CN100349921C

CN100349921C - Fusion protein of tetanus toxin T cell expressing bit polypeptide and human bata lymph cell stimulating factor and preparing thereof

Info

Publication number: CN100349921C
Application number: CNB200510096372XA
Authority: CN
Inventors: 张英起; 薛晓畅; 颜真; 赵宁
Original assignee: Fourth Military Medical University FMMU
Current assignee: Fourth Military Medical University FMMU
Priority date: 2005-11-17
Filing date: 2005-11-17
Publication date: 2007-11-21
Anticipated expiration: 2025-11-17
Also published as: CN1793178A

Abstract

The present invention discloses tetanotoxin T cell epi-position peptide, fusion protein of human B lymphocyte stimulating factors and a preparation method. The TT epi position peptide-QYIKANSKFIGITE-(glutamine-tyrosine-isoleucine-lysine-alanine-asparagine-serine-lysine-phenylalanine-isoleucine-glycine-isoleucine-threonine-glutamate) can stimulate the reaction of CD4<+>T cells of an organism and promote the humoral immunity level of the organism. Step-by-step cloning is used for constructing fusion genes for TT and BLyS, and the high-level expression of the fusion genes for TT and BLyS is realized in colibacillus. Through the fusion of TT and BLyS, the immune tolerance of the organism is overcome, so that a high-level humoral immunity reaction occurs in the organism, and generated BLyS polyclonal neutralizing antibodies can neutralize the stimulating activity of B lymphocytes of high-level natural BLyS in the organism; consequently, the action of autoimmune disease resistance is performed.

Description

Tetanus toxin t cell epitope polypeptide and human B lymphocyte stimulating factor fusion rotein and preparation thereof

Technical field

Technology such as the purifying of the expression of the gene clone, gene recombination, foreign gene that the invention belongs to the medical biotechnology field in prokaryotic cell prokaryocyte, target protein, the active determination in vitro of recombinant protein vaccine and physico-chemical property evaluation, relate to a kind of anti-autoimmune disorder medicine and preparation method thereof, particularly bone-marrow-derived lymphocyte stimulating factor recombinant protein vaccine of tetanus toxin t cell epitope polypeptide and human B lymphocyte stimulating factor fusion rotein (TT-BLyS) formation and preparation method thereof.

Background technology

1.1B the correlative study of lymphocyte stimulating factor

The bone-marrow-derived lymphocyte stimulating factor is tumour necrosis factor (TumorNecrosis Factor, TNF) family newcomer of being found by Switzerland and U.S. scientist in 1999.This molecule found simultaneously by a plurality of different research groups, and be named as BAFF respectively, THANK, TALL-1, zTNF4, TNFRSF19 etc., it is named so far also not by unification.

Different with the most of member of TNF family is, BLyS is expressed in the activatory immunocyte, and it has the expression of composition in monocyte and scavenger cell, mainly by the emiocytosis in medullary system source.IFN-γ has very strong rise effect to its expression, a little less than the rise effect slightly of IL-10.

1.1.1 the biologic activity of Interferon, rabbit

Similar with other molecules of TNF family, the activity form of BLyS is also to be homotrimer, changes molecule and plays an important role in body's immunological function is regulated.

Bone-marrow-derived lymphocyte is regulated: BLyS has very strong B cytotaxis, and as a kind of lymphocytic costimulating factor, it has the hormesis that intensive promotes propagation and differentiation to activatory B cell, and rather similar to the effect of CD40L.In vitro tests shows, to normal B cell, utilize the pre-activation of anti-IgM after, BLyS can induce all types of immunoglobulin (Ig)s of its a large amount of propagation justacrines, wherein IgM and IgA ratio are higher, and BLyS is not obvious to the cell effect of tranquillization.External, BLyS is to the activation of the outer B cell of capsule and the secretion of antigen-specific IgM; The classification conversion of immunoglobulin (Ig) and the formation of spleen germinal center have vital role.

The T lymphocyte is regulated: the research report is arranged, and in the BLyS transgenic mice, though the T total cellular score does not obviously rise in the spleen, CD4 ⁺And CD8 ⁺Cell all presents active state; And the CD44 level of these cell surfaces rises and the decline of LECAM-1 level, in addition, flow cytometry analysis shows that BLyS can combine (avidity be about B cell 5%) with a small amount of activated T cell, and activating T cell has the expression of TACI, and these all point out the BLyS can be indirectly to the T cytosis.Another discovers that the T total cellular score of mouse has increased twice, and germinal center increases increase under the situation of not carrying out immunity, and lymphoid organ increases, and has also found the rheumatism factor.Difference on these details may be because the expression regulation difference of BLyS causes.

1.1.2B the clinical application present situation of lymphocyte stimulating factor

2.1.2.1 common variable immunodeficient disease

Though the BLyS molecule finds only have several years, molecular biology research to prove that in a short period of time it has irreplaceable vital role in immunity system.Because of this, (Human GenomeSciences HGS) has formulated the plan of BLyS treatment albumen, and decision utilizes genetic engineering means development BLyS related preparations to be used for the immunodeficient disease treatment in U.S. Human Genome Sciences.The said firm announces the U.S. FDA approved, and it is treated common variable immunodeficient disease (commonvariable immunodeficiency, medicine-BLyS CVID) is rare sick curative.

CVID is quite common and one group of syndrome that clearly do not understand.The men and women all can get involved, and age of onset did not wait at 15～35 years old, can be congenital or acquired.Its immune deficiency is involved scope and can be changed with stadium, shows as hypogammag lobulinemia during onset, along with disease progression can concurrent cellular immunity deficiency.Be characterized in: 1. hypogammag lobulinemia, immunoglobulin (Ig) total amount and IgG all reduce; 2. the B cell quantity is normal in 2/3 patient's circulation of blood, but can not be divided into plasmocyte, in lymphoglandula, spleen, the alimentary canal Lymphoid tissue B hyperplasia obvious, but lack plasmocyte.3. the patient mainly shows as respiratory tract, gastral lasting chronic inflammatory diseases, and as pneumonia, bronchitis and nasal sinusitis etc., the sickness rate of autoimmune disease is also higher.

The researchist of the said firm finds that the BLyS recombinant protein can promote some to separate from CVID patient's B cell and produce immunoglobulin (Ig), thereby has strengthened the anti-infection ability of body.BLyS now is in the middle of the assessment of I clinical trial phase.

1.1.2.2 first type immunoglobulin deficiency disease

The low patient of first type immunoglobulin (Ig) can not produce the IgA of normal quantity, and IgA is that the intravital mucosal tissue of machine (lung, small intestine, oral cavity, urogenital tract) provides the first line of defence of resisting foreign matter, is one of important factor of body defence infection.The IgA level lowly is one of common disease of body antibody forming system, and one of its reason is that intravital plasmocyte level is low.Patient's cardinal symptom is the severe infections that shows effect repeatedly, can involve gi tract, lung and fistula, also allergy and tumour can be arranged.

Also not having effective ways for this treatment of diseases at present, mainly is the injection alloantibody.BLyS can stimulate the multiple antibody of B emiocytosis, and it is used for the treatment of this disease and may helps patient to produce the antibody of oneself, thereby fundamentally changes patient's symptom.Preclinical test shows that BLyS can be so that the low patient of part IgA produces the IgA that is higher than normal level.It is clinical that this research also is in the I phase.

1.1.2.3B cell malignancies

The BLyS of radioisotope labeling is useful on the potential possibility of treatment Malignant B cell tumour.Because at present for some tumours to responding property of radioactive rays, radiotherapy still compares tradition and effective means.If make a kind of medicine of forming by BLyS molecule and radioactive source, this medicine is because the selectivity distribution of BLyS acceptor will be specific in conjunction with the B cell, so the result of selective binding causes the enrichment of medicine, thereby make the effective dose of medicine killing tumor cell reduce, reduce the side effect that radioactive rays bring.HGS company is assessing the feasibility of this technology at present.

As non_hodgkin lymphoma (non-Hodgkin ' s lymphoma), lymphocytic leukemia (chronic lymphocytic leukemia, CLL), multiple myeloma (multiple myeloma) waits in some specific tumours, bone-marrow-derived lymphocyte worsens, and grows in a kind of mode out of control.These tumours are very big to the mankind's threat.Non_hodgkin lymphoma occupies the 5th in U.S.'s kinds of tumor, also be very common a kind of children's cancer; CLL is modal leukemia; Multiple myeloma is a kind of fatal B cell tumour, and 5 annual survival rates have only 28%.So, also need new therapy to improve patient's survival rate and curative ratio for this class tumour.

Because the B glucagonoma is relatively more responsive to radiotherapy, I ¹³¹The precedent that is used to treat thyroid carcinoma is arranged.HGS company is with the radioactive I of BLyS ¹³¹The treatment that mark is used for multiple myeloma, CLL, non_hodgkin lymphoma, mantle cell lymphoma (mantle cell lymphoma), folliculus type B lymphoma (follicular B-cell lymphomas) later on and suddenly restrains lymphomas (Burkitt ' s lymphoma).Preclinical test is the result show, this protein can specificly combine and enrichment with the B cell, utilizes the radioactivity of iodine to come the kill tumor cell then.This medicine is named as LymphoRad ¹³¹, at present, having gone through, it is clinical to enter the I phase.

Preliminary pharmacokinetic data shows the LymphoRad of initial injection ¹³¹Its transformation period has only 18～23 hours, and is shorter than the time that the CD20 antibody institute with mark reports.Decomposition run and radioactivity measurement result show that healthy tissues (as liver, lung, kidney) only is subjected to the radiation within the tolerance interval of being in of low dosage in the body.Moreover, since the pre-B cell not can with LymphoRad ¹³¹Take place in conjunction with and survive and can be used for additional killed improper B cell, this has also alleviated LymphoRad ¹³¹Side effect.

1.1.2.4BLyS the anti-agent of a word used in person's names is used for the treatment of autoimmune disorder

Under the normal circumstances, the human immune system has possessed autologous tissue in growth course and the exotic disease substance tolerates respectively or the ability of immune attack.If self tolerance has taken place unusually, thereby immunity system will be attacked the tissue system generation autoimmune disorder of self.(systemiclupus erythematosus, SLE) and in the rheumatoid arthritis (rheumatoid arthritis), the B cell all plays an important role in a lot of autoimmune disorders such as systemic lupus erythematous.

Because BLyS has the strong impulse effect to the B cell, therefore, in a lot of experiments, find that the BLyS and the autoimmune disorder of high expression level have dependency.In view of this, the effect of inhibition BLyS may be the effective way of a treatment autoimmune disorder.The scientist of HGS company has obtained the monoclonal antibody LymphoStat-B of people BLyS, and this medicine is in I phase clinical study.

Summary of the invention

The objective of the invention is to, a kind of tetanus toxin t cell epitope polypeptide and human B lymphocyte stimulating factor fusion rotein (TT-BLyS) and preparation method thereof are provided.Tetanus toxin t cell epitope polypeptide and the human B lymphocyte stimulating factor fusion rotein of the present invention preparation can overcome the limitation of monoclonal antibody, minimizing dosage and medication number of times.

To achieve these goals, the technical scheme that the present invention takes is, tetanus toxin t cell epitope polypeptide and human B lymphocyte stimulating factor fusion rotein, it is characterized in that, select tetanus toxin epitope peptide Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile-Thr-Glu-(glutamine-tyrosine-Isoleucine-Methionin-L-Ala-l-asparagine-Serine-Methionin-phenylalanine-Isoleucine-glycine-Isoleucine-Threonine-L-glutamic acid) and human B lymphocyte stimulating factor to make up the fusion gene of TT and bone-marrow-derived lymphocyte stimulating factor, and in intestinal bacteria, obtain to efficiently express, obtain the TT-BLyS fusion rotein after purified.

Its TT-BLyS fusion rotein of the present invention can stimulate body CD4 ⁺Cell response helps to cause high-caliber humoral immune reaction.Can prevent the generation of EAE after this vaccine inoculation mouse, model mice is had good protective action.

Expression product is through the purifying of N,O-Diacetylmuramidase cracking, inclusion body washing, S-300 gel permeation chromatography post, and purity of protein can reach more than 95%.Through subcutaneous injection TT-BLyS immune animal, find that this recombiant vaccine has good immunogenicity to new zealand rabbit, rat and mouse, can bring out the reaction of high titer antibody, the polyclonal antibody of generation can in and natural B lyS molecule to the activity that stimulates proliferation of mouse bone-marrow-derived lymphocyte.

Tetanus toxin t cell epitope polypeptide of the present invention and human B lymphocyte stimulating factor fusion rotein, its preparation method is, the substep cloning has made up recombinant plasmid pGEM-3zf (-)-TT-BLyS, comprises the clone of TT gene and the structure of BLyS and TT fusion gene cloning carrier; Construction of prokaryotic expression vector and expression of recombinant proteins; The evaluation of expression product; The purifying of recombinant protein; TT-BLyS induces the research of polyclone neutralizing antibody and TT-BLyS that the provide protection of EAE and RA model mice is studied in different classes of animal.

TT epitope polypeptide of the present invention and human B lymphocyte stimulating factor fusion rotein (TT-BLyS) by the fusion of polypeptide and BLyS, have overcome the restriction of BLyS molecule self-tolerance, also can guide immune response into CD4 simultaneously ⁺T cell rather than CD8 ⁺The T cell.This molecule is different from monoclonal antibody, and low dose acts on the autoimmune disorder patient can guide immunity system into the BlyS neutralizing antibody that produces high titre, and neutralizing antibody oppositely reverse feedback is regulated immune level, finally makes body reach balance.Thereby be further to reduce the clinical application amount, reduce toxic side effect and lay the foundation.

Description of drawings

Fig. 1 is the sequencing result figure of pGEM-3Zf (-)-TT-BLyS;

Fig. 2 is PCR product agarose electrophoresis figure, 1:DNA marker among the figure; 2: goal gene;

Fig. 3 is that the enzyme of pKKH-TT-BLyS recombinant plasmid is cut qualification result figure; 1:DNA marker among the figure; 2-4:pKKH-TT-BlyS.

Fig. 4 is that the abduction delivering and the Western-blot of TT-BLyS fusion rotein identifies; Among the figure 1: do not induce bacterium protein; Bacterium protein after 2:IPTG induces;

Fig. 5 is the purification result of TT-BLyS; A:TT-BLyS purifying color atlas among the figure; B-C: the SDS-PAGE of purified product identifies;

Fig. 6 is the BLyS polyclonal antibody titre detection after TT-BLyS is used for immune animal; A among the figure: new zealand rabbit; B: rat; C: mouse;

Fig. 7 is the clinical manifestation scoring of EAE rat model;

Fig. 8 is the provide protection of TT-BLyS to EAE model mice spinal cord; A-C among the figure: normal group; The D-F:TT-BLyS group; The G-I:EAE model group; A, D, G: * 40; B, E, H: * 100; C, F, I: * 200;

Fig. 9 is the HE colored graph of TT-BLyS to EAE model mice cerebellum; A among the figure: normal group; The B:TT-BLyS group; C: model group; 1,2: * 100; 3,4: * 200; 1,3:HE dyeing; 2,4: fast blue dyeing;

Figure 10 is that TT-BLyS is to the kneed X-photoimaging of RA model mice figure, A among the figure: normal group; The B:TT-BLyS group; The C:RA model group.

For a more clear understanding of the present invention, the present invention is described in further detail for the embodiment that finishes below in conjunction with the contriver.

Embodiment

According to technical scheme of the present invention, the t cell epitope polypeptide of tetanus toxin and human B lymphocyte stimulating factor fusion rotein, select the t cell epitope polypeptide-Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile-Thr-Glu-(glutamine-tyrosine-Isoleucine-Methionin-L-Ala-l-asparagine-Serine-Methionin-phenylalanine-Isoleucine-glycine-Isoleucine-Threonine-L-glutamic acid) of tetanus toxin, it can stimulate the CD4 of body ⁺Cell response.Adopt the substep gene clone, make up the fusion gene of TT-BLyS, and in intestinal bacteria, obtain to efficiently express.Expression product is through the purifying of N,O-Diacetylmuramidase cracking, inclusion body washing and gel permeation chromatography post, and purity of protein can reach more than 95%.After the subcutaneous injection TT-BLyS, all induced the high-level antibody reaction in economic mouse new zealand rabbit, rat, this polyclonal antibody can in and natural B LyS to the effect of stimulating proliferation of mouse bone-marrow-derived lymphocyte.Can prevent the generation of EAE after this vaccine inoculation mouse, model mice is had good protective action.

Realize the t cell epitope polypeptide and the human B lymphocyte stimulating factor fusion rotein (TT-BLyS) of above-mentioned tetanus toxin, its preparation method carries out according to the following steps:

3.1 the substep clone, the structure of recombinant plasmid pGEM-3zf (-)-TT-BLyS

3.1.1TT the clone of epitope peptide gene

The design of the oligonucleotide fragment of coding TT is as follows, has introduced the EcoRI restriction enzyme site at this 5 ' end to oligonucleotide fragment, and 3 ' end has been introduced the BamHI restriction enzyme site:

S1(51nt)5’-AAT?TCA?TGC?AGT?ACA?TCA?AAG?CTA?ACT?CCA?AAT?TCA?TCG?GTA?TCACTG?AAG-3’

S2(51nt)5’-GAT?CCT?TCA?GTG?ATA?CCG?ATG?AAT?TTG?GAG?TTA?GCT?TTG?ATG?TACTGC?ATG-3’

Get equivalent S1 and S2, boiled 5 minutes, naturally cool to room temperature, anneal.Cloning vector pGEM-3Zf (-) is through EcoRI and BamHI double digestion and reclaim big fragment.The annealing product of TT and carrier enzyme are cut the big fragment that reclaims the back and are carried out ligation through the T4 ligase enzyme.Connect product transformed competence colibacillus cell DH5 α, picking mono-clonal overnight incubation is extracted plasmid, cuts evaluation through enzyme, obtains the insertion fragment of expection size, and (Fig. 1) checks order.Correct plasmid called after pGEM-3Zf (-)-TT checks order

3.1.2BLyS structure with TT peptide fusion gene cloning carrier

PCR reaction design of primers is as follows, and translation initiation sign indicating number ATG and BamHI restriction enzyme site are introduced 5 ' end primer, and terminator codon TGA and PstI restriction enzyme site are introduced 3 ' end primer:

P1 (5 ' end primer 31nt) 5 '-GCG GGA TCC ATG GCC GTT CAG GGT CCA GAA G-3 '

P2 (3 ' end primer 31nt) 5 '-GCG CTG CAG TCA CAG CAG TTT CAA TGC ACC A-3 '

Pcr amplification is carried out by following condition in human leukocyte cDNA library.

The pcr amplification reaction pipe is formed:

CDNA template 1 μ l

2.5mM?dNTPs 4μl

25mM?MgCl ₂ 23μl

50μM?P1 1μl

50μM?P2 1μl

5.0U/ μ l Taq enzyme 1 μ l

10×PCR?Buffer 5μl

H ₂O 34μl

Amount to 50 μ l.

The preparation of reaction tubes is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive components, 95 ℃ of reaction 5min add the Taq enzyme again, carry out the PCR reaction then,

The loop parameter of pcr amplification is: 94 ℃ of 1min, 52 ℃ of 45s, 72 ℃ of 1min, totally 30 circulations; At last, extend 10min in 72 ℃ again; The PCR product carries out 0.8% agarose gel electrophoresis, the dna fragmentation (Fig. 2) of visible expection size.

Plasmid pGEM-3Zf (-)-TT reclaims big fragment behind BamH I and PstI double digestion, the fragment of recovery and above-mentioned PCR product carry out ligation through the recovery product of same double digestion.Connect product transformed competence colibacillus cell DH5 α, picking mono-clonal overnight incubation is extracted plasmid, cuts through enzyme and identifies the insertion fragment that obtains the expection size, and (Fig. 1) checks order.Correct plasmid called after pGEM-3Zf (-)-TT-BLyS checks order.

3.1.2 construction of prokaryotic expression vector and expression of recombinant proteins

Plasmid pGEM-3Zf (-)-TT-BLyS reclaims small segment behind EcoRI and PstI double digestion, protokaryon non-fusion expression carrier pKKH also reclaims big fragment behind above-mentioned same double digestion, and both carry out ligation.Connect product transformed competence colibacillus cell DH5 α, picking mono-clonal overnight incubation is extracted plasmid, cuts through enzyme and identifies correct plasmid called after pKKH-TT-BLyS.The bacillus coli DH 5 alpha that will contain recombinant plasmid pKKH-TT-BLyS is in 37 ℃ of activation jolting overnight incubation in the LB substratum, m seq, be inoculated in the LB substratum in 1: 100 ratio after, continue 37 ℃ of cultivations 3 hours to logarithmic growth later stage, OD _650nm=0.4 o'clock, add 1mM IPTG, cultivated 4 hours, collect thalline.Through N,O-Diacetylmuramidase cracking, centrifugal collecting precipitation.

3.1.3 the evaluation of expression product

Expression product is carried out Western-blot to be identified.The bacterioprotein sample is after the SDS-polyacrylamide gel electrophoresis separates, according to the Bio-Rad description of product, near negative electrode one side, close anode one side of nitrocellulose (NC) film, 1 hour electrophoresis of 100V constant voltage is transferred to albumen on the NC film in the transfering buffering liquid of precooling with gel.After electrophoresis finishes, take out the NC film, washings TBST clean the back immerse in the confining liquid (TBST that contains 2%BSA) 37 ℃ 1 hour, the TBST room temperature is washed 3 times (5min/ time), adds goat-anti people's BLyS antibody (Santa Cruz:sc-5743), hatches 1 hour for 37 ℃, TBST washes 3 times, add the anti-sheep IgG-AP of rabbit, hatched 1 hour for 37 ℃, TBST washes 3 times, wash 3 times with TBS again, the NC film immerses in the colour developing liquid, room temperature lucifuge colour developing appropriate time, distilled water flushing termination reaction.

Needing has transfering buffering liquid (25mmol/L Tris, 192mmol/L Glycine, 20% methanol), TBS (20mmol/L Tris-HCl pH7.5,150mmol/L NaCl) and TBST (TBS contain 0.05%Tween20) with reagent

3.1.4 the purifying of recombinant protein

By 1 the gram thalline add 5ml split the bacterium damping fluid (0.1mol/L Tris, pH 7.0; 1mmol/L EDTA) ratio adds the N,O-Diacetylmuramidase of 1.5mg/mL with thalline resuspended (volume is V), stirs 30 minutes under the room temperature; Add the DNase I of 10 μ g/mL and the MgCl2 of 20mmol/L then, continuation effect 30 minutes; Adding final concentration is 1.5mol/L NaCl, 2%Triton X-100 and 20mmol/L EDTA (3V), 4 ℃ were stirred 30 minutes, centrifugal under 4 ℃ of conditions, 12000rpm, 20min, collecting precipitation were with 4mol/L Urea, 2%Triton X-100,20mol/L EDTA, 0.1mol/L TrispH 8.0 (3V) room temperature washing 30 minutes, and precipitation is used 20mol/L EDTA again, 0.1mol/L Tris pH 7.0 (8V) room temperature washing 30 minutes, centrifugal collection inclusion body; Inclusion body dissolves with 7M Guanidinium hydrochloride, 10mg/L DTT, centrifugal back supernatant carries out gel permeation chromatography with Sephacryl 300, and elutriant is the 7M Guanidinium hydrochloride, collects elutriant, carry out quality evalution respectively to 4M urea, 2M urea and the water renaturation of dialysing, and to purified product.

3.2 the immunogenicity determining of vaccine

3.2.1. experiment material

3.2.1.1 medicine

TT-BLyS is provided by The Fourth Military Medical University's biotechnology center, and 80 μ g/ prop up, and-20 ℃ of preservations add physiological saline solution before the use.

3.2.1.2 control drug

Injection physiological saline

3.2.1.3BLyS antibody test reagent

BLyS standard substance (20ug): be cushioned the liquid dissolving with bag during use

1 of enzyme labelled antibody (120 μ l): do 100 times of dilutions with diluent during use

1 of negative control sera (200 μ l);

1 of ABTS substrate.

3.2.2 animal and grouping:

1. new zealand rabbit is 5, ♂, 2.5kg.One gives physiological saline, and 4 are carried out vaccine (1mg/ only) immunity.

2. SD rat, 15, ♀, 250g is divided into 3 groups at random, is respectively normal control group, physiological saline group and TT-BLyS immune group.

3. Balb/c mouse (about 18g) is 70, and ♂ is divided into 7 groups at random.TT-BLyS ^{Low, high, low+adjuvant, high+adjuvant}, GST-BLyS ^{Low, high}With the physiological saline control group.Wherein high dosage is 80 μ g/, and low dosage is 8 μ g/.

3.2.3 experimental technique

The multi-point injection immunity of the equal back of animal of all categories for new zealand rabbit, has been used the Fu Shi immunological adjuvant, and wherein Freund's complete adjuvant is used in immunity for the first time, is Freund for the second time; The SD rat is not used immunological adjuvant; The Balb/c mouse carries out immunity according to grouping.Every treated animal immunity 4 times whenever biweekly, is an abdominal injection for the last time, eyeball blood sampling after 10 days, and 4 ℃ solidify, low-temperature centrifugation (10000rpm, 4 ℃) 30min, separation of serum, 4 ℃ of preservations are carried out the detection that the BLyS polyclonal antibody is tired according to the ELISA method.

3.2.4 the detection of antibody titer

3.2.4.1 bag quilt

The BLyS standard substance that dilution is good add elisa plate, and 36-48h is placed for 4 ℃ in 100 μ l/ holes.

3.2.4.2 add serum to be checked

Wash elisa plate 3 times with washings, add test serum sample, negative control and the blank of 1: 100,1: 1000,1: 10000,1: 100000 gradient dilution, 100 μ l/ holes, 37 ℃, 1h.

3.2.4.3 add enzyme labelled antibody

Wash plate 3 times with washings, add the good enzyme labelled antibody of dilution, 100 μ l/ holes, 37 ℃, 45min.

3.2.4.4 colour developing

Washings is washed plate 3 times, adds the ABTS colour developing liquid for preparing, 100 μ l/ holes, color development at room temperature 15-30min.

3.2.4.5ELISA readout instrument is surveyed the 410nm OD of place value, as shown in Figure 6.

3.3TT-BLyS provide protection to EAE and RA rat model

3.3.1EAE model

3.3.1.1 animal and grouping

Six the week age SD rat, 250g, ♀ is divided into 3 groups at random: normal control group, model group and treatment group.

3.3.1.2 experimental technique

3.3.1.2.1 model is induced

The treatment group goes out neutrality antibody with the TT-BLyS immune induction in advance, carries out inducing of disease model after mensuration is tired.Spinal induction is adopted in the foundation of MS disease model.Get the physiological saline homogenate of spinal cord preparation 50% (weight/volume) of normal rat, mix, make emulsion with the equal-volume complete Freund's adjuvant; In the every sufficient sole of the foot intradermal injectable emulsion 0.1mL of rat, inject 0.4mL altogether for every then.Carry out booster immunization with the subcutaneous injection of 0.1mL back after two weeks.Observe clinical indication in the immunologic process, the brain of delivery type animal and myeloid tissue's sections observation damage after finishing.

3.3.1.2.2 study of behaviour evaluation

Clinical score is with reference to the correction standard of Weiner etc.: 0 grade is not morbidity; 1 grade is the decline of afterbody mobility; 2 grades are the afterbody paralysis; 3 grades are affected for one or both sides hind leg activity; 4 grades is acroparalysis behind the both sides; 5 grades is all paralysis or moribund condition or death of front and back limb.

3.3.1.2.3 morphological observation

1) the SD rat is poured into 4% Paraformaldehyde 96, brain and spinal cord fixing 6h-8h in formaldehyde phosphoric acid buffer stationary liquid (pH 7.15 for the PBS that contains 4% formaldehyde, 0.05mol/L), 4 ℃ of temperature.

2) with phosphoric acid buffer (0.05mol/L, pH 7.28) flushing 2 times, the time is each 40min-60min, 4 ℃ of temperature.

3) in 5% sucrose-PBS liquid, spend the night 4 ℃ of temperature.

4) 50% acetone fixed 30-45min.

5) go into pure acetone, change 2 times, time 4h, 4 ℃ of temperature, last 1h-2h can at room temperature carry out, and goes in the dimethylbenzene, changes 3 times, each 20min.

6) saturating wax

Go in the wax cup that has melted, change 2 times, soak 90min, 54 ℃-56 ℃ of temperature, paraffin embedding.

7) cut into slices after repairing piece, thickness is 5 μ m, the warm water tank that fills about 40 ℃ is put in section launched, and being attached on the slide glass (has adhesion promotor).

8) behind the paster, blot, put 37 ℃ of incubator inner dryings, behind the 12h, after the dewaxing treatment, carry out tissue incubation's reaction routinely with filter paper.

9) with phosphoric acid buffer (PBS, pH 7.4) flushing 3 times, each 5min.

10) carry out HE dyeing and observe spinal cord and cerebellum inflammatory cell infiltration situation; Perhaps fast blue (Fast Luxol Blue)-cresyl viollet myelin staining is observed the myelin integrity.

11) image analyzer microimaging.

3.3.2RA model

3.3.2.1 animal and grouping

3.3.2.2 experimental technique

The treatment group goes out neutrality antibody with the TT-BLyS immune induction in advance, carries out inducing of disease model after mensuration is tired.The foundation of RA disease model adopts adjuvant to induce.Complete Freund's adjuvant (CFA) faces with preceding abundant mixing, gets 0.1mL and is injected in the right back sufficient sole of the foot intracutaneous of SD rat and induces sacroiliitis to take place, and gets 0.125mL (2u/mL) TNF-α again respectively at right ankle joint of rat and infrapatellar fat pad injection booster immunization.After model is induced successfully hind leg is taken the X-mating plate, observe the inflammation situation in joint.

3.4 effect of the present invention

1) by the substep cloning, the oligonucleotide fragment of the coding TT of synthetic is connected with the 5 ' end of BLyS, made up cloning vector pGEM-3Zf (-)-TT-BLyS of TT-BLyS fusion gene, the determined dna sequence confirmation is entirely true.

2) made up the prokaryotic expression carrier pKKH-TT-BLyS of TT-BLyS fusion gene, induced through IPTG, SDS-PAGE and Western-blot confirm to obtain to express in intestinal bacteria.

3) purifying the TT-BLyS fusion rotein.Purity is greater than 95% (referring to Fig. 5).

4) the TT-BLyS immune animal can induce the BLyS polyclone neutralizing antibody of high titre in different classes of animal (new zealand rabbit, rat and mouse) body.This vaccine all has good protective action to EAE and RA rat model.

5) with three kinds of fusion roteins in addition of similar methods clone promptly: PADRE-BLyS, BLyS-TT have obtained similar result (referring to Fig. 6) with BLyS-PADRE.

Claims

1. the preparation method of tetanus toxin t cell epitope polypeptide and human B lymphocyte stimulating factor fusion rotein TT-BLyS is characterized in that carrying out according to the following steps:

1) construction of recombinant plasmid

1. the clone of TT epitope peptide gene

Coding TT830-843 has introduced the EcoRI restriction enzyme site at this 5 ' end to oligonucleotide fragment, and 3 ' end has been introduced the BamHI restriction enzyme site:

S1?5’-AAT?TCA?TGC?AGT?ACA?TCA?AAG?CTA?ACT?CCA?AAT?TCA?TCG?GTA?TCA?CTG?AAG-3’

S2?5’-GAT?CCT?TCA?GTG?ATA?CCG?ATG?AAT?TTG?GAG?TTA?GCT?TTG?ATG?TAC?TGC?ATG-3’

Get equivalent S1 and S2, boiled 5 minutes, naturally cool to room temperature, anneal; Cloning vector pGEM-3Zf (-) is through EcoRI and BamHI double digestion and reclaim big fragment; The annealing product of TT and carrier enzyme are cut the big fragment that reclaims the back and are carried out ligation through the T4 ligase enzyme; Connect product transformed competence colibacillus cell DH5 α, picking mono-clonal overnight incubation is extracted plasmid, carries out double digestion and determined dna sequence and identifies;

2. the structure of human B lymphocyte stimulating factor and TT fusion gene cloning carrier

Obtain people BLyS gene by the PCR reaction, simultaneously the BamHI restriction enzyme site is introduced 5 ' end primer, the PstI restriction enzyme site is introduced 3 ' end primer; Primer sequence is as follows:

P1 (5 ' end primer 31nt) 5 '-GCG GGA TCC ATG GCC GTT CAG GGT CCA GAA G-3 '

P2 (3 ' end primer 31nt) 5 '-GCG CTG CAG TCA CAG CAG TTT CAA TGC ACC A-3 '

Pcr amplification is carried out in human leukocyte cDNA library; The preparation of pcr amplification reaction pipe is all carried out in ice bath, and the PCR reaction system is: do not contain Mg ²⁺10 * PCR buffer, 5 μ L, 2.5mmol/L dNTP 4 μ L, each 1 μ L of primer, 25mmol/LMgCl ₂3 μ L, cDNA template 1 μ L, Taq archaeal dna polymerase 1 μ L adds water to cumulative volume 50 μ L; PCR reaction conditions: 94 ℃ of 1min, 52 ℃ of 45s, 72 ℃ of 1min, totally 30 circulations; At last, 72 ℃ are extended 10min; The PCR product carries out 0.8% agarose gel electrophoresis, the dna fragmentation of visible expection size;

Plasmid pGEM-3Zf (-)-TT reclaims big fragment behind BamHI and PstI double digestion, the fragment of recovery and above-mentioned PCR product carry out ligation through the recovery product of same double digestion; Connect product transformed competence colibacillus cell DH5 α, picking mono-clonal overnight incubation is extracted plasmid, cuts through enzyme and identifies the insertion fragment that obtains the expection size, checks order, and the result is consistent with expection;

2) construction of prokaryotic expression vector and expression of recombinant proteins

Plasmid pGEM-3Zf (-)-TT-BLyS reclaims small segment behind EcoRI and PstI double digestion, protokaryon non-fusion expression carrier pKKH also reclaims big fragment behind above-mentioned same double digestion, and both carry out ligation; Connect product transformed competence colibacillus cell DH5 α, picking mono-clonal overnight incubation, extract plasmid, enzyme is cut and is identified correct plasmid called after pKKH-TT-BLyS, the bacillus coli DH 5 alpha that will contain recombinant plasmid pKKH-TT-BLyS is inserted activation jolting overnight incubation in the LB substratum in 37 ℃, m seq, be inoculated in the LB substratum in 1: 100 ratio after, continue 37 ℃ and cultivate 3h to logarithmic growth later stage, OD _650nm=0.4 o'clock, add 1mM IPTG, cultivate 4h, collect thalline; Ultrasonic split bacterium after, centrifugal collection inclusion body;

3) evaluation of expression product

Adopt Western blot to identify to the evaluation of expression product, detect the atopic of itself and BLyS monoclonal antibody, step is as follows:

1. split the bacterium product after pKKH-TT-BLyS/DH5 α induces and carry out SDS-PAGE;

2. after the electrophoretic separation, according to the Bio-Rad description of product, near negative electrode one side, close anode one side of nitrocellulose filter, 1 hour electrophoresis of 100V constant voltage is transferred to albumen on the NC film in the transfering buffering liquid of precooling with gel;

3. after electrophoresis finishes, take out the NC film, washings TBST clean the back immerse among the TBST that contains 2%BSA 37 ℃ 1 hour, the TBST room temperature is washed 3 times, 5min/ time, adds goat-anti people BLyS antibody, hatched 1 hour for 37 ℃, TBST washes 3 times, adds the anti-sheep IgG-AP of rabbit, hatched 1 hour for 37 ℃, TBST washes 3 times, washes 3 times with TBS again, and the NC film immerses in the colour developing liquid, room temperature lucifuge colour developing appropriate time, the distilled water flushing termination reaction; Observe the specific reaction of induced product and BLyS antibody;

Simultaneously, purified product is carried out the-terminal amino acid order-checking, method is: be transferred on the pvdf membrane behind the protein s DS-PAGE electrophoresis of purifying, and R-250 dyeing, the decolouring back uses the Edman edman degradation Edman at 492 type Procise ^TMAnalyze the N terminal amino acid sequence on the cLC protein sequencing instrument;

4) purifying of recombinant protein

Per 1 gram thalline adds 5ml and splits the bacterium damping fluid, and both are V by mixed volume, and thalline is resuspended, and the prescription that splits the bacterium damping fluid is: 0.1mol/L Tris, pH7.0; 1mmol/L EDTA, and the N,O-Diacetylmuramidase of adding 1.5mg/mL stirred 30 minutes under the room temperature; Add the DNase I of 10 μ g/mL and the MgCl of 20mmol/L then ₂, continuation effect 30 minutes; Adding final concentration is the 20mmol/L EDTA of 1.5mol/L NaCl, 2% Triton X-100 and 3V, 4 ℃ were stirred 30 minutes, centrifugal under 4 ℃ of conditions, 12000rpm, 20min, 4mol/L Urea, 2% Triton X-100,20mol/L EDTA, the 0.1mol/LTris pH8.0 room temperature washing of collecting precipitation usefulness 3V 30 minutes, precipitation is used 20mol/L EDTA, the 0.1mol/L Tris pH7.0 of 8V again, room temperature washing 30 minutes, centrifugal collection inclusion body; Inclusion body dissolves with 7M Guanidinium hydrochloride, 10mg/L DTT, centrifugal back supernatant carries out gel permeation chromatography with Sephacryl 300, and elutriant is the 7M Guanidinium hydrochloride, collects elutriant, carry out quality evalution respectively to 4M urea, 2M urea and the water renaturation of dialysing, and to purified product.

2. the preparation method of tetanus toxin epitope peptide as claimed in claim 1 and human B lymphocyte stimulating factor fusion rotein TT-BLyS is characterized in that, described pcr amplification reaction pipe consists of:

cDNA 1μl

2.5mM?dNTPs 4μl

25mM?MgCl ₂ 3μl

50μM?P1 1μl

50μM?P2 1μl

5.0U/ μ l Taq enzyme 1 μ l

10×PCR?Buffer 5μl

H ₂O 34μl

Amount to 50 μ l.

3. the tetanus toxin t cell epitope polypeptide that obtains of claim 1 or 2 described preparation methods and human B lymphocyte stimulating factor merge egg TT-BLyS, it is characterized in that, select tetanus toxin epitope peptide Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile-Thr-Glu-and human B lymphocyte stimulating factor to make up the fusion gene of TT and bone-marrow-derived lymphocyte stimulating factor, and in intestinal bacteria, obtain to efficiently express, obtain the TT-BLyS fusion rotein after purified.