therapeutic vaccine has become a focus, relates to a lot of diseases, like chronic viral infection, tumor, Alzheimer, diabetes, hypertension, obesity and rheumatic arthritis etc.

Therapeutic vaccine makes people's immune system help reaction.Most vaccine can be divided into two big types: one type is to induce body generation humoral immune reaction, produces antibody; Another kind of is to induce body generation cell immune response, produces cytotoxic T cell (CTLs).The one type of therapeutic vaccine in back is mainly used in the treatment of tumor and disease of viral infection.

All through inducing production of antibodies to protect body, this just proves through inducing antibody to treat infectious disease is a kind of efficacious therapy method to most preventative vaccines.Compare with preventative vaccine, the development of therapeutic vaccine is slow relatively, but monoclonal antibody was indicating in the immense success that is obtained aspect the treatment disease that therapeutic vaccine had vast potential for future development in recent years.In fact, it is possible that the endogenous specific antibody that has had animal experiment to show to induce certain level is treated disease, as: the vaccine to angiotensin can be treated hypertension; Vaccine to IL-9 can be treated Eosinophilia's disease; Vaccine to IL-5 can be treated asthma; Vaccine to N-methyl-D-aspartate receptor-1 (NMDAR1) can be treated apoplexy.In addition, the immunity to some gonadal hormone such as human chorionic gonadotropin (human chorionic gonadotro-pin HCG) can reduce the intravital hormonal readiness of women and reach contraceptive effect; Vaccine to gonadotropin-releasing hormone (GnRH) can be used to treat advanced prostate cancer; Among the cancer of pancreas patient, the antibody that utilizes therapeutic vaccine to induce to gastrin (gastrin) can prolong patient's life late.

So, is there which problem in the research of therapeutic vaccine? As if this problem is answered in the development process what happens of the therapeutic vaccine that is directed against Alzheimer recently.Alzheimer is a kind of very suitable disease of treating with therapeutic vaccine that seems, the pathogenic process of this disease reaches several years even many decades.If can induce long-term antibody with therapeutic vaccine, be undoubtedly a kind of ideal Therapeutic Method so, especially this patient often forgets and takes medicine.

The characteristic of Alzheimer is the deposition of speckle in the brain, contains the A beta-peptide in this speckle, and this A beta-peptide is that (amyloid precursor protein APP) derives for precursor protein from amyloid.In the gene of coding APP was undergone mutation the crowd that the generation that causes the A beta-peptide increases, Alzheimer was just beginning to have taken place in one's early years.Expressing in the transgenic mice brain of APP of this sudden change has a large amount of plaque deposition, and the speckle of finding in this speckle and the Alzheimer patient brain is similar.Add that with the A beta-peptide strong immunological adjuvant comes immune this transgenic mice that the speckle in the mouse brain is reduced, the spiritual expression of mice is clearly better.Subsequently; A kind of therapeutic vaccine to Alzheimer has begun clinical trial; After it has good tolerability in clinical I phase evidence; The clinical II phase that comprises more than 300 patient has been followed by having begun, but unexpected be 6% subjects owing to produce the aseptic encephalitis and be compelled to stop to test.Research subsequently confirms that it doesn't matter for the titre of this side effect and antibody, and in fact, the patient that tried who does not produce antibody response also got the aseptic encephalitis.In addition, in a patient's brain, find to have a large amount of lymphocytic infiltrations.These results of study and a kind of hypothesis are consistent, and that side effect of in patient, finding is because A beta-peptide specific T lymphocyte causes, rather than since antibody cause.This just requires to develop the autoimmune second filial generation vaccine that does not cause the T cell mediated.So; How is the effect of this therapeutic vaccine to Alzheimer? During the clinical II phase of mentioning in front tests; In the patient who induces anti-A beta-peptide antibody some cognitive power weaken certain postponement, some patients are arranged in addition in this year of receiving treatment state of consciousness improvement has been arranged.If when vaccine design, can solve its safety issue, so in the near future, will occur to the therapeutic vaccine of Alzheimer.

Another kind of slightly different vaccine is lower to the requirement of safety, and that is exactly the vaccine to habit-forming medicine.Vaccine to cocaine and nicotine in animal experiment has reduced the level of these medicines in brain, has eliminated their addiction symptom, and experimental animal is being accepted immune then medicine no longer the dependence.In testing in the clinical I phase, the cocaine vaccine has been proved to be good tolerability, and can induce good antibody response, is carrying out validation verification at present.

2.2 induce the theoretical research of autoantibody

Is therapeutic vaccine to oneself protein how to induce the autoimmune system to produce the antibody to oneself protein? The specific antibody that produces sufficiently high titre is with the treatment relevant disease, and therapeutic vaccine must overcome three obstacles: T cell tolerance, Blymphocyte tolerance, under the situation that does not have adjuvant and antigen durative action preparation, induce antibody.As everyone knows, the human immune system is mainly to external invador's attack, and body itself is not attacked, and this possibly be because body has the ability that can discern " nonego " and " oneself ".Immune this specific character is commonly called tolerance or anergy.Tolerance occurs in B cell and T cellular level.In general, the T cell tolerance is stricter.Concerning many antigens, when the T cell tolerance took place, normal B cell strain but existed in vivo.In fact, have three kinds of mechanism to cause immunologic tolerance: cell strain is rejected, and promptly specific lymphocyte is thoroughly rejected from lymphocyte populations; Immunity is incompetent, and promptly specific lymphocyte exists, but its function can not be activated; Immunity is ignored, and the lymphocyte that promptly has immunologic function exists, but owing to do not run into the autoantigen that exists with the antigen form, so can not be activated.Concerning the T cell, inducing tolerance and incompetent major organs is thymus (central tolerance), but induces tolerance also can carry out in periphery.Blymphocyte tolerance is mainly induced in bone marrow, but also can induce in periphery.Usually, illustrate more easily for the antigenic immunologic tolerance of generally expressing of enriching.

In being directed against the immunoreation of exotic antigen, T cell and B cell are worked in coordination and could be produced antibody effectively: when receiving the exotic antigen immunity, specific B cell conjugated antigen produces initial activation signal.In addition, B cell endocytic antigen presents the complex of antigenic peptides and MHC II quasi-molecule on its surface.Usually, the B cell can not activate the TH cell.Activate the TH cell, BMDC is absolutely necessary, the BMDC antigen, and, activate the TH cell at the complex of its cell surface antigen-presenting peptide and MHC II quasi-molecule.The antigenic peptides that TH cell recognition B cell surface after the activation appears and the complex of MHC II quasi-molecule cause other conversion of B cell proliferation, production of antibodies and antibody class.If lack the synergism of TH cell because of immunologic tolerance, so just can not produce antibody.In the design process of the vaccine that is directed against oneself protein; If autoantigen is merged with foreign protein or peptide carrier or is coupled at; Just might walk around the TH cell tolerance: the specific B cell of autoantigen just can absorb this autoantigen and coupled carrier protein; And present the complex of carrier peptides and MHC II quasi-molecule on its surface; Because the TH cell does not have immunologic tolerance to carrier protein, thus can be activated, thus the collaborative specific antibody that is directed against autoantigen that produces from the B of antigenic specificity cell.

Compare with the T cell tolerance, it is much loose that Blymphocyte tolerance is wanted.In fact, in many cases, autospecific B cell occurs with normal frequency in vivo, will be activated if receive the combined effect of antigen and TH cell.So to many solubility oneself proteins, have its specific b cells strain in the body, especially when this albumen is not very expression more than needed especially like this.For this albuminoid or polypeptide, it is linked to each other with carrier protein, walk around the T cell tolerance, just might produce effective vaccine.In fact, utilize this strategy, derived to the antibody response of multiple self hormone.

3.TNF-the progress of α autovaccine

At present, TNF-α vaccine research mainly concentrates on screening TNF-α epitope, amalgamation protein vaccine and the several aspects of dna vaccination.Personnel selection such as Sioud TNF purification rheumatoid arthritis patient's antiserum; Obtain having among the TNF-α and active TNF-α autoantibody; With this antibody screening phage random nonapeptide storehouse; Phage particle immune mouse with screening obtains has brought out mouse-anti-human T NF-Alpha antibodies, and research shows that recombinant phage can be used as the effective tool of vaccine research.Dalum etc. are with the inductive arthritis mouse model of protein immunization two Collagen Type VIs of t helper cell epi-position and TNF-alpha fusion expression, and the symptom of mice has obtained alleviation as a result, for rheumatoid arthritis and chronic infection treatment of diseases have been opened up a new way.Blank etc. are with the mice of the anti-phospholipid syndrome of dna immunization of coding TNF-α; In the mice body, brought out anti-TNF-Alpha antibodies; These antibody can block the endothelial cell activity that TNF-α causes; Suppress mononuclear cell to the sticking of activated endothelial cells, compared the symptom that significantly to improve anti-phospholipid syndrome mice with matched group.Waterston etc. have compared TNF-alpha monoclonal antibodies and the TNF-α autovaccine effect in the treatment neoplasm metastasis, and the result shows the transfer of the minimizing lung tumor that both can be successful in the mice body.Recent Waterston etc. have assessed the immunogenicity and the safety of TNF-α autovaccine through a clinical trial phase; Although unfortunately the serum antibody that brings out of vaccine can be discerned humanTNF-'s molecule of degeneration; But but can not in humanTNF-'s molecule of native state, analyzing reason possibly be because make the reason of antigen degeneration in the antigenic purge process.

More than research shows; Though TNF-α autovaccine also is the good method of the too high relevant disease of a kind of TNF-of treatment alpha expression; But screen effective T cell is assisted epi-position, and suitable epi-position on position and suitable purification process all become the critical problem of TNF-α autovaccine research.

To above problem, we are model with the mice in the work in early stage, have screened the on position in mice TNF-alpha molecule of the auxiliary epi-position of external source T cell to the TNF-alpha molecule of mice, and screening is than the auxiliary epi-position of right different T cell.The result finds the on position of mice TNF-alpha molecule 128-140 amino acids for best exogenous molecules, and PADRE is for the most effectively inserting epi-position.To above research basis, the autovaccine that the present invention is primarily aimed at its corresponding humanTNF-'s molecule makes up, expression, purification research, is intended to obtain a kind of effectively to the autovaccine of humanTNF-'s molecule.

Summary of the invention

The objective of the invention is to, a kind of method of the humanTNF-'s of preparation autovaccine molecule is provided, this molecule can induce the specific antibody to humanTNF-'s molecule, for the too high relevant disease of TNF-alpha expression provides a kind of new medicine.

To achieve these goals, the technical scheme that the present invention adopted is: induce the construction method to the protein vaccine of the autoantibody of humanTNF-'s molecule in a kind of body, it is characterized in that, with fusion gene (TNF-α _1-128One PADRE-TNF-α _142-157) be cloned between the Nde I and Sal I MCS of prokaryotic expression carrier pET22b; With the recombinant expression carrier transformed into escherichia coli BL21 that obtains, inducing the back expression product is inclusion body, washs through inclusion body; Gel filtration chromatography; The dialysis renaturation can obtain the purpose fusion rotein of purification, with inducing the protein vaccine to the autoantibody of humanTNF-'s molecule in this a kind of body; Immune mouse can produce the anti-humanTNF-'s autoantibody of high titre under the situation of not using adjuvant, and wherein said fusion gene sequence is:

5’- CATATGGTCAGATCATCTTCTCGAACCCCGAGTGACAAGCCTGTAGCCCATGTTGTAGCAAA

CCCTCAAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTCCTGGCCAATGG

CGTGGAGCTGAGAGATAACCAGCTGGTGGTGCCATCAGAGGGCCTGTACCTCATCTACTCCCAG

GTCCTCTTCAAGGGCCAAGGCTGCCCCTCCACCCATGTGCTCCTCACCCACACCATCAGCCGCA

TCGCCGTCTCCTACCAGACCAAGGTCAACCTCCTCTCTGCCATCAAGAGCCCCTGCCAGAGGGA

GACCCCAGAGGGGGCTGAGGCCAAGCCCTGGTATGAGCCCATCTATCTGGGAGGGGTCTTCCA

GCTGCAATTG GCTAAATTCGTTGCTGCATGGACTCTGAAAGCTGCAGCTCTCGACTTTGCCGAG

AGTGGGCAGGTCTACTTTGGGATCATTGCCCTGTGA GTCGAC-3’

The structure of its gene is accomplished according to the following steps:

According to humanTNF-'s aminoacid sequence and PADRE aminoacid sequence (AKFVAAWTLKA), design hTNF-PADRE gene order, the insertion site of PADRE is 128 back of TNF-α in the gene order, promptly replaces people TNF with the PADRE coded sequence _129-141The coded sequence of amino acids;

Wherein the 1-128 amino acids coded sequence of TNF-α obtains through pcr amplification, and introduces Nde I restriction enzyme site (CATATG) at the upper reaches, wherein contains translation initiation codon; Mun I restriction enzyme site (CAATTG) is introduced in downstream; Synthetic PADRE-TNF-α _142-157Coded sequence, upstream and downstream is introduced Mun I and Sal I (GTCGAC) restriction enzyme site respectively, in synthetic fragment, gene has been carried out two point mutation (T439 → A, C440 → G) in addition;

Two fragment genes connect through Mun I restriction enzyme site, and fusion gene cloning is gone between the Nde I and Sal I restriction enzyme site of carrier pET22b;

With above-mentioned expression vector transformed into escherichia coli BL21, the plasmid called after pET22b-hTNF-PADRE that checks order correct, engineering bacteria called after pET22b-hTNF-PADRE/BL21; Its said gene order is following:

5’- CATATGGTCAGATCATCTTCTCGAACCCCGAGTGACAAGCCTGTAGCCCATGTTGTAGCAAA

Nde?I

CCCTCAAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTCCTGGCCAATGG

CGTGGAGCTGAGAGATAACCAGCTGGTGGTGCCATCAGAGGGCCTGTACCTCATCTACTCCCAG

GTCCTCTTCAAGGGCCAAGGCTGCCCCTCCACCCATGTGCTCCTCACCCACACCATCAGCCGCA

TCGCCGTCTCCTACCAGACCAAGGTCAACCTCCTCTCTGCCATCAAGAGCCCCTGCCAGAGGGA

GACCCCAGAGGGGGCTGAGGCCAAGCCCTGGTATGAGCCCATCTATCTGGGAGGGGTCTTCCA

GCTGCAATTG GCTAAATTCGTTGCTGCATGGACTCTGAAAGCTGCAGCTCTCGACTTTGCCGAG

Mun?I PADRE

AGTGGGCAGGTCTACTTTGGGATCATTGCCCTGTGA GTCGAC-3’。

Mutational site Sal I

The protein expression of said gene constructed engineering bacteria; Accomplish according to the following steps: picking engineering bacteria list colony inoculation is in the fresh LB culture medium of 5mL; Described LB culture fluid contains Amp100mg/L, 37 ℃ of shaking table overnight incubation, and next day, the ratio with 1: 100 was transferred in the LB culture fluid once more; Cultivate 3h at 37 ℃ of shaking tables, to logarithmic growth OD in mid-term ₆₀₀Be 0.4～0.6, add 1mM IPTG and induce, continue to cultivate 4h, centrifugal collection thalline.

The purification renaturation of expressed fusion protein, accomplish according to the following steps:

Get the bacterial strain that contains the pET22b-hTNF-PADRE recombiant plasmid and induce bacterium liquid in a large number, the centrifugal 10min of 5000rpm receives bacterium, ultrasonicly splits bacterium; 4 ℃, 12000rpm, centrifugal 20min collects supernatant and deposition respectively;

Get 10g fermentation thalline,, thalline thoroughly is resuspended in 100mM Tris-HCl (7.0), among the 1mM EDTA, add the 1.5mg/mL lysozyme, 10ug/mL DNaseI, 20mM MgCl in the ratio of 1g weight in wet base to 7ml ₂Hang and carry out cracking; Add 1.5M NaCI, 2%Triton X-100,20mM EDTA to 3 times of first volumes, room temperature washing 30min uses the 4M Urea of 3 times of volumes after centrifugal more respectively; 2% Triton X-100,20mM EDTA, 0.1M Tris; With the 20mM EDTA of 8 times of volumes, 0.1M Tris washing; The inclusion body of warp washing is dissolved in 50mmol/LTris.Cl pH 7.0, the 5mmol/L beta-mercaptoethanol with the ratio of 1g weight in wet base to 10ml; 1mmol/L EDTA, in the buffer of 7mol/L guanidine hydrochloride, 4 ℃ of stirred overnight; The centrifugal 30min of 12000r/min collects supernatant, is the destination protein crude extract; (gel chromatography of 1.6cm * 100cm) is with working solution (7mol/L guanidine hydrochloride, 50mmol/L Tris.Cl pH 7.0 for Sephacryl S-100; 1mmol/L EDTA, 100mmol/L NaCl) fully after the balance, destination protein crude extract 2ml upper prop; With the working solution eluting, flow velocity 1ml/min, fraction collection; Measure protein content, and confirm the destination protein position, collect the higher elution fraction of purity with SDS-PAGE; Purification destination protein component is carried out gradient dialysis renaturation to carbamide, and gradient does, 4M carbamide, and 2M carbamide, 1M carbamide carries out dialysis distilled water at last, the centrifugal supernatant that stays, lyophilizing; HPLC detects refolded protein purity.

The fusion rotein that said method is obtained, immune mouse can produce the anti-humanTNF-of high titre self neutrality polyclonal antibody under the situation of not using adjuvant.This antibody has excellent protection and therapeutical effect for mouse models such as the inductive rheumatoid arthritis of II Collagen Type VI, the inductive dyscrasia of LPS.The antibody that this vaccine produces is expected to be used for prevention and treatment rheumatoid arthritis and dyscrasia etc.

Make up a kind of hTNF-α autologous protein vaccine, it is characterized in that this vaccine that makes contains hTNF-α complete sequence, wherein the coded sequence of 129-141 amino acids is enhanced the TT of vaccine immunogenicity _830-844(QYIKANSKFIGITEL), HEL _46-61(NTDGSTDYGILQ INSR) and PADRE t helper cell epitope peptides such as (AKFVAAWTLKA) are replaced.

This vaccine can induce body to produce the antibody of the hTNF-alpha specific of high titre, this antiserum external can in the lethal effect of humanTNF-to the L929 cell.Can bring out the anti-humanTNF-'s autoantibody of high titre, for the immunization therapy of the too high relevant disease of TNF-alpha expression provides a kind of new pharmaceutical grade protein.

Above-mentioned hTNF-α is characterized in that from the method for preparing of body polypeptide vaccine, the epitope peptide domain of and hTNF-α truncate expression analysis hTNF-α auxiliary according to computer software finally is template with humanTNF-, amplification TNF-α _1-128The coded sequence of amino acids; Synthetic TT _830-844-, HEL _46-61-, PADRE-and TNF-α _142-157Genetic fragment connects and is cloned between the Nde I and Sal I MCS of prokaryotic expression carrier pET22b through introducing Mun I (making the 129-141 amino acids of TNF-α not introduce any exogenous amino acid outside being substituted by PADRE etc.) restriction enzyme site between amplified fragments and the synthetic fragment.Because wild type gene is not expressed,, make the expression of the genes of interest that script is not expressed may be up to 20% of bacterial protein so the secondary structure of mRNA after we transcribe through analysis has been introduced a samesense mutation (two point mutation).Expression product is an inclusion body; Through inclusion body washing, gel filtration chromatography, dialysis renaturation and purification; Can obtain the destination protein PADRE-hTNF-α of purification; This vaccine can induce body to produce the antibody of the hTNF-alpha specific of high titre, and this antibody can neutralize natural hTNF-α for the killing activity of L929 cell, when being used for animal, has good protective action for dyscrasia and rheumatoid arthritis mouse model.

The particular content of preparation is (is example with h TNF-PADRE):

1. synthetic TNF-α _1-128-PADRE-TNF-α _142-157Gene order

Its aminoacid sequence is:

VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

The gene order of the hTNF-α that announces according to Gene-bank, 5 ' end inserts Nde I restriction enzyme site, and 3 ' end also inserts Mun I restriction enzyme site, and according to the codon of escherichia coli preference, the synthetic primer amplification obtains genes of interest hTNF-α _1-128

5’- CATATGGTCAGATCATCTTCTCGAACCCCGAGTGACAAGCCTGTAGCCCATGTTGTAGCAAA

CCCTCAAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTCCTGGCCAATGG

CGTGGAGCTGAGAGATAACCAGCTGGTGGTGCCATCAGAGGGCCTGTACCTCATCTACTCCCAG

GTCCTCTTCAAGGGCCAAGGCTGCCCCTCCACCCATGTGCTCCTCACCCACACCATCAGCCGCA

TCGCCGTCTCCTACCAGACCAAGGTCAACCTCCTCTCTGCCATCAAGAGCCCCTGCCAGAGGGA

GACCCCAGAGGGGGCTGAGGCCAAGCCCTGGTATGAGCCCATCTATCTGGGAGGGGTCTTCCA

GCTG CAATTG-3’

Synthetic PADRE-TNF-α _142-157Gene order (two ends add Mun I and Sal I restriction enzyme site respectively) as follows, 3 ' end is introduced termination codon TGA:

5’- AATTGGCTAAATTCGTTGCTGCATGGACTCTGAAAGCTGCAGCTCTCGACTTTGCCGAGTCT

GGGCAGGTCTACTTTGGGATCATTGCCCTGTGA G-3’

Synthetic simultaneously T cell is assisted the gene order of epi-position PADRE and is introduced EcoR I and BamHI restriction enzyme site at two ends, introduces start codon ATG at 5 ' end.Synthetic sequence is following:

2. construction recombination plasmid pET22b-PADRE-TNF-α

With Nde I and two digestion with restriction enzyme pET22b of Sal I carrier, the big fragment of 1.0% agarose gel electrophoresis Separation and Recovery enzyme action, and with hTNF-α through Nde I and Mun I enzyme action _1-128PCR product and synthetic PADRE-TNF-α _142-157The annealing product connect, the T4 ligase spends the night 16 ℃ of connections, transformed into escherichia coli BL21 competent cell, bed board, cultivation, the picking monoclonal carries out enzyme action and identifies.Obtain containing the pulsating positive colony of genes of interest, be called pET22b-TPT.

3. the abduction delivering of recombiant protein, purification and evaluation

3.1pET-22b-PADRE-TNF-the abduction delivering of α/BL21 engineering bacteria

Picking engineering bacteria list colony inoculation in 5mL LB culture fluid (containing Amp100mg/L), 37 ℃ of shaking table overnight incubation, next day, the ratio with 1: 100 was transferred in the LB culture fluid that contains Amp, cultivated 3h to logarithmic growth (OD in mid-term at 37 ℃ of shaking tables ₆₀₀Be 0.4～0.6); 1h, 2h, 3h, 4h, 5h, 6h collect thalline respectively after adding 1mM IPTG abduction delivering; To confirm suitable inducing the back time, small-scale is cultivated and is induced the centrifugal collection thalline in back, with proteic expression of SDS-PAGE testing goal and existence form.

3.2pET-22b-PADRE-TNF-the fermentation of α/BL21 engineering bacteria

Select single colony inoculation in the test tube that contains LB from activatory LB flat board, cultivate 10h for 37 ℃, change then and plant to the triangular flask that contains 200ml LB, 37 ℃ of overnight incubation.Next day, seed liquor is inoculated in the 5L fermentation tank.37 ℃ of fermentation temperatures of control, rotating speed 250～450rpm/min, ventilation 3～5L/min, pH7.3.Be cultured to OD ₆₀₀During=8 left and right sides, add 3mM IPTG and induce, continue to cultivate 4h, stop fermentation, collect thalline, weigh ,-20 ℃ of refrigerators are preserved subsequent use.After the thalline that takes a morsel is handled, with the proteic expression of SDS-PAGE testing goal.

3.3 the separation of destination protein inclusion body and washing

Get 10g fermentation thalline,, thalline thoroughly is resuspended in 100mM Tris-HCl (7.0), among the 1mM EDTA, add the 1.5mg/mL lysozyme, 10ug/mL DNaseI, 20mM MgCl in the ratio of 1g weight in wet base to 7ml ₂Hang and carry out cracking; Add 1.5M NaCl, 2%Triton X-100,20mM EDTA to 3 times of first volumes, room temperature washing 30min uses the 4M Urea of 3 times of volumes after centrifugal more respectively; 2% Triton X-100,20mM EDTA, 0.1M Tris; With the 20mM EDTA of 8 times of volumes, 0.1M Tris washing.Inclusion body after centrifugal dissolves (beta-mercaptoethanol that adds 20mM) with the 7M guanidine hydrochloride.Final inclusion body carries out purification through the Sephacryl-100 gel permeation chromatography, and the protein behind the purification carries out carbamide gradient centrifugation renaturation and obtained natural structure, thereby can induce the neutralizing antibody of high-affinity.

3.4 purification and renaturation under the expression product degeneration condition

The inclusion body of warp washing is dissolved in 50mmol/L Tris.Cl pH 7.0, the 5mmol/L beta-mercaptoethanol with the ratio of 1g weight in wet base to 10ml; 1mmol/L EDTA, in the buffer of 7mol/L guanidine hydrochloride, 4 ℃ of stirred overnight; The centrifugal 30min of 12000r/min collects supernatant, is the destination protein crude extract.(gel chromatography of 1.6cm * 100cm) is with working solution (7mol/L guanidine hydrochloride, 50mmol/L Tris.Cl pH 7.0 for SephacrylS-100; 1mmol/L EDTA, 100mmol/L NaCl) fully after the balance, destination protein crude extract 2ml upper prop; With the working solution eluting, flow velocity 1ml/min, fraction collection; Measure protein content, and confirm the destination protein position, collect the higher elution fraction of purity with SDS-PAGE.Purification destination protein component is carried out gradient dialysis renaturation to carbamide, and gradient does, 4M carbamide, and 2M carbamide, 1M carbamide carries out dialysis distilled water at last, the centrifugal supernatant that stays, lyophilizing.HPLC detects refolded protein purity.

3.5 the evaluation of recombiant protein

3.5.1 the evaluation of immunogenicity and purity

The purified proteins sample is through 15%SDS-PAGE, then from the gel electrotransfer to nitrocellulose membrane, after the BSA sealing; Combine 1h with one anti-(mouse anti human TNF-alpha monoclonal antibodies); Two anti-(goat anti-mouse IgG-HRP) combine 30min, at last with the colour developing of ECL system, distilled water flushing cessation reaction.The result shows hTNF-TT, and what hTNF-HEL, hTNF-PADRE can be special combines with mouse anti human TNF-alpha monoclonal antibodies.

The TNF-TT that purification obtains, TNF-HEL, TNF-PADRE carry out purity to be identified.Chromatographic column is exclusion chromatography post (7.5mm * 300mm G2000SW column (TOSOH)), and mobile phase liquid is PBS, and flow velocity is 0.5ml/min, and the detection wavelength is 280nm.Purity is all greater than 95% as a result.

3.5.2 residual natural TNF-α cell killing activity experiment

The mice L929 cell that growth conditions is good transfers to 10 ⁶The cell suspension of/ml, every hole adds 100 μ l cell suspension, 50ml/lCO in 96 well culture plates ₂, 37 ℃ of overnight incubation, abandon supernatant, every hole adds the TNF-TT after the renaturation of serial dilution, 5%CO ₂, 37 ℃ cultivate 16h, abandon supernatant, the concentration that every hole adds 30 μ L is 500 μ g/mL violet staining 3-5min, carefully washes away crystal violet with flowing water, dries residual moisture, every hole adds destaining solution 100 μ L, ELIASA 570nm place mensuration light absorption value.The humanTNF-is as positive control, and the GST albumen of purification is as negative control.The result shows that TNF-TT, TNF-HEL, TNF-PADRE all do not have natural humanTNF-'s cytotoxicity.

3.5.3 animal immune

3.5.3.1Balb/c mice group and immunization protocol

Divide into groups: 25 male Balb/c mices are divided into 5 groups, are respectively the adjuvant group, hTNF group, hTNF-TT group, hTNF-HEL group and hTNF-PADRE group.Pluck the eyeball blood sampling in back 10 days in the 4th immunity, measure hTNF antibody titer in the serum.Immunization protocol: the subcutaneous multi-point injection in back; 30 μ g antigens/only; Use Freund's complete adjuvant the 1st time, the 2nd, 3 time and the 4th use Freund, the assist agent solution cumulative volume is 200 μ L/, respectively injection every other week.

3.5.3.2 antiserum ELISA identifies

In the 4th immunity blood sampling in back 10 days, collect antiserum, measure serum anti hTNF antibody titer through indirect ELISA.

3.5.4TNF-PADRE treatment to the inductive cachexia model of TNF-α

3.5.4.1 divide into groups and immune mouse

The Balb/c mice, male, totally 30, body weight 18-20g divides two groups at random by body weight, and per 10 one group, body weight statistics zero difference between group.The A group: the TNF-PADRE group, antigen is dissolved in the normal saline, and 50 μ g/ml, 200 μ l/ are only.B group: normal saline group.Immunization protocol: the subcutaneous multiple spot immunity in first three time back, the 4th peritoneal immunity, immune liquor capacity are 200 μ l/, respectively injection every other week.

3.5.4.2 inducing of model

Mice immunized is got 5 by every group of body weight, body weight 27-28g, and body weight no difference of science of statistics between group, every day, intraperitoneal injection of mice TNF-α 1 * 10 ⁵IU/, weighing every day mice body weight.

Mice immunized is got 10 by every group of body weight, body weight 27-30g, and body weight no difference of science of statistics between group, every day, intraperitoneal injection of mice TNF-α 2 * 10 ⁵IU/ only adds up the life cycle of mice.

3.5.5TNF-PADRE treatment to the inductive rheumatoid arthritis mouse model of II Collagen Type VI

3.5.5.1 the preparation of derivant

The II Collagen Type VI is dissolved in the solution that 0.1M acetic acid is processed 4mg/mL; Mix the back with isopyknic complete Freund's adjuvant (incomplete Freund adds the bacillus calmette-guerin vaccine of final concentration 2mg/mL) and push away suction emulsifying repeatedly with syringe, being mixed with II Collagen Type VI final concentration is the Emulsion of 2mg/mL.

3.5.5.2 the immunity of animal

The Si Laike animal center is bought 40 of the DBA-1 inbred mouses in 5 ages in week from Shanghai, is divided into four groups at random, 10 every group.First group of immunization method is subcutaneous multiple spot immunity, and immunizing dose is that 100 μ L normal saline/only, immunity every other week once is total to immunity four times.Second group of immunization method is identical with first group.The 3rd group of immunization method is subcutaneous multiple spot immunity hTNF-PADRE, and immunizing dose is 50ug/.The 4th group is that model induces immunity on the same day to be used to study the therapeutic effect of vaccine.Immunity every other week once is total to immunity four times.

3.5.5.3 the inducing and treating of model

Back 10 days of immunity, every root of the tail portion of first group of mice intradermal injection 100 μ L normal saline; Its every root of the tail portion of excess-three group mice intradermal injection 100 μ L derivants.Induce after 21 days every root of the tail portion of first group of mice Intradermal to inject 50 μ L normal saline once more; Its every root of the tail portion of excess-three group mice Intradermal is injected the identical derivant of 50 μ L once more.

Mouse invasion rate statistics: induce the back promptly to begin to add up the sickness rate of the inductive mice rheumatoid arthritis of II Collagen Type VI next day for the second time.The result is as shown in Figure 8, compares the sickness rate that TNF-PADRE can reduce the inductive mice rheumatoid arthritis of II Collagen Type VI with contrast.

Mouse arthritis pathological changes standards of grading: promptly begin to observe front and back foot swelling degree and scoring next day after inducing for the second time, is the acute arthritis mark with every Mus front and back arthropathy scoring.Arthritis index (arthritis index, AI) standards of grading are following: 0 minute-do not have red and swollen; 1 minute-joint is swollen slightly; 2 minutes-joint is slightly red, swollen, hot; 3 minutes-joint moderate is red, swollen, hot, slight dysfunction; 4 minutes-joint severe is red, swollen, hot, and severe dysfunction can't be born a heavy burden late deformity.The score of 4 ankle joint of accumulation is the joint scoring of every mice.Two tail T check check group differences.The result is as shown in Figure 8, compares the mouse arthritis pathological changes scoring that TNF-PADRE can reduce the inductive mice rheumatoid arthritis of II Collagen Type VI with contrast.

4. effect of the present invention

Following through experiment confirm effect of the present invention:

(1) screens and made up people TNF-PADRE amalgamation protein vaccine.

(2) this vaccine has kept the immunogenicity of TNF, has removed the natural bioactive of TNF.

(3) obtained the purifying process of this vaccine.

(4) confirm that through zoopery this fusion rotein can induce high titre antibody in the mice body.

(5) proved that tentatively this albumen can alleviate the symptom of the inductive cachexia mouse model of TNF to a certain extent as tumor vaccine, alleviates the symptom of the inductive rheumatoid arthritis mice of two Collagen Type VIs.

Description of drawings

Fig. 1 a is the mRNA structure (the part labyrinth amplifies) of natural TNF.

Fig. 1 b is the mRNA structure of introducing after two point mutation are optimized.

Fig. 2 a is PADRE-hTNF _142-157Gene is synthetic, and wherein, " 1 " is the cloning vehicle cleavage map, and small fragment wherein is an aim sequence, and " 2 " are DNAMarker.

Fig. 2 b is hTNF _1-128Amplification figure, wherein, " 1 " is DL2000DNA marker, and " 2 " are negative controls, and " 3 " are hTNF _1-128Positive amplified band (shown in the arrow).

Fig. 3 is pET22b-hTNF-TT/HEL/PADRE expression vector plasmid enzyme restriction figure, and wherein, " 1 " is DL2000DNAmarker, and " 2 "～" 4 " are pET22b-hTNF-TT/HEL/PADRE plasmid Nde I and Sal I enzyme action result.

Fig. 4 is pET22b-hTNF-TT/BL21; PET22b-hTNF-HEL/BL21 and pET22b-hTNF-PADRE/BL21 abduction delivering result; Wherein, " 1 ", " 3 ", " 5 " are that not induce bacterial protein, " 2 ", " 4 " be that pET22b-hTNF-TT/BL21 and pET22b-hTNF-HEL/BL21 induce, and " 6 "～" 8 " are that pET22b-hTNF-PADRE/BL21 induced 1～3 hour.

Fig. 5 is the purification result of hTNF-PADRE, and wherein, " 1 ", " 3 ", " 5 " swimming lane are the later result of inclusion body washing, and " 2 ", " 4 ", " 6 " swimming lane are result behind the purification.

Fig. 6 is that the Western-blot of hTNF-TT, hTNF-HEL, hTNF-PADRE purifying protein identifies that wherein, " M " is protein standard molecular weight marker, and " 1 ", " 2 ", " 3 " are that hTNF-TT/HEL/PADRE induces the back bacterial protein.

Fig. 7 is that the remaining natural bioactive of hTNF-TT, hTNF-HEL, hTNF-PADRE is identified.

Fig. 8 is the protective effect of hTNF-PADRE to the dyscrasia model mice.

Fig. 9 is the protective effect of hTNF-PADRE autovaccine molecule to the inductive rheumatoid arthritis mice of two Collagen Type VIs; Wherein, TNF-PADRE1 is the vaccine effect of the prevention RA of immunity in advance, and TNF-PADRE2 is the therapeutic effect that after inducing the RA model, gives vaccine.

Below in conjunction with accompanying drawing and embodiment the present invention is made further detailed description.

The specific embodiment

According to the people TNF therapeutic autovaccine of technical scheme preparation of the present invention, with coding TT _830-844(QYIKANSKFIGITEL), HEL _46-61(NTDGSTDYGILQINSR) and 129 nucleotide sequences of the amino acid whose nucleotide sequence of PADRE (AKFVAAWTLKA) replacement humanTNF-to 141 amino acids; Thereby obtain hTNF-TT, hTNF-HEL, hTNF-PADRE fusion rotein; Immune mouse behind the purification; Relatively these three kinds of molecules induce to the titre of hTNF-Alpha antibodies with in the ability of hTNF-alpha active, filter out hTNF-PADRE and have best immune effect.Not only can reduce alleviating of the inductive cachexia mice of natural TNF-α body weight with hTNF-PADRE recombiant protein immune mouse, can also prolong the life cycle of the inductive cachexia mice of mTNF-α simultaneously; Prolong the life cycle of the inductive acute lung injury mice of LPS; Alleviate the symptom of the inductive rheumatoid arthritis of II Collagen Type VI.Secondly thereby recombined human TNF-PADRE fusion rotein might produce anti-humanTNF-'s autoantibody in human body, for the immunization therapy of the too high relevant disease of TNF-alpha expression provides a kind of new pharmaceutical grade protein.

Realize the screening of vaccine of the present invention, vaccine construction and purification, immune animal, antibody calibrating and zoopery work, accomplish according to the following steps:

1. the preparation of vaccine and screening

We are model with the mice in the work in early stage, have screened the on position in mice TNF-alpha molecule of the auxiliary epi-position of external source T cell to the TNF-alpha molecule of mice, and screening is than the auxiliary epi-position of right different T cell.The result finds the on position of mice TNF-alpha molecule 128-141 amino acids for best exogenous molecules, and PADRE is for the most effectively inserting epi-position.We use coding TT to above research basis _830-844(QYIKANSKFIGITEL), HEL _46-61(NTDGSTDYGILQINSR) and 129 nucleotide sequences of the amino acid whose nucleotide sequence of PADRE (AKFVAAWTLKA) replacement humanTNF-to 141 amino acids; And (Fig. 1 a is the mRNA structure of natural TNF to introduce two point mutation at 439,440 of people's tnf gene; Fig. 1 b is the mRNA structure of introducing after two point mutation are optimized); Thereby obtain hTNF-TT, hTNF-HEL, hTNF-PADRE fusion rotein, concrete grammar is following:

1.1pET22b-TNF-TT, the structure of TNF-HEL, TNF-PADRE/BL21 engineering bacteria

1.1.1TNF-TT, the amplification of TNF-HEL, TNF-PADRE gene and synthetic

The epitope peptide domain of and hTNF-α truncate expression analysis hTNF-α auxiliary according to computer software finally is template with humanTNF-, amplification TNF-α _1-128The coded sequence of amino acids; Synthetic TT _830-844-, HEL _46-61-, PADRE-(substitutes TNF-α _129-141) and TNF-α _142-157Genetic fragment is introduced Mun I restriction enzyme site (making the 129-141 amino acids of TNF-α not introduce any exogenous amino acid outside being substituted by PADRE etc.) between amplified fragments and the synthetic fragment.Recombiant vaccine fusion gene (the TNF-α that obtains at last _1-128-PADRE-TNF-α _142-157) be cloned between the Nde I and Sal I MCS of prokaryotic expression carrier pET22b.Because the mRNA secondary structure more complicated after wild type gene is transcribed; Be unfavorable for the expression of recombiant vaccine; So we transcribe the secondary structure of back mRNA through analysis; A samesense mutation (two point mutation T439 → A, C440 → G), make the mRNA structure of genes of interest more be tending towards simplifying and low-energy state (Fig. 1 a is the mRNA structure of natural TNF, and Fig. 1 b introduces two mRNA structures after the point mutation optimizations) have been introduced.Gene order is seen 1.1.4.

1.1.2BL21 the preparation of competent cell

Get in the LB culture medium that BL21 glycerol strain inoculates in 1: 100 ratio, 37 ℃ of shaken cultivation are spent the night, and transfer once next day, continues to be cultured to OD ₆₀₀About about 0.4.(sterile working) with bacterium liquid ice bath 10min, centrifugal (3000rpm * 5min, 4 ℃) are supernatant discarded afterwards, adds the 100mmol/L CaCl of 1/2 volume pre-cooling ₂, blow afloat deposition gently, ice bath 40min, centrifugal (3000rpm * 5min, 4 ℃) are abandoned the 100mmol/L CaCl that contains 25% glycerol that adds 1/25 volume behind the supernatant ₂, blow afloat deposition, divide in the Eppendorf pipe of packing into ,-70 ℃ of preservations are subsequent use.

1.1.3 the conversion of competent cell, cultivation

Competent escherichia coli cell is taken out from-70 ℃, and ice bath melted 5-10 minute, added the pET22b contain genes of interest, and slight mixing continues ice bath 30min, 42 ℃ of heat shock 45s then, ice bath 5min more at last.Bed board then, 37 ℃ of overnight incubation.

1.1.4 the extraction of plasmid and evaluation

Transform picking neat in edge on the culture dish of back 37 ℃ of incubated overnight at plasmid; The clone that growth conditions is good; Be inoculated into 10ml and contain in the LB culture medium of Amp, 37 ℃, 200rpm are cultivated 8h, extract plasmid with plasmid extraction kit; Behind Nde I and Sal I double digestion; Whether row agarose gel electrophoresis is observed has the segment of inserting segment and insertion whether consistent with the length of expection (Fig. 2 a, Fig. 2 b is the structure of plasmid, in order to obtain destination protein hTNF-PADRE, at first synthesize PADRE and hTNF according to the escherichia coli preference codon _142-156The coded sequence of amino acids<fig. 2 a>, synthetic primer amplification hTNF then _1-128The coded sequence of amino acids<fig. 2 b>Two fragment clonings are gone into the expression plasmid that the pET22b carrier obtains recombiant vaccine.Fig. 3 is the restriction enzyme mapping of protokaryon non-fusion expression plasmid pET22b-hTNF-PADRE.Two fragments among Fig. 2 a, Fig. 2 b are connected with the pET22b carrier of process Nde I and Sal I enzyme action, and the picking positive colony extracts plasmid behind the conversion BL21 competent cell.Recombiant plasmid identifies that through restricted enzyme Nde I and Sal I enzyme action it is consistent with the expection size to obtain corresponding segment), enzyme action is identified that male clone serves Hai Boya company and carries out dna sequencing.Corresponding positive engineering bacteria is called after pET22b-hTNF-TT/BL21, pET22b-hTNF-HEL/BL21 and pET22b-hTNF-PADRE/BL21 respectively.

The gene order of total length people TNF-TT (the line part is the nucleotide sequence of TT epi-position, and thickened portion is respectively NdeI, Mun I and Nde I restriction enzyme site)

5’- CATATGTGGTCAGATCATCTTCTCGAACCCCGAGTGACAAGCCTGTAGCCCATGTTGTAGCAAA

CCCTCAAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTCCTGGCCAATGG

CGTGGAGCTGAGAGATAACCAGCTGGTGGTGCCATCAGAGGGCCTGTACCTCATCTACTCCCAG

GTCCTCTTCAAGGGCCAAGGCTGCCCCTCCACCCATGTGCTCCTCACCCACACCATCAGCCGCA

TCGCCGTCTCCTACCAGACCAAGGTCAACCTCCTCTCTGCCATCAAGAGCCCCTGCCAGAGGGA

GACCCCAGAGGGGGCTGAGGCCAAGCCCTGGTATGAGCCCATCTATCTGGGAGGGGTCTTCCA

GCTGCAATTG CAGTACATCAAAGCTAACTCCAAATTCATCGGTATCACTCGAACTTCTC

GACTTTGCCGAGAGTGGGCAGGTCTACTTTGGGATCATTGCCCTGTGAGTCGAC-3’

The gene order of total length people TNF-HEL is (the line part is that the nucleotide sequence of HEL epi-position, thickened portion are respectively Nde I, Mun I and Nde I restriction enzyme site)

5’- CATATGGTCAGATCATCTTCTCGAACCCCGAGTGACAAGCCTGTAGCCCATGTTGTAGCAAA

CCCTCAAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTCCTGGCCAATGG

CGTGGAGCTGAGAGATAACCAGCTGGTGGTGCCATCAGAGGGCCTGTACCTCATCTACTCCCAG

GTCCTCTTCAAGGGCCAAGGCTGCCCCTCCACCCATGTGCTCCTCACCCACACCATCAGCCGCA

TCGCCGTCTCCTACCAGACCAAGGTCAACCTCCTCTCTGCCATCAAGAGCCCCTGCCAGAGGGA

GACCCCAGAGGGGGCTGAGGCCAAGCCCTGGTATGAGCCCATCTATCTGGGAGGGGTCTTCCA

GCTGCAATTG AACACTGACGGTTCCACCGATTACGGCATTCTGCAGATCAACTCCCG

CCTCGACTTTGCCGAGAGTGGGCAGGTCTACTTTGGGATCATTGCCCTGTGAGTCGAC-3’

The gene order of total length people TNF-PADRE is (the line part is that the nucleotide sequence of PADRE epi-position, thickened portion are respectively Nde I, Mun I and Nde I restriction enzyme site)

5’- CATATGGTCAGATCATCTTCTCGAACCCCGAGTGACAAGCCTGTAGCCCATGTTGTAGCAAA

CCCTCAAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTCCTGGCCAATGG

CGTGGAGCTGAGAGATAACCAGCTGGTGGTGCCATCAGAGGGCCTGTACCTCATCTACTCCCAG

GTCCTCTTCAAGGGCCAAGGCTGCCCCTCCACCCATGTGCTCCTCACCCACACCATCAGCCGCA

TCGCCGTCTCCTACCAGACCAAGGTCAACCTCCTCTCTGCCATCAAGAGCCCCTGCCAGAGGGA

GACCCCAGAGGGGGCTGAGGCCAAGCCCTGGTATGAGCCCATCTATCTGGGAGGGGTCTTCCA

GCTGCAATTG GCTAAATTCGTTGCTGCATGGACTCTGAAAGCTGCAGCTCTCGACTTTGCCGAG

AGTGGGCAGGTCTACTTTGGGATCATTGCCCTGTGA GTCGAC-3’

1.2 recombiant vaccine protein expression, purification and evaluation

1.2.1pET-22b-PADRE-hTNF the abduction delivering of α/BL21 engineering bacteria

The e. coli bl21 list colony inoculation that contains recombinant expression plasmid is in the fresh LB culture fluid of 5ml (containing ampicillin 100mg/L); 37 ℃ of overnight incubation of shaking table; Next day, the ratio with 1: 100 was inoculated in the fresh LB culture fluid (containing ampicillin 100mg/L), and 37 ℃ are continued to be cultured to bacterial density and reach OD ₆₀₀=0.4～0.6; Add 1mMIPTG; Difference 1h, 2h, 3h, 4h collect thalline behind the abduction delivering, to confirm suitable induction time, cultivate and induce the centrifugal collection thalline in back on a small scale; With proteic expression of SDS-PAGE testing goal and existence form, the result is illustrated in the theoretical molecular position has nascent protein band (Fig. 4).

1.2.2pET-22b-PADRE-hTNF the fermentation of α/BL21 engineering bacteria

Select single colony inoculation in the test tube that contains LB from activatory LB flat board, cultivate 10h for 37 ℃, change then and plant to the triangular flask that contains 200ml LB, 37 ℃ of overnight incubation.Next day, seed liquor is inoculated in the 5L fermentation tank.37 ℃ of fermentation temperatures of control, rotating speed 250～450rpm/min, ventilation 3～5L/min, pH7.3.Be cultured to OD ₆₀₀During=8 left and right sides, add 1mM IPTG and induce, continue to cultivate 4h, stop fermentation, collect thalline, weigh ,-20 ℃ of refrigerators preserve subsequent use.After the thalline that takes a morsel is handled, with the proteic expression of SDS-PAGE testing goal.The result shows that protein is with the inclusion body formal representation.

1.2.3 the purification and the renaturation of reorganization inclusion body protein

Get 10g fermentation thalline,, thalline thoroughly is resuspended in 100mM Tris-HCl (7.0), among the 1mM EDTA, add the 1.5mg/mL lysozyme, 10ug/mL DNaseI, 20mM MgCl in the ratio of 1g weight in wet base to 7ml ₂Hang and carry out cracking; Add 1.5M NaCl, 2%Triton X-100,20mM EDTA to 3 times of first volumes, room temperature washing 30min uses the 4M Urea of 3 times of volumes after centrifugal more respectively; 2%Triton X-100,20mM EDTA, 0.1M Tris; With the 20mM EDTA of 8 times of volumes, 0.1M Tris washing.The inclusion body of warp washing is dissolved in 50mmol/LTris.Cl pH 7.0, the 5mmol/L beta-mercaptoethanol with the ratio of 1g weight in wet base to 10ml; 1mmol/L EDTA, in the buffer of 7mol/L guanidine hydrochloride, 4 ℃ of stirred overnight; The centrifugal 30min of 12000r/min collects supernatant, is the destination protein crude extract.(gel chromatography of 1.6cm * 100cm) is with working solution (7mol/L guanidine hydrochloride, 50mmol/L Tris.Cl pH 7.0 for Sephacryl S-100; 1mmol/L EDTA, 100mmol/L NaCl) fully after the balance, destination protein crude extract 2ml upper prop; With the working solution eluting, flow velocity 1ml/min, fraction collection; Measure protein content, and confirm the destination protein position, collect the higher elution fraction of purity with SDS-PAGE.Purification destination protein component is carried out gradient dialysis renaturation to carbamide, and gradient does, 4M carbamide, and 2M carbamide, 1M carbamide carries out dialysis distilled water at last, the centrifugal supernatant that stays, lyophilizing.SDS-PAGE and HPLC detect refolded protein purity (Fig. 5).

2. the evaluation of recombinant protein

2.1 the evaluation of immunogenicity (Western blot) and purity (HPLC)

The purified proteins sample is through 15% PAGE; Pull down gel after electrophoresis finishes, according to the Bio-Rad description of product, with gel near negative electrode one side, pvdf membrane near anode one side; Transfering buffering liquid (25mmol/L Tris-HCl in pre-cooling; 192mmol/L Glycine, 20% methanol) middle 100V constant voltage transfer 1h, albumen is transferred on the pvdf membrane.After shifting end, take out pvdf membrane, (20mmol/L TrisHCl pH7.5,150mmol/LNaCl 0.05%Tween20) clean the back and immerse the middle incubated at room 1h of confining liquid (TBST that contains 2%BSA) cleaning mixture TBST.With one anti-(mouse anti human TNF-alpha monoclonal antibodies, dilution in 1: 5000) incubated at room 1h, TBST washes film 3 times, each 5min; With two anti-(goat anti-mouse IgG-HRP, dilution in 1: 2000) incubated at room 30min, TBST washes film 3 times again, each 5min, and TBS (not containing Tween20) washes film 3 times, each 5min.At last with the colour developing of ECL system, distilled water flushing cessation reaction.The result shows hTNF-TT, and what hTNF-HEL, hTNF-PADRE can be special combines (Fig. 6) with mouse anti human TNF-alpha monoclonal antibodies.

TNF-TT, TNF-HEL, TNF-PADRE that purification is obtained carry out the purity evaluation.Chromatographic column is exclusion chromatography post (7.5mm * 300mm G2000SW column (TOSOH)), and mobile phase is PBS, and flow velocity is 0.5ml/min, and the detection wavelength is 280nm.Purity is all greater than 95% as a result.

2.2 residual natural TNF-α cell killing activity experiment

The mice L929 cell that growth conditions is good transfers to 10 ⁶The cell suspension of/ml, every hole adds 100 μ l cell suspension, 50ml/lCO in 96 well culture plates ₂, 37 ℃ of overnight incubation, abandon supernatant, every hole adds the TNF-TT after the renaturation of serial dilution, 5%CO ₂, 37 ℃ cultivate 16h, abandon supernatant, the concentration that every hole adds 30 μ L is 500 μ g/mL violet staining 3-5min, carefully washes away crystal violet with flowing water, dries residual moisture, every hole adds destaining solution 100 μ L, ELIASA 570nm place mensuration light absorption value.The humanTNF-is as positive control, and the GST albumen of purification is as negative control.The result shows that TNF-TT, TNF-HEL, TNF-PADRE all do not have natural humanTNF-'s cytotoxicity (Fig. 7).

3. animal immune

3.1Balb/c mice group and immunization protocol and antiserum ELISA identify

The preparation of required solution:

Encapsulate buffer: carbonate buffer solution

Na ₂CO ₃(0.32g final concentration 0.015mol/L)

NaHCO ₃(0.59g final concentration 0.035mol/L)

Pure water is settled to 200ml (pH 9.6) effect duration: two weeks

Cleaning mixture: the PBS (pH 7.4) that contains 0.05%Tween-20

Confining liquid: the cleaning mixture that contains 1%BSA

Diluent: the cleaning mixture that contains 0.5%BSA

Substrate buffer solution: Na ₂HPO ₄.12H ₂O 1.84g

Citric acid 0.51g

Pure water is settled to 100ml (pH 5.0)

Substrate colour developing liquid: OPD 8mg

30％H ₂O ₂ 30μl

Substrate buffer solution 10ml

Stop buffer: H ₂SO ₄1mol/L

Operational approach:

Encapsulate: get 96 hole elisa plates, antigen is dissolved in encapsulates (2-5 μ g/ml) in the buffer, add in the plate hole (100 μ l/ hole), 4 ℃ encapsulate and spend the night.→ outwell coating buffer, add confining liquid, 37 ℃ of sealing 2h; → discard confining liquid, wash 6 times each 2min with cleaning mixture; → adding the antiserum (from 100 times) of 10 times of doubling dilutions, 1h is incubated for 37 ℃ in 100 μ l/ holes; → discard antiserum, wash 6 times each 2min with cleaning mixture; Two of the anti-mice of → adding HRP labelling resists, and 1h is hatched for 37 ℃ in 100 μ l/ holes; → discard two to resist, wash 6 times each 2min with cleaning mixture; → add substrate colour developing liquid, 100 μ l/ holes, 37 ℃, 10-20min develops the color; → adding stop buffer, 100 μ l/ holes, cessation reaction; → ELIASA reading is measured the light absorption value of every hole at 490nm.

3.2TNF-PADRE treatment to the inductive cachexia model of TNF-α

3.2.1 divide into groups and immune mouse

3.2.2 inducing of model

Mice immunized is got 10 by every group of body weight, body weight 27-30g, and body weight no difference of science of statistics between group, every day, intraperitoneal injection of mice TNF-α 2 * 10 ⁵IU/ only compares with the normal saline group life cycle (Fig. 8) of statistics mice, and this recombiant vaccine has significant protective effect for the dyscrasia model of the inductive DBA mice of TNF.The body weight of mice does not have significance to change (negative control group loses weight); Survival rate obviously is superior to the normal saline group.

3.3TNF-PADRE treatment to the inductive rheumatoid arthritis mouse model of II Collagen Type VI

3.3.1 the preparation of derivant

3.3.2 the immunity of animal

3.3.3 the inducing and treating of model

Mouse invasion rate statistics: induce the back promptly to begin to add up the sickness rate of the inductive mice rheumatoid arthritis of II Collagen Type VI next day for the second time.The result is as shown in Figure 9, compares the sickness rate that TNF-PADRE can reduce the inductive mice rheumatoid arthritis of II Collagen Type VI with contrast.

Mouse arthritis pathological changes standards of grading: promptly begin to observe front and back foot swelling degree and scoring (Fig. 9) next day after inducing for the second time, is the acute arthritis mark with every Mus front and back arthropathy scoring.Arthritis index (arthritis index, AI) standards of grading are following: 0 minute-do not have red and swollen; 1 minute-joint is swollen slightly; 2 minutes-joint is slightly red, swollen, hot; 3 minutes-joint moderate is red, swollen, hot, slight dysfunction; 4 minutes-joint severe is red, swollen, hot, and severe dysfunction can't be born a heavy burden late deformity.The score of 4 ankle joint of accumulation is the joint scoring of every mice.Two tail T check check group differences.The result is as shown in Figure 9, compares the mouse arthritis pathological changes scoring that TNF-PADRE can reduce the inductive mice rheumatoid arthritis of II Collagen Type VI with contrast.

Claims

1. induce construction method in the body, it is characterized in that, fusion gene (TNF-α to the protein vaccine of the autoantibody of humanTNF-'s molecule _1-128-PADRE-TNF-α _142-157) be cloned between the Nde I and Sal I MCS of prokaryotic expression carrier pET22b; With the recombinant expression carrier transformed into escherichia coli BL21 that obtains, inducing the back expression product is inclusion body, washs through inclusion body; Gel filtration chromatography; The dialysis renaturation can obtain the purpose fusion rotein of purification, with inducing the protein vaccine to the autoantibody of humanTNF-'s molecule in this a kind of body; Immune mouse can produce the anti-humanTNF-'s autoantibody of high titre under the situation of not using adjuvant, and wherein said fusion gene sequence is:

5’- CATATGGTCAGATCATCTTCTCGAACCCCGAGTGACAAGCCTGTAGCCCATGTTGTAGCAAA

CCCTCAAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTCCTGGCCAATGG

CGTGGAGCTGAGAGATAACCAGCTGGTGGTGCCATCAGAGGGCCTGTACCTCATCTACTCCCAG

GTCCTCTTCAAGGGCCAAGGCTGCCCCTCCACCCATGTGCTCCTCACCCACACCATCAGCCGCA

TCGCCGTCTCCTACCAGACCAAGGTCAACCTCCTCTCTGCCATCAAGAGCCCCTGCCAGAGGGA

GACCCCAGAGGGGGCTGAGGCCAAGCCCTGGTATGAGCCCATCTATCTGGGAGGGGTCTTCCA

GCTGCAATTG GCTAAATTCGTTGGTGCATGGACTCTGAAAGCTGCAGCTCTCGACTTTGCCGAG

AGTGGGCAGGTCTACTTTGGGATCATTGCCCTGTG AGTCGAC-3’

2. the method for claim 1 is characterized in that, the structure of its gene is accomplished according to the following steps:

5’- CATATGGTCAGATCATCTTCTCGAACCCCGAGTGACAAGCCTGTAGCCCATGTTGTAGCAAA

Nde?I

CCCTCAAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTCCTGGCCAATGG

CGTGGAGCTGAGAGATAACCAGCTGGTGGTGCCATCAGAGGGCCTGTACCTCATCTACTCCCAG

GTCCTCTTCAAGGGCCAAGGCTGCCCCTCCACCCATGTGCTCCTCACCCACACCATCAGCCGCA

TCGCCGTCTCCTACCAGACCAAGGTCAACCTCCTCTCTGCCATCAAGAGCCCCTGCCAGAGGGA

GACCCCAGAGGGGGCTGAGGCCAAGCCCTGGTATGAGCCCATCTATCTGGGAGGGGTCTTCCA

GCTGCAATTG GCTAAATTCGTTGCTGCATGGACTCTGAAAGCTGCAGCTCTCGACTTTGCCGAG

MunI PADRE

AGTGGGCAGGTCTACTTTGGGATCATTGCCCTGTGA GTCGAC-3’。

Mutational site Sal I

3. the method for claim 1 is characterized in that, by the protein expression of the said gene constructed engineering bacteria of claim 2; Accomplish according to the following steps: picking engineering bacteria list colony inoculation is in the fresh LB culture medium of 5mL; Described LB culture fluid contains Amp100mg/L, 37 ℃ of shaking table overnight incubation, and next day, the ratio with 1: 100 was transferred in the LB culture fluid once more; Cultivate 3h at 37 ℃ of shaking tables, to logarithmic growth OD in mid-term ₆₀₀Be 0.4～0.6, add 1mM IPTG and induce, continue to cultivate 4h, centrifugal collection thalline.

4. the method for claim 1 is characterized in that, by the purification renaturation of claim 3 expressed fusion protein, accomplishes according to the following steps:

Get 10g fermentation thalline, in the ratio of 1g weight in wet base to 7ml, thalline thoroughly is resuspended in 100mM Tris-HCl (7.0), among the 1mM EDTA, add the 1.5mg/mL lysozyme, 10ug/mL DNaseI, 20mM MgCl2 hang and carry out cracking; Add 1.5M NaCl, 2% Triton X-100,20mM EDTA to 3 times of first volumes, room temperature washing 30min uses the 4M Urea of 3 times of volumes after centrifugal more respectively; 2% Triton X-100,20mM EDTA, 0.1M Tris; With the 20mM EDTA of 8 times of volumes, 0.1M Tris washing; The inclusion body of warp washing is dissolved in 50mmol/LTris.Cl pH 7.0, the 5mmol/L beta-mercaptoethanol with the ratio of 1g weight in wet base to 10ml; 1mmol/L EDTA, in the buffer of 7mol/L guanidine hydrochloride, 4 ℃ of stirred overnight; The centrifugal 30min of 12000r/min collects supernatant, is the destination protein crude extract; (gel chromatography of 1.6cm * 100cm) is with working solution (7mol/L guanidine hydrochloride, 50mmol/L Tris.Cl pH 7.0 for Sephacryl S-100; 1mmol/L EDTA, 100mmol/L NaCl) fully after the balance, destination protein crude extract 2ml upper prop; With the working solution eluting, flow velocity 1ml/min, fraction collection; Measure protein content, and confirm the destination protein position, collect the higher elution fraction of purity with SDS-PAGE; Purification destination protein component is carried out gradient dialysis renaturation to carbamide, and gradient does, 4M carbamide, and 2M carbamide, 1M carbamide carries out dialysis distilled water at last, the centrifugal supernatant that stays, lyophilizing; HPLC detects refolded protein purity.

5. the method for claim 1 is characterized in that, by the fusion rotein that the said method of claim 4 is obtained, immune mouse can produce the anti-humanTNF-of high titre self neutrality polyclonal antibody under the situation of not using adjuvant.This antibody has excellent protection and therapeutical effect for mouse models such as the inductive rheumatoid arthritis of II Collagen Type VI, the inductive dyscrasia of LPS.The antibody that this vaccine produces is expected to be used for prevention and treatment rheumatoid arthritis and dyscrasia etc.