CN102370979B - Building method for autovaccine by aiming at human TNF(Tumor Necrosis Factor)-alpha molecule - Google Patents

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 of the autovaccine for humanTNF-α'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 for the protein vaccine of humanTNF-α's molecule autoantibody.

Background technology

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

1.1TNF-alpha molecule

The discoveries such as Carswell in 1975, in the mice serum that bacillus calmette-guerin vaccine and bacterial endotoxin infect a kind of highly active tumor cytotoxicity factor is arranged, can make tumor generation hemorrhagic necrosis, but the normal tissue cell is without killing activity, with its called after tumor necrosis factor---TNF-α.HumanTNF-α'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 comprised of 232 aminoacid, contains 76 amino acid whose signal peptides.The mice gene is positioned at chromosome No. 17, is comprised of 4 exons and 3 introns, and precursor is comprised of 235 aminoacid.People and mice TNF-α precursor have 79% homology, and the adult form humanTNF-α is comprised 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 (people 69,101; Mus 69,100), can form intramolecular disulfide bond.The TNF-α of separation and purification from cell strain culture supernatant molecular size range under non-reduced condition does not wait, and molecular weight is 17KDa under reducing condition, and is consistent with theoretical molecular.This may 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 comprised 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 the several 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) brings into play cytotoxicity by being combined with the surface receptor of tumor cell TNF-α.Known receptor has two kinds at present, and TNFR I and TNFRII are transmembrane glycoprotein.TNFR I is by the apoptosis of dead plot structure territory (death domain, DD) the mediation tumor cell of about 80 amino acid residues of its cytoplasmic domain.

(2) by 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 the ischemia of tumor tissues and necrosis.

(3) kill tumor effector lymphocyte's effect and the inflammatory reaction of induced tumor part by mediation, promote to kill tumor effector lymphocyte's effect.

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 to viruses such as VSV, HSV, but HIV had facilitation.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 monocytes adhesion on 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 TNF alpha antibody treatment

Research finds, TNF-α continues in vivo, play an important role in various diseases 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 for the treatment of the overexpression relevant disease of TNF-α in the body mainly contains: nonsteroidal anti-inflammatory agent (NSAIDS), the antirheumatic (DMARD) of alleviation 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 obvious toxic and side effects.

At present research 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.First three is planted medicine and ratifies by FDA.Etanercept is the fusion rotein of gene recombinaton sTNFR75 and human IgG Fc section, can suppress simultaneously TNF-α and TNF-β, and be used for the treatment of teenager RA and psoitis by the FDA approval in November, 1998.Infliximab is TNF-α neutrality people Mus chimeric antibody, is used for treating RA by FDA approval in 1999, after be used for again treating the Crohn disease.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 TNF alpha antibody obtained preferably curative effect at clinical treatment RA.The RA treatment updated Guidelines of Americanism damp disease association issue in 2002 points out that early stage RA patient and the patient who uses DMARDS to fail to respond to any medical treatment are effective to etanercept.Use the out of contior RA patient who carries out sexual activity of capacity methotrexate, etanercept and infliximab unite and use effectively, recommend at present to use separately 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.

Although TNF-alpha monoclonal antibodies and soluble recepter have preferably curative effect clinically,, these medicines have dosage large, need prolonged and repeated use, easily 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, the monoclonal antibody for mankind itself's albumen has demonstrated good effect in the process of the acute and chronic disease for the treatment of.But, the costliness of cost and the inconvenience of using have limited the extensive use of monoclonal antibody, therefore, turn to the active immunity vaccine of seeking for mankind itself's albumen by accepting passively this antibody protein, namely the therapeutic modality with active immunity substitutes passive immunity, becomes the developing direction of protein drug.At present, the research of therapeutic vaccine has become a focus, relates to a lot of diseases, such as chronic viral infection, tumor, Alzheimer, diabetes, hypertension, obesity and rheumatic arthritis etc.

Therapeutic vaccine makes people's immune system be conducive to reaction.Most vaccine can be divided into two large classes: a class is to induce the immunoreation of body artificial body for generating liquid, produces antibody; Another kind of is to induce body generation cell immune response, produces cytotoxic T cell (CTLs).A rear class therapeutic vaccine is mainly used in the treatment of tumor and disease of viral infection.

Most preventative vaccines all are to protect body by the generation of inducing antibody, and this just proves by inducing antibody to treat infectious disease is a kind of effective Therapeutic Method.Compare with preventative vaccine, the development of therapeutic vaccine is relatively slow, but monoclonal antibody was indicating in obtained immense success 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 for angiotensin can be treated hypertension; Vaccine for IL-9 can be treated Eosinophilia's disease; Vaccine for IL-5 can be treated asthma; Vaccine for N-methyl-D-aspartate receptor-1 (NMDAR1) can be treated apoplexy.In addition, the immunity for some gonadal hormone such as human chorionic gonadotropin (human chorionic gonadotro-pin HCG) can reduce the hormonal readiness in the human female and reach contraceptive effect; Vaccine for gonadotropin-releasing hormone (GnRH) can be used for the treatment of advanced prostate cancer; In the Patients with Pancreatic Cancer, the antibody that utilizes therapeutic vaccine to induce for 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 for the therapeutic vaccine of Alzheimer recently.Alzheimer is a kind of disease for the treatment of with therapeutic vaccine of seeming to be fit to very much, and the pathogenic process of this disease reaches several years even many decades.If can induce long-term antibody with therapeutic vaccine, be undoubtedly so a kind of desirable Therapeutic Method, especially this patient often forgets and takes medicine.

The feature 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 the precursor protein (amyloid precursor protein, APP) from amyloid derives.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 occured 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 Ahl tribulus sea silent sickness 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 for Alzheimer has begun clinical trial, after it has good toleration 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 forced to stop to test.Studies confirm that subsequently it doesn't matter for the titre of this side effect and antibody, in fact, a tested patient 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 are consistent with a kind of hypothesis, and that side effect of finding in patient 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 for 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 for the therapeutic vaccine of Alzheimer.

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

2.2 induce the theoretical research of autoantibody

Is therapeutic vaccine for oneself protein how to induce the generation of autoimmune system for the antibody of 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, induce antibody in the situation that does not have adjuvant and antigen durative action preparation.As everyone knows, the human immune system is mainly to external invader's offensive attack, and body itself is not attacked, and this may be because body has the ability that can identify " 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 occured, normal B cell strain but existed in vivo.In fact, have three kinds of mechanism to cause immunologic tolerance: cell strain is rejected, and namely specific lymphocyte is thoroughly rejected from lymphocyte populations; Immunity is incompetent, and namely specific lymphocyte exists, but its function can not be activated; Immunity is ignored, and the lymphocyte that namely 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, for easier the illustrating of the immunologic tolerance of enriching antigen of generally expressing.

In the immunoreation for exotic antigen, T cell and B cell are worked in coordination and could effectively be produced antibody: when being subject to the exotic antigen immunity, specific B Cell binding 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, dendritic cell is absolutely necessary, the dendritic cell antigen uptaking, and at the complex of its cell surface antigen-presenting peptide and MHC II quasi-molecule, activate the TH cell.The antigenic peptides that TH cell recognition B cell surface after the activation presents and the complex of MHC II quasi-molecule cause the generation of B cell proliferation, antibody and the conversion of antibody isotype.If lack the synergism of TH cell because of immunologic tolerance, so just can not produce antibody.In the design process for the vaccine of oneself protein, if autoantigen merged with foreign protein or peptide carrier or be 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 peptide and MHC II quasi-molecule on its surface, because the TH cell does not have immunologic tolerance to carrier protein, so can be activated, thus collaborative from the specific antibody of the B of antigenic specificity cell generation for autoantigen.

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 be subject to 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 be not when having more than needed very much expression 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, be induced out for 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.The antiserum of the employment TNF purification Patients With Rheumatoid Arthritis such as Sioud, obtain having among the TNF-α and active TNF-α autoantibody, with this antibody screening phage random nonapeptide storehouse, the phage particle immune mouse that obtains with screening, brought out mouse-anti-human T NF-Alpha antibodies, studies show that recombinant phage can be used as the effective tool of vaccine research.The arthritis mouse model that the protein immunization Type Ⅱ collagen of the usefulness t helper cell epi-positions such as Dalum and TNF-alpha fusion expression is induced, the symptom of mice has obtained alleviation as a result, for a new way has been opened up in the treatment of rheumatoid arthritis and chronic infectious diseases.Blank etc. use the mice of the anti-phospholipid syndrome of dna immunization of coding TNF-α, in Mice Body, brought out anti-TNF-α antibody, these antibody can block the endothelial cell activity that TNF-α causes, suppress mononuclear cell to the sticking of the endotheliocyte of activation, compared the symptom that significantly to improve anti-phospholipid syndrome mice with matched group.Waterston etc. have compared TNF-alpha monoclonal antibodies and TNF-α autovaccine in the effect for the treatment of neoplasm metastasis, and the result shows the transfer of the minimizing lung tumor that both can be successful in Mice Body.Immunogenicity and the safety of TNF-α autovaccine that recent Waterston etc. have passed through the first phase clinical trial assessment, although unfortunately the serum antibody that brings out of vaccine can be identified humanTNF-α's molecule of degeneration, but but can not in and humanTNF-α's molecule of native state, analyzing reason may be because make the reason of antigen degeneration in the purge process of antigen.

More than studies show that, although TNF-α autovaccine also is the good method of the too high relevant disease of a kind of TNF-for the treatment of alpha expression, but, screening the auxiliary epi-position of effective T cell, suitable epi-position on position and suitable purification process all become the critical problem of TNF-α autovaccine research.

For above problem, we take mice as model, have screened the on position in mice TNF-alpha molecule of the auxiliary epi-position of external source T cell for the TNF-alpha molecule of mice in the work in early stage, and screening is than the auxiliary epi-position of right different T cell.Found that mice TNF-alpha molecule 128-140 amino acids is the on position of best exogenous molecules, PADRE is for the most effectively inserting epi-position.For above Research foundation, the present invention mainly for the autovaccine of its corresponding humanTNF-α's molecule make up, the research of expression, purification, be intended to obtain a kind of effectively for the autovaccine of humanTNF-α's molecule.

Summary of the invention

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

To achieve these goals, the technical solution adopted in the present invention is: induce the construction method for the protein vaccine of the autoantibody of humanTNF-α's molecule in a kind of body, it is characterized in that, with fusion gene (TNF-α _1-128-PADRE-TNF-α _142-157) be cloned between the Nde I and Sal I multiple clone site of prokaryotic expression carrier pET22b, the recombinant expression carrier that obtains is transformed e. coli bl21, inducing rear expression product is inclusion body, wash through inclusion body, gel filtration chromatography, dialysis renaturation, can obtain the purpose fusion rotein of purification, with the protein vaccine that induces in this a kind of body for the autoantibody of humanTNF-α's molecule, immune mouse can produce the anti-humanTNF-α's autoantibody of high titre in 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 finished according to the following steps:

According to humanTNF-α's aminoacid sequence and PADRE aminoacid sequence (AKFVAAWTLKA), design hTNF-PADRE gene order, the insertion point of PADRE is 128 back of TNF-α in the gene order, namely 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 by pcr amplification, and introduces Nde I restriction enzyme site (CATATG) in the upstream, wherein contains translation initiation codon; Mun I restriction enzyme site (CAATTG) is introduced in the downstream; Synthetic PADRE-TNF-α _142-157Coded sequence, upstream and downstream is introduced respectively Mun I and Sal I (GTCGAC) restriction enzyme site, gene has been carried out in addition two point mutation (T439 → A, C440 → G) in synthetic fragment;

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

Above-mentioned expression vector is transformed e. coli bl21, the plasmid called after pET22b-hTNF-PADRE that checks order correct, engineering bacteria called after pET22b-hTNF-PADRE/BL21; Its described gene order is as follows:

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 described gene constructed engineering bacteria, finish 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, next day, the ratio with 1: 100 was transferred in the LB culture fluid again, 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 rotein, finish according to the following steps:

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

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 MgCl 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 respectively the 4M Urea of 3 times of volumes after centrifugal again, 2%Triton X-100,20mM EDTA, 0.1M Tris, with the 20mM EDTA of 8 times of volumes, 0.1M Tris washing; Inclusion body through 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 stirrings are spent the night, the centrifugal 30min of 12000r/min collects supernatant, is the destination protein crude extract; Sephacryl S-100 (the gel chromatography of 1.6cm * 100cm), with working solution (7mol/L guanidine hydrochloride, 50mmol/L Tris.Cl pH 7.0,1mmol/L EDTA, 100mmol/L NaCl) after the abundant balance, destination protein crude extract 2ml upper prop, with the working solution eluting, flow velocity 1ml/min, fraction collection, measure protein content, and determine the destination protein position with SDS-PAGE, collect the higher elution fraction of purity; Purification destination protein component is carried out the gradient dialysis renaturation to carbamide, and gradient is, 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 described method is obtained, immune mouse can produce the anti-humanTNF-α of high titre self neutrality polyclonal antibody in the situation of not using adjuvant.The mouse models such as dyscrasia that the rheumatoid arthritis that this antibody is induced for the II Collagen Type VI, LPS induce have good protection and therapeutical effect.The antibody that this vaccine produces is expected to 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 _46-61The t helper cell epitope peptide such as (NTDGSTDYGILQ INSR) and PADRE (AKFVAAWTLKA) is replaced.

This vaccine can induce body to produce the antibody of the hTNF-alpha specific of high titre, this antiserum external can in and the humanTNF-α to the lethal effect of L929 cell.Can bring out the anti-humanTNF-α'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 preparation method of body polypeptide vaccine, the epitope peptide domain of and hTNF-α truncate expression analysis hTNF-α auxiliary according to computer software, and finally take the humanTNF-α as template, 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 multiple clone site of prokaryotic expression carrier pET22b by introducing Mun I (so that the 129-141 amino acids of TNF-α is not introduced any exogenous amino acid outside being substituted by PADRE etc.) restriction enzyme site between amplified fragments and the synthetic fragment.Because wild type gene do not express, so we transcribe the secondary structure of rear mRNA by analysis, introduced a samesense mutation (two point mutation), so that the expression of the genes of interest of originally not expressing may be up to 20% of bacterial protein.Expression product is inclusion body; wash through inclusion body; 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, has good protective effect for dyscrasia and rheumatoid arthritis mouse model when being used for animal.

The particular content of preparation is (take hTNF-PADRE as example):

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

Its aminoacid sequence is:

VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVL

FKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGD

RLSAEINRPDYLDFAESGQVYFGIIAL

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 following (two ends add respectively Mun I and Sal I restriction enzyme site), 3 ' end is introduced termination codon TGA:

5’- AATTGGCTAAATTCGTTGCTGCATGGACTCTGAAAGCTGCAGCTCTCGACTTTGCCGAGTCT

GGGCAGGTCTACTTTGGGATCATTGCCCTGTGA G-3’

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

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

With Nde I and two digestion with restriction enzyme pET22b of Sal I carrier, 1.0% agarose gel electrophoresis Separation and Recovery enzyme action large fragment, 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, transforms the e. coli bl21 competent cell, bed board, cultivation, the picking monoclonal carries out enzyme action and identifies.Obtain containing the positive colony of genes of interest segment, 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 determine suitable inducing the rear time, cultivate and induce on a small scale rear centrifugal collection thalline, with SDS-PAGE testing goal protein expression 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 the LB flat board of activation, cultivate 10h for 37 ℃, then transferred species 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 controlled fermentation temperature, 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 Refrigerator stores are for subsequent use.After the thalline that takes a morsel is processed, with SDS-PAGE testing goal protein expression.

3.3 the separation of destination protein inclusion body and washing

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 MgCl 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 respectively the 4M Urea of 3 times of volumes after centrifugal again, 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 by the Sephacryl-100 gel permeation chromatography, and the protein behind the purification carries out the centrifugal renaturation of Urea Gradient and obtained natural structure, thereby can induce the neutralizing antibody of high-affinity.

3.4 the purification under the expression product Denaturing and renaturation

Inclusion body through 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 stirrings are spent the night, the centrifugal 30min of 12000r/min collects supernatant, is the destination protein crude extract.SephacrylS-100 (the gel chromatography of 1.6cm * 100cm), with working solution (7mol/L guanidine hydrochloride, 50mmol/L Tris.Cl pH 7.0,1mmol/L EDTA, 100mmol/LNaCl) after the abundant balance, destination protein crude extract 2ml upper prop, with the working solution eluting, flow velocity 1ml/min, fraction collection, measure protein content, and determine the destination protein position with SDS-PAGE, collect the higher elution fraction of purity.Purification destination protein component is carried out the gradient dialysis renaturation to carbamide, and gradient is, 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 protein sample of purification is through 15%SDS-PAGE, then from the gel electrotransfer to nitrocellulose membrane, after the BSA sealing, with primary antibodie (mouse anti human TNF-alpha monoclonal antibodies) in conjunction with 1h, two anti-(goat anti-mouse IgG-HRP) in conjunction with 30min, at last with the colour developing of ECL system, distilled water flushing cessation reaction.The result shows hTNF-TT, and hTNF-HEL, hTNF-PADRE can be special and the combination of mouse anti human TNF-alpha monoclonal antibodies.

The TNF-TT that purification obtains, TNF-HEL, TNF-PADRE carry out Purity.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 Mouse L929 cell that growth conditions is good transfers to 10 ⁶The cell suspension of/ml, every hole adds 100 μ l cell suspension, 50ml/l CO 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, microplate reader 570nm place mensuration light absorption value.The humanTNF-α is as positive control, and the GST albumen of purification is as negative control.The result shows that TNF-TT, TNF-HEL, TNF-PADRE are all without natural humanTNF-α's cytotoxicity.

3.5.3 animal immune

3.5.3.1Balb/c mice group and immunization protocol

Grouping: 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 rear 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 Freunds incomplete adjuvant, the assist agent solution cumulative volume is 200 μ L/, respectively every other week injection.

3.5.3.2 antiserum ELISA identifies

In the 4th immunity blood sampling in rear 10 days, collect antiserum, measure the anti-hTNF antibody titer of serum by indirect ELISA.

3.5.4TNF-PADRE the treatment of the cachexia model that TNF-α is induced

3.5.4.1 grouping 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 every other week injection.

3.5.4.2 inducing of model

The mice of immunity 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 Mouse Weight.

The mice of immunity 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 the treatment of the rheumatoid arthritis mouse model that the II Collagen Type VI is induced

3.5.5.1 the preparation of derivant

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

3.5.5.2 the immunity of animal

Buy 40 of the DBA-1 inbred mouses in 5 ages in week from Shanghai Si Laike animal center, be divided at random four groups, 10 every group.First group of immunization method is subcutaneous multiple spot immunity, and immunizing dose is that 100 μ L normal saline/only, every other week immunity 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 for the therapeutic effect of research vaccine.Every other week immunity once is total to immunity four times.

3.5.5.3 the inducing and treating of model

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

Mouse invasion rate statistics: induce the sickness rate that namely begins to add up the mice rheumatoid arthritis that the II Collagen Type VI induces rear next day for the second time.The result as shown in Figure 8, TNF-PADRE can reduce the sickness rate of the mice rheumatoid arthritis that the II Collagen Type VI induces compared with the control.

Mouse arthritis pathological changes standards of grading: induce the second time namely to begin to observe front and back foot swelling degree and scoring rear next day, take every Mus front and back arthropathy scoring as the acute arthritis mark.Arthritis index (arthritis index, AI) standards of grading are as follows: 0 minute-without red and swollen; 1 minute-joint is slightly swollen; 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 as shown in Figure 8, TNF-PADRE can reduce the mouse arthritis pathological changes scoring of the mice rheumatoid arthritis that the II Collagen Type VI induces compared with the control.

4. effect of the present invention

As follows 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 by zoopery this fusion rotein can induce high titre antibody in Mice Body.

(5) preliminary proof this albumen can alleviate to a certain extent the symptom of the cachexia mouse model that TNF induces as tumor vaccine, alleviate the symptom of the rheumatoid arthritis mice that Type Ⅱ collagen induces.

Description of drawings

Fig. 1 a is the mRNA structure (the some of complex structure is amplified) 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 aim sequence, and " 2 " are DNAMarker.

Fig. 2 b is hTNF _1-128Amplification figure, wherein, " 1 " is DL2000DNAmarker, 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 DL2000DNA marker, 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 not induce bacterial protein, " 2 ", " 4 " are 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 rear bacterial protein.

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

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

The protective effect of Fig. 9 rheumatoid arthritis mice that to be hTNF-PADRE autovaccine molecule induce Type Ⅱ collagen; wherein; TNF-PADRE1 is the in advance effect of the prevention RA of immunity of vaccine, and TNF-PADRE2 is the therapeutic effect that gives vaccine after inducing the RA model.

The present invention is described in further detail below in conjunction with drawings and Examples.

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 the amino acid whose nucleotide sequence of PADRE (AKFVAAWT LKA) replace 129 nucleotide sequences to 141 amino acids of humanTNF-α, thereby obtain hTNF-TT, hTNF-HEL, hTNF-PADRE fusion rotein, immune mouse behind the purification, relatively these three kinds of molecules induce for the titre of hTNF-Alpha antibodies and in and the ability of hTNF-alpha active, filter out hTNF-PADRE and have best immune effect.Not only can reduce alleviating of cachexia Mouse Weight that natural TNF-α induces with hTNF-PADRE recombiant protein immune mouse, can also prolong the life cycle of the cachexia mice that mTNF-α induces simultaneously; The life cycle of the mice with acute lung injury that prolongation LPS induces; Alleviate the symptom of the rheumatoid arthritis that the II Collagen Type VI induces.Secondly thereby recombined human TNF-PADRE fusion rotein might produce anti-humanTNF-α'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, finish according to the following steps:

1. the preparation of vaccine and screening

We take mice as model, have screened the on position in mice TNF-alpha molecule of the auxiliary epi-position of external source T cell for the TNF-alpha molecule of mice in the work in early stage, and screening is than the auxiliary epi-position of right different T cell.Found that mice TNF-alpha molecule 128-141 amino acids is the on position of best exogenous molecules, PADRE is for the most effectively inserting epi-position.We use coding TT for above Research foundation _830-844(QYIKANSKFIGITEL), HEL _46-61(NTDGSTDYGILQINSR) and the amino acid whose nucleotide sequence of PADRE (AKFVAAWTLKA) replace 129 nucleotide sequences to 141 amino acids of humanTNF-α, 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 as follows:

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 take the humanTNF-α as template, 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 (so that the 129-141 amino acids of TNF-α is not introduced 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 multiple clone site 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 rear mRNA by analysis, a samesense mutation (two point mutation T439 → A, C440 → G) have been introduced, so that the mRNA structure of genes of interest more is tending towards simplifying and low-energy state (Fig. 1 a is the mRNA structure of natural TNF, and Fig. 1 b is the mRNA structure of introducing after two point mutation are optimized).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 gently precipitation, ice bath 40min, centrifugal (3000rpm * 5min, 4 ℃) abandon the 100mmol/L CaCl that contains 25% glycerol that adds 1/25 volume behind the supernatant ₂, blow afloat precipitation, be distributed in the Eppendorf pipe ,-70 ℃ save backup.

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, at last ice bath 5min again.Then bed board, 37 ℃ of overnight incubation.

1.1.4 the extraction of plasmid and evaluation

Picking neat in edge on the culture dish of 37 ℃ of incubated overnight behind the Plasmid Transformation, the clone that growth conditions is good, be inoculated in the LB culture medium that 10ml contains 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 according to the synthetic PADRE of escherichia coli preference codon and hTNF _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 fragments are cloned 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 be connected the pET22b carrier through Nde I with the SalI enzyme action and be connected, transform picking positive colony extraction plasmid behind the 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 positive 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’- CATATGGTCAGATCATCTTCTCGAACCCCGAGTGACAAGCCTGTAGCCCATGTTGTAGCAAA

CCCTCAAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTCCTGGCCAATGG

CGTGGAGCTGAGAGATAACCAGCTGGTGGTGCCATCAGAGGGCCTGTACCTCATCTACTCCCAG

GTCCTCTTCAAGGGCCAAGGCTGCCCCTCCACCCATGTGCTCCTCACCCACACCATCAGCCGCA

TCGCCGTCTCCTACCAGACCAAGGTCAACCTCCTCTCTGCCATCAAGAGCCCCTGCCAGAGGGA

GACCCCAGAGGGGGCTGAGGCCAAGCCCTGGTATGAGCCCATCTATCTGGGAGGGGTCTTCCA

GCTGCAATTG CAGTACATCAA?AGCTAACTCCAAATTCATCGGTATCACTGAACTTCTC

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 Characterization

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

Contain the e. coli bl21 list colony inoculation of recombinant expression plasmid 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, 1h, 2h, 3h, 4h collect thalline respectively behind the abduction delivering, to determine suitable induction time, cultivate and induce on a small scale rear centrifugal collection thalline, with SDS-PAGE testing goal protein expression and existence form, the result shows in theoretical molecular weight position 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 the LB flat board of activation, cultivate 10h for 37 ℃, then transferred species 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 controlled fermentation temperature, 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 Refrigerator stores are for subsequent use.After the thalline that takes a morsel is processed, with SDS-PAGE testing goal protein expression.The result shows that protein is with the inclusion body formal representation.

1.2.3 purification and the renaturation of restructuring inclusion body protein

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 MgCl 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 respectively the 4M Urea of 3 times of volumes after centrifugal again, 2%Triton X-100,20mM EDTA, 0.1M Tris, with the 20mM EDTA of 8 times of volumes, 0.1M Tris washing.Inclusion body through 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 stirrings are spent the night, the centrifugal 30min of 12000r/min collects supernatant, is the destination protein crude extract.Sephacryl S-100 (the gel chromatography of 1.6cm * 100cm), with working solution (7mol/L guanidine hydrochloride, 50mmol/L Tris.Cl pH 7.0,1mmol/L EDTA, 100mmol/LNaCl) after the abundant balance, destination protein crude extract 2ml upper prop, with the working solution eluting, flow velocity 1ml/min, fraction collection, measure protein content, and determine the destination protein position with SDS-PAGE, collect the higher elution fraction of purity.Purification destination protein component is carried out the gradient dialysis renaturation to carbamide, and gradient is, 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 protein sample of purification is through 15% polyacrylamide gel electrophoresis, electrophoresis is pulled down gel after finishing, according to the Bio-Rad description of product, with close negative electrode one side of gel, close anode one side of pvdf membrane, transfering buffering liquid (25mmol/L Tris-HCl in pre-cooling, 192mmol/L Glycine, 20% methanol) the 100V constant voltage shifts 1h in, with protein delivery to pvdf membrane.After shifting end, take out pvdf membrane, immerse the middle incubated at room 1h of confining liquid (TBST that contains 2%BSA) after cleaning mixture TBST (20mmol/L TrisHCl pH7.5,150mmol/LNaCl, 0.05%Tween20) cleans.With primary antibodie (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 hTNF-HEL, hTNF-PADRE can be special with mouse anti human TNF-alpha monoclonal antibodies in conjunction with (Fig. 6).

TNF-TT, TNF-HEL, TNF-PADRE that purification is obtained carry out Purity.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 Mouse L929 cell that growth conditions is good transfers to 10 ⁶The cell suspension of/ml, every hole adds 100 μ l cell suspension, 50ml/l CO 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, microplate reader 570nm place mensuration light absorption value.The humanTNF-α is as positive control, and the GST albumen of purification is as negative control.The result shows that TNF-TT, TNF-HEL, TNF-PADRE are all without natural humanTNF-α's cytotoxicity (Fig. 7).

3. animal immune

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

Grouping: 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 rear 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 Freunds incomplete adjuvant, the assist agent solution cumulative volume is 200 μ L/, respectively every other week injection.

In the 4th immunity blood sampling in rear 10 days, collect antiserum, measure the anti-hTNF antibody titer of serum by indirect ELISA.

The preparation of required solution:

Coated 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 nitrite ion: OPD 8mg

30％H ₂O ₂ 30μl

Substrate buffer solution 10ml

Stop buffer: H ₂SO ₄1mol/L

Operational approach:

Coated: as to get 96 hole elisa plates, antigen is dissolved in the coated buffer (2-5 μ g/ml), add in the plate hole (100 μ l/ hole), 4 ℃ of coated spending 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 the substrate nitrite ion, 100 μ l/ holes, 37 ℃, colour developing 10-20min; → adding stop buffer, 100 μ l/ holes, cessation reaction; → microplate reader reading is measured every hole at the light absorption value of 490nm.

3.2TNF-PADRE the treatment of the cachexia model that TNF-α is induced

3.2.1 grouping 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 every other week injection.

3.2.2 inducing of model

The mice of immunity 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 Mouse Weight.

The mice of immunity 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 DBA mice that TNF induces.The body weight of mice does not have significance to change (negative control group loses weight); Survival rate obviously is better than the normal saline group.

3.3TNF-PADRE the treatment of the rheumatoid arthritis mouse model that the II Collagen Type VI is induced

3.3.1 the preparation of derivant

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

3.3.2 the immunity of animal

Buy 40 of the DBA-1 inbred mouses in 5 ages in week from Shanghai Si Laike animal center, be divided at random four groups, 10 every group.First group of immunization method is subcutaneous multiple spot immunity, and immunizing dose is that 100 μ L normal saline/only, every other week immunity 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 for the therapeutic effect of research vaccine.Every other week immunity once is total to immunity four times.

3.3.3 the inducing and treating of model

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

Mouse invasion rate statistics: induce the sickness rate that namely begins to add up the mice rheumatoid arthritis that the II Collagen Type VI induces rear next day for the second time.The result as shown in Figure 9, TNF-PADRE can reduce the sickness rate of the mice rheumatoid arthritis that the II Collagen Type VI induces compared with the control.

Mouse arthritis pathological changes standards of grading: induce the second time namely to begin to observe front and back foot swelling degree and scoring (Fig. 9) rear next day, take every Mus front and back arthropathy scoring as the acute arthritis mark.Arthritis index (arthritis index, AI) standards of grading are as follows: 0 minute-without red and swollen; 1 minute-joint is slightly swollen; 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 as shown in Figure 9, TNF-PADRE can reduce the mouse arthritis pathological changes scoring of the mice rheumatoid arthritis that the II Collagen Type VI induces compared with the control.

Sequence table

＜110〉the Fourth Military Medical University of P.L.A

＜120〉a kind of construction method of the autovaccine for humanTNF-α's molecule

<130>2011

<160>9

<170>PatentIn?version?3.3

<210>1

<211>486

<212>DNA

＜213〉artificial sequence

<400>1

catatggtca?gatcatcttc?tcgaaccccg?agtgacaagc?ctgtagccca?tgttgtagca 60

aaccctcaag?ctgaggggca?gctccagtgg?ctgaaccgcc?gggccaatgc?cctcctggcc 120

aatggcgtgg?agctgagaga?taaccagctg?gtggtgccat?cagagggcct?gtacctcatc 180

tactcccagg?tcctcttcaa?gggccaaggc?tgcccctcca?cccatgtgct?cctcacccac 240

accatcagcc?gcatcgccgt?ctcctaccag?accaaggtca?acctcctctc?tgccatcaag 300

agcccctgcc?agagggagac?cccagagggg?gctgaggcca?agccctggta?tgagcccatc 360

tatctgggag?gggtcttcca?gctgcaattg?gctaaattcg?ttgctgcatg?gactctgaaa 420

gctgcagctc?tcgactttgc?cgagagtggg?caggtctact?ttgggatcat?tgccctgtga 480

gtcgac 486

<210>2

<211>11

<212>PRT

＜213〉artificial sequence

<400>2

Ala?Lys?Phe?Val?Ala?Ala?Trp?Thr?Leu?Lys?Ala

1 5 10

<210>3

<211>15

<212>PRT

＜213〉artificial sequence

<400>3

Gln?Tyr?Ile?Lys?Ala?Asn?Ser?Lys?Phe?Ile?Gly?Ile?Thr?Glu?Leu

1 5 10 15

<210>4

<211>16

<212>PRT

＜213〉artificial sequence

<400>4

Asn?Thr?Asp?Gly?Ser?Thr?Asp?Tyr?Gly?Ile?Leu?Gln?Ile?Asn?Ser?Arg

1 5 10 15

<210>5

<211>157

<212>PRT

＜213〉artificial sequence

<400>5

Val?Arg?Ser?Ser?Ser?Arg?Thr?Pro?Ser?Asp?Lys?Pro?Val?Ala?His?Val

1 5 10 15

Val?Ala?Asn?Pro?Gln?Ala?Glu?Gly?Gln?Leu?Gln?Trp?Leu?Asn?Arg?Arg

20 25 30

Ala?Asn?Ala?Leu?Leu?Ala?Asn?Gly?Val?Glu?Leu?Arg?Asp?Asn?Gln?Leu

35 40 45

Val?Val?Pro?Ser?Glu?Gly?Leu?Tyr?Leu?Ile?Tyr?Ser?Gln?Val?Leu?Phe

50 55 60

Lys?Gly?Gln?Gly?Cys?Pro?Ser?Thr?His?Val?Leu?Leu?Thr?His?Thr?Ile

65 70 75 80

Ser?Arg?Ile?Ala?Val?Ser?Tyr?Gln?Thr?Lys?Val?Asn?Leu?Leu?Ser?Ala

85 90 95

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

100 105 110

Pro?Trp?Tyr?Glu?Pro?Ile?Tyr?Leu?Gly?Gly?Val?Phe?Gln?Leu?Glu?Lys

115 120 125

Gly?Asp?Arg?Leu?SerAla?Glu?Ile?Asn?Arg?Pro?Asp?Tyr?Leu?Asp?Phe

130 135 140

Ala?Glu?Ser?Gly?Gln?Val?Tyr?Phe?Gly?Ile?Ile?Ala?Leu

145 150 155

<210>6

<211>390

<212>DNA

＜213〉artificial sequence

<400>6

catatggtca?gatcatcttc?tcgaaccccg?agtgacaagc?ctgtagccca?tgttgtagca 60

aaccctcaag?ctgaggggca?gctccagtgg?ctgaaccgcc?gggccaatgc?cctcctggcc 120

aatggcgtgg?agctgagaga?taaccagctg?gtggtgccat?cagagggcct?gtacctcatc 180

tactcccagg?tcctcttcaa?gggccaaggc?tgcccctcca?cccatgtgct?cctcacccac 240

accatcagcc?gcatcgccgt?ctcctaccag?accaaggtca?acctcctctc?tgccatcaag 300

agcccctgcc?agagggagac?cccagagggg?gctgaggcca?agccctggta?tgagcccatc 360

tatctgggag?gggtcttcca?gctgcaattg 390

<210>7

<211>96

<212>DNA

＜213〉artificial sequence

<400>7

aattggctaa?attcgttgct?gcatggactc?tgaaagctgc?agctctcgac?tttgccgagt 60

ctgggcaggt?ctactttggg?atcattgccc?tgtgag 96

<210>8

<211>492

<212>DNA

＜213〉artificial sequence

<400>8

catatggtca?gatcatcttc?tcgaaccccg?agtgacaagc?ctgtagccca?tgttgtagca 60

aaccctcaag?ctgaggggca?gctccagtgg?ctgaaccgcc?gggccaatgc?cctcctggcc 120

aatggcgtgg?agctgagaga?taaccagctg?gtggtgccat?cagagggcct?gtacctcatc 180

tactcccagg?tcctcttcaa?gggccaaggc?tgcccctcca?cccatgtgct?cctcacccac 240

accatcagcc?gcatcgccgt?ctcctaccag?accaaggtca?acctcctctc?tgccatcaag 300

agcccctgcc?agagggagac?cccagagggg?gctgaggcca?agccctggta?tgagcccatc 360

tatctgggag?gggtcttcca?gctgcaattg?cagtacatca?aagctaactc?caaattcatc 420

ggtatcactg?aacttctcga?ctttgccgag?agtgggcagg?tctactttgg?gatcattgcc 480

ctgtgagtcg?ac 492

<210>9

<211>495

<212>DNA

＜213〉artificial sequence

<400>9

catatggtca?gatcatcttc?tcgaaccccg?agtgacaagc?ctgtagccca?tgttgtagca 60

aaccctcaag?ctgaggggca?gctccagtgg?ctgaaccgcc?gggccaatgc?cctcctggcc 120

aatggcgtgg?agctgagaga?taaccagctg?gtggtgccat?cagagggcct?gtacctcatc 180

tactcccagg?tcctcttcaa?gggccaaggc?tgcccctcca?cccatgtgct?cctcacccac 240

accatcagcc?gcatcgccgt?ctcctaccag?accaaggtca?acctcctctc?tgccatcaag 300

agcccctgcc?agagggagac?cccagagggg?gctgaggcca?agccctggta?tgagcccatc 360

tatctgggag?gggtcttcca?gctgcaattg?aacactgacg?gttccaccga?ttacggcatt 420

ctgcagatca?actcccgcct?cgactttgcc?gagagtgggc?aggtctactt?tgggatcatt 480

gccctgtgag?tcgac 495

Claims (5)

1. induce the construction method for the protein vaccine of the autoantibody of humanTNF-α's molecule in the body, it is characterized in that, with fusion gene TNF-α _1-128-PADRE-TNF-α _142-157Be cloned between the Nde I and SalI multiple clone site of prokaryotic expression carrier pET22b, the recombinant expression carrier that obtains is transformed e. coli bl21, inducing rear expression product is inclusion body, wash through inclusion body, gel filtration chromatography, dialysis renaturation can obtain the purpose amalgamation protein vaccine of purification, this protein vaccine immune mouse in the situation of not using adjuvant can be produced the anti-humanTNF-α's autoantibody of high titre, and wherein said fusion gene sequence is:

5’- CATATGGTCAGATCATCTTCTCGAACCCCGAGTGACAAGCCTGTAGCCCATGTTGTAGCAAACCCTCAAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTCCTGGCCAATGGCGTGGAGCTGAGAGATAACCAGCTGGTGGTGCCATCAGAGGGCCTGTACCTCATCTACTCCCAGGTCCTCTTCAAGGGCCAAGGCTGCCCCTCCACCCATGTGCTCCTCACCCACACCATCAGCCGCATCGCCGTCTCCTACCAGACCAAGGTCAACCTCCTCTCTGCCATCAAGAGCCCCTGCCAGAGGGAGACCCCAGAGGGGGCTGAGGCCAAGCCCTGGTATGAGCCCATCTATCTGGGAGGGGTCTTCCAGCTGCAATTG GCTAAATTCGTTGCTGCATGGACTCTGAAAGCTGCAGCTCTCGACTTTGCCGAG? AGTGGGCAGGTCTACTTTGGGATCATTGCCCTGTGA GTCGAC-3’?。

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

According to humanTNF-α's aminoacid sequence and PADRE aminoacid sequence AKFVAAWTLKA, design hTNF-PADRE gene order, the insertion point of PADRE is 128 back of TNF-α in the gene order, namely 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 by pcr amplification, and introduces Nde I restriction enzyme site CATATG in the upstream, wherein contains translation initiation codon; MunI restriction enzyme site CAATTG is introduced in the downstream; Synthetic PADRE-TNF-α _142-157Coded sequence, upstream and downstream is introduced respectively MunI and SalI GTCGAC restriction enzyme site, in synthetic fragment gene has been carried out two point mutation T439 → A, C440 → G in addition;

Two fragment genes connect by Mun I restriction enzyme site, and fusion gene cloning enters between the Nde I and SalI restriction enzyme site of carrier pET22b;

Above-mentioned expression vector is transformed e. coli bl21, the plasmid called after pET22b-hTNF-PADRE that checks order correct, engineering bacteria called after pET22b-hTNF-PADRE/BL21; Its described gene order is as follows:

5’- CATATGGTCAGATCATCTTCTCGAACCCCGAGTGACAAGCCTGTAGCCCATGTTGTAGCAAA

Nde?I

CCCTCAAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTCCTGGCCAATGGCGTGGAGCTGAGAGATAACCAGCTGGTGGTGCCATCAGAGGGCCTGTACCTCATCTACTCCCAGGTCCTCTTCAAGGGCCAAGGCTGCCCCTCCACCCATGTGCTCCTCACCCACACCATCAGCCGCA?TCGCCGTCTCCTACCAGACCAAGGTCAACCTCCTCTCTGCCATCAAGAGCCCCTGCCAGAGGGAGACCCCAGAGGGGGCTGAGGCCAAGCCCTGGTATGAGCCCATCTATCTGGGAGGGGTCTTCCAGCTGCAATTG GCTAAATTCGTTGCTGCATGGACTCTGAAAGCTGCAGCTCTCGACTTTGCCGAG

Mun?I PADRE

AGTGGGCAGGTCTACTTTGGGATCATTGCCCTGTGA GTCGAC-3’。

Mutational site SalI

3. method as claimed in claim 2, it is characterized in that, the abduction delivering step of described engineering bacteria is as follows: picking engineering bacteria list colony inoculation is in the fresh LB culture fluid of 5mL, described LB culture fluid contains Amp100mg/L, 37 ℃ of shaking table overnight incubation, next day, the ratio with 1:100 was transferred in the LB culture fluid again, cultivated 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 is with SDS-PAGE testing goal protein expression.

4. method as claimed in claim 3 is characterized in that, the purification renaturation of described destination protein is finished 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, pH is among 7.0, the 1mM EDTA, adds the 1.5mg/mL lysozyme, 10ug/mL DNaseI, 20mM MgCl ₂Carry out cracking; Add 1.5M NaCl, 2%Triton X-100,20mM EDTA to 3 times of first volumes, room temperature washing 30min uses respectively the 4M Urea of 3 times of volumes after centrifugal again, 2%Triton X-100,20mM EDTA, 0.1M Tris, with the 20mM EDTA of 8 times of volumes, 0.1M Tris washing; Inclusion body through washing is dissolved in 50mmol/L Tris.Cl pH7.0, 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 stirrings are spent the night, the centrifugal 30min of 12000r/min collects supernatant, is the destination protein crude extract; SephacrylS-100, specification is 1.6cm * 100cm, gel chromatography, to contain the 7mol/L guanidine hydrochloride, 50mmol/L Tris.Cl pH7.0,1mmol/L EDTA, after the abundant balance of the working solution of 100mmol/L NaCl, destination protein crude extract 2ml upper prop, with the working solution eluting, flow velocity 1ml/min, fraction collection, measure protein content, and determine the destination protein position with SDS-PAGE, collect the higher elution fraction of purity; Purification destination protein component is carried out the gradient dialysis renaturation to carbamide, and gradient is, 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. method as claimed in claim 4 is characterized in that, the mice rheumatoid arthritis that the fusion rotein that obtains is induced the II Collagen Type VI has protection and therapeutical effect.

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