CN105906717A

CN105906717A - Preparation method and application of Brucella multi-epitope fusion protein vaccine

Info

Publication number: CN105906717A
Application number: CN201610277587.XA
Authority: CN
Inventors: 徐坤; 李娟�; 李丽; 殷德辉; 宋秀玲; 王娟; 刘玉申; 宋丹丹; 曲笑锋; 鞠文; 赵超; 庞博; 孟祥君; 张惠雯; 翟玥; 暴昊
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2016-04-29
Filing date: 2016-04-29
Publication date: 2016-08-31

Abstract

The invention discloses a Brucella multi-epitope fusion protein antigen. Amino acid sequences of dominant epitopes of Brucella outer membrane proteins BP26, OMP31, OMP16 and OMP2b are in series connection to construct a fusion protein gene to express the proteins, and a Brucella multi-epitope fusion protein antigen vaccine is prepared. Mice challenge tests show that the Brucella multi-epitope fusion protein antigen vaccine plays a role in immune protection in Brucella infection.

Description

The preparation of brucella Multi-Epitope Fusion Protein vaccine and application

Technical field

The present invention relates to Protocols in Molecular Biology and field of immunology.It is specifically related to a kind of brucella multi-epitope and merges egg Bai Kangyuan and the application in terms of preparation brucellosis Seedling thereof.

Background technology

In recent years, along with the fast development of economic globalization, new infectious disease constantly occurs, and some old infectious disease are also opened Begin revivable, especially the most serious to mankind's harm brucellosis, sickness rate was in the trend increased year by year in recent years, Huge public health problem and economic loss is brought to countries in the world.

Brucellosis is to be invaded, by brucella, a kind of Arbo infectious disease that body causes.Population infection cloth Lu Shi Can cause the systemic symptoms such as heating, hyperhidrosis, weak, migrans arthralgia after bacterium, there is liver spleen and swollen testis in severe patient, closes Joint deformation, final disability and fertility；Animal is after infecting brucella, and male animal there will be swollen testis, shadow Ringing breeding and breeding, female animal is likely to occur miscarriage, premature labor, and aborted fetus, afterbirth, vaginal secretions etc. containing substantial amounts of carefully Bacterium, if sterilized not in time, it is also possible to causes same group's zoogenetic infection or infects the mankind.Brucella mainly exists after entering body Cytozoicus, and breed, therefore, general medicine is difficult to play drug effect, and relapse rate is the highest, currently without preferably controlling Treating brucella medicine, vaccine prevention is the maximally efficient means of this disease of prevention and control.Brucella vaccine kind is more at present, Mainly include attenuated live vaccines, inactivated vaccine, mutant vaccine, DNA vaccination etc.^[1].Owing to these vaccines all also exist animal stream The shortcomings such as product, immune effect are bad, the most also do not have especially desirable brucella vaccine to be applied to sick the preventing of cloth Lu Shi Control.Therefore development of new, effective vaccine are study hotspots sick for current prevention and control cloth Lu Shi.

Genetic engineering subunit vaccine and amalgamation protein vaccine are important directions of brucellosis vaccine research, Its principle is to use DNA recombinant technique, by the channel genes engineering bacteria (recipient bacterium) of coding pathogenic microorganism specific antigen epitope Or cell so that it is efficiently expressing in recipient cell, make vaccine after being subsequently adding immunological adjuvant, having an advantage in that can Extend protective period limit, do not have attenuated live vaccine change pathogenicity and cause human or animal to be caused a disease, prevent animal occur miscarriage, Good stability etc..Therefore, this research is intended utilizing bioinformatics technique to predict the B cell of brucella main protection antigen, T Cell dominant antigen epi-position, utilizes immunological technique to screen preferable epi-position, and Dominant Epitopes is entered by recycling fusion protein technology Row series system is for going out new generation vaccine that have autonomous innovation, that immunogenicity is good, and prevention and control for brucellosis carry For good means.

Summary of the invention

It is an object of the present invention to provide a kind of brucella Multi-Epitope Fusion Protein antigen.

Brucella Multi-Epitope Fusion Protein antigen, its aminoacid sequence is as shown in sequence table SQ ID No.1.

Brucella Multi-Epitope Fusion Protein antigen, its base sequence is as shown in sequence table SQ ID No.2.

Brucella Multi-Epitope Fusion Protein antigen vaccine, it is that the brucella multi-epitope described in claim 1 merges Proteantigen.

The invention provides brucella Multi-Epitope Fusion Protein antigen, be by brucella outer membrane protein BP26, The aminoacid sequence of OMP31, OMP16, OMP2b dominant antigen epi-position is connected, and constructs antigen-4 fusion protein gene, have expressed egg In vain, brucella Multi-Epitope Fusion Protein antigen vaccine it is prepared for.Mice challenge viral dosage shows to have Infected with Brucella to exempt from Epidemic disease protectiveness.

Accompanying drawing explanation

Fig. 1 brucella rMEP expresses and location (M: albumen Marker；1: before induction；The supernatant of 2:37 DEG C of induction 4h； The precipitation of 3:37 DEG C of induction 4h)；

Fig. 2 brucella rMEP nickel agarose affinity chromatography SDS-PAGE result (M:Protein marker；1: loading；2: stream Go out；3:10mmol/L Imidazole elution fraction；4:20 mmol/L Imidazole elution fraction；5-8:50 mmol/L Imidazole elution fraction；9:500 mmol/L Imidazole elution fraction)；

Fig. 3 brucella rMEP anion-exchange chromatography SDS-PAGE result (M:Protein marker；1: loading；2: flow out； 3:50 mmol/L NaCl elution fraction；4-7:100 mmol/L NaCl elution fraction；8-10:150 mmol/L NaCl washes De-component；11:200 mmol/L NaCl elution fraction；12:300 mmol/L NaCl elution fraction；13:1 mol/L NaCl Elution fraction)；

Fig. 4 Bacillus brucellae rMEP expresses SDS-PAGE qualification result, and (M is protein Marker；1 merges egg for Bacillus brucellae multi-epitope In vain)；

Fig. 5 Bacillus brucellae rMEP expresses Western Blot qualification result, and (M is protein Marker；1 melts for Bacillus brucellae multi-epitope Hop protein)；

Specific antibody level measurement result in tri-groups of mice serums of Fig. 6；

Subtype-specific antibody measurement result in tri-groups of mice serums of Fig. 7；

In tri-groups of mouse boosting cell culture supernatant of Fig. 8, cytokine IFN-γ and IL-6 measures；

Tri-groups of mice CTL activity testing results of Fig. 9；

Tri-groups of mouse T cell hypotype testing results of Figure 10.

Detailed description of the invention

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.Unreceipted concrete bar in enforcement The experimental program of part, generally according to normal condition, or is carried out according to the condition proposed by manufacturer.

The design of embodiment 1 brucella multi-epitope fusion gene and the structure of plasmid

From Pubmed data base, transfer the aminoacid sequence of brucella advantage outer membrane protein, use BepiPred (http://www.cbs.dtu.dk/services/Bepipred/), ABCpred (http:// www. imtech. Res. in/ raghava/abcpred/) and COBEpro (http: // scratch. proteomics. ics. uci. Etc. edu/) the B cell epi-position of brucella outer membrane protein is predicted by software, uses IEDB (http: // tools. Iedb.org/main/tcell/) t cell epitope is predicted by the t cell epitope forecasting tool of website, uses BLASTP Analysis carries out similarity analysis, selection advantage epitope to epitope, adds one section of connection between adjacent epi-position Peptide sequence, uses DNA Star to be optimized the series sequence of epitope and the kind of connection peptides, selects immunogenicity good Sequence as the aminoacid sequence of purpose amalgamation protein vaccine, for expression and the application of next step brucella rMEP vaccine Research.

Use DNAStar to the designed α spiral of brucella rMEP vaccine, β-pleated sheet, flexibility, hydrophilic, antigenicity It is predicted Deng amynologic parameter, carries out codon optimized according to the aminoacid sequence of the brucella rMEP vaccine designed After, synthetic or PCR method construction of fusion protein gene order are as shown in SEP ID NO.2, by constructed fusion protein Gene is connected to the polyclone enzyme action site of PET-28b carrier.

The advantage outer membrane protein chosen in above-mentioned steps is brucella BP26, OMP31, OMP16 and OMP2b.

Epitope in above-mentioned steps is shown in Table 1.

The coding " GGGS " of above-mentioned connection peptides sequence Linker.

The expression of embodiment 2 brucella rMEP

Expressive host bacterium e. coli bl21 (DE3) strain is preserved by sanitary inspection teaching and research room of HSPH of Jilin University； Expression vector pET-28b plasmid containing brucella rMEP vaccine base sequence is by constructed in embodiment 1；Various molecular biosciences Learn reagent and be all purchased from biological reagent company.

The pET-28b plasmid transformation escherichia coli BL21(DE3 that will obtain in embodiment 1) expression bacterium, 42 DEG C of thermal shock 90s, It is coated with LA flat board (containing 30 μ g/mL kanamycin), 37 DEG C of overnight incubation after standing 2min on ice.The single bacterium colony of picking expression strain In 4mL LB culture medium (containing 30 μ g/mL kanamycin), 37 DEG C, 220r/min cultivates 18 ~ 24h.Take 0.4mL culture to add Continue to cultivate, as the OD of bacterium solution in LB culture medium (containing 30 μ g/mL kanamycin) fresh for 4mL₆₀₀When reaching about 0.6, add Adding the IPTG of final concentration of 0.5mmol/L, 37 DEG C, 220r/min induces 4h, concurrently sets the conduct being not added with IPTG derivant Negative control.12000r/min is centrifuged 10min, collects thalline.Use the expression of SDS-PAGE detection brucella rMEP vaccine Situation and location, result is as shown in Figure 1.Result display Host Strains all has purpose to melt after induction in bacterial precipitation with supernatant Hop protein is expressed, and expression is the highest in bacterial sediment, and the brucella fusion protein expressed by explanation is inclusion body protein.

In order to obtain the brucella rMEP of solubility, by collect thalline with disruption buffer (0.05mol/L Tris, 0.3 mol/LNaCl, 0.1% Trition X-100, pH=8.0) resuspended after, ultrasonication thalline in ice bath, ultrasonic power is The ultrasonic 2s of 400W, 20min(, suspend 6s), ultrasonic after, 12000r/min, 4 DEG C of centrifugal 20min, abandon supernatant, precipitation inclusion body Dissolve buffer (0.008mol/L carbamide, 0.05mol/L Tris, 0.3 mol/LNaCl, 0.1%Triton X-100, pH= 8.0) dissolving, ultrasonication thalline in ice bath, ultrasonic power is the ultrasonic 2s of 400W, 20min(, suspends 6s), 12000r/min, 4 DEG C centrifugal 20min, abandons precipitation, containing purpose fusion protein in supernatant, carries out next step purification.

The purification of embodiment 3 brucella rMEP

Take the supernatant after above-described embodiment 2 ultrasonication, containing brucella solubility rMEP in described bacterial supernatant, adopt With nickel agarose affinity chromatography and anion-exchange chromatography, brucella solubility rMEP in supernatant is purified, specifically Refined solution and purification process are prepared according to the description of related kit and are operated, by respectively washing of the purifying protein liquid of collection High-purity component, after SDS-PAGE detects, is carried out dialysis in PBS (containing 0.1%SKL, pH=7.4) by de-component, and 4 DEG C thoroughly Analyse overnight, the 0.45 degerming rear subpackage of μm membrane filtration, it is placed in-80 DEG C of refrigerators and preserves for a long time.Use SDS-PAGE electrophoresis and Fusion protein after purification is detected by Western Blot detection, and as shown in figures 4 and 5, expressed melts result Hop protein molecular weight is positioned at about 60kD, and band is clear and does not has miscellaneous band.Through SK3071 non-interference type determination of protein concentration reagent Box measures protein concentration, and standard curve equation is y=-0.0054x+0.9946, R²=0.9946, it is computed, albumen after purification Concentration is 1.39mg/mL.

The preparation of embodiment 4 brucella rMEP vaccine and mouse immune

Taking the brucella rMEP of above-described embodiment 3 purification, adjusting protein concentration with sterile PBS buffer is 600 μ g/mL, with Isopyknic Freund's complete adjuvant (or incomplete Freund's adjuvant) mixes to complete emulsified state, is brucella rMEP epidemic disease Seedling.Taking brucella vaccine strain M5-90, adjusting bacteria concentration is 2 × 10⁹CFU/mL, with isopyknic Freund's complete adjuvant (or not Family name's Freund's incomplete adjuvant) mix to complete emulsified state, it is control vaccine.Take aseptic PBS, with isopyknic Freund's complete adjuvant (or incomplete Freund's adjuvant) mixes to complete emulsified state, is negative vaccine.

Taking BALB/c mouse 30, after labelling, adaptability is raised one week, the most without exception, and mice is randomly divided into three groups, the One group of mouse immune brucella rMEP vaccine, second group of mouse immune control vaccine, the 3rd group of mouse immune feminine gender vaccine, make For blank group.Mouse immune approach is lumbar injection.Initial immunity uses Freund's complete adjuvant vaccine to carry out immunity, for the first time Immunity uses immunity incomplete Freund's adjuvant vaccine booster immunization in latter 15 days, 30 days and 45 days, and immunization route is lumbar injection, Dosage is only 100 μ L/, all uses the mode of tail vein blood to collect serum before each immunity, and-70 DEG C save backup.

The immune protective effect evaluation of embodiment 5 brucella rMEP vaccine and response analysis

(1) in mice serum, specific antibody level measures

The change of specific antibody level in mice serum before and after employing indirect elisa method detection immunity, concrete operation step is: By the brucella rMEP of embodiment 3 purification, brucella vaccine strain M5-90 coated elisa plate, 100 μ L/ holes, 4 DEG C of mistakes respectively At night, PBST claps after washing three times, closes 37 DEG C with 1%BSA and closes 2h, 300 μ L/ holes, adds 1:200 and start the serum of doubling dilution Sample, 100 μ L/ holes, to hatch after 1h, PBST clap and wash three times for 37 DEG C, the sheep anti mouse two of the HRP labelling adding 1:10000 dilution resists, 100 μ L/ holes, 37 DEG C hatch 1h, PBST clap wash three times after, add TMB nitrite ion, 100 μ L/ holes, room temperature lucifuge reaction 15min, Add 2M H₂SO₄, 50 μ L/ holes terminate reaction, and microplate reader reads OD450.Result as shown in Figure 6, immunity brucella rMEP and After brucella vaccine strain M5-90, in Mice Body, all can produce specific antibody, and the mice of immunity brucella rMEP The internal time mice early than immunity brucella vaccine strain M5-90 that specific antibody occurs.

(2) in mice serum, subtype-specific antibody measures

Antibody subtype is used to measure test kit (Cat.5300-5;Southern Biotech, Birmingham, AL, USA) Total IgG, the level of IgM, IgG1, IgG2a, IgG2b and IgG3 in detection mice positive serum, concrete operation step is: with real Execute the brucella rMEP of example 3 purification, brucella vaccine strain M5-90 and capture antibody coated elisa plate, 100 μ L/ holes, 4 DEG C Overnight, PBST claps after washing three times, closes 37 DEG C with 1%BSA and closes 1h, 300 μ L/ holes, adds the Mouse Blood final proof of 1:1000 dilution Product, 100 μ L/ holes, hatch after 1h, PBST clap and wash three times, add the subclass antibodies of HRP labelling of 1:500 dilution, 100 μ L/ for 37 DEG C Hole, 37 DEG C hatch 1h, PBST clap wash three times after, add ABTS nitrite ion, 100 μ L/ holes, room temperature lucifuge reaction 10 ~ 20 min, add Enter 2M H₂SO₄, 50 μ L/ holes terminate reaction, and microplate reader reads OD450.Result as it is shown in fig. 7, immunity brucella rMEP vaccine After vaccine strain M5-90, IgM, IgG1, IgG2a, IgG2b and the IgG3 in mice serum all rises brilliant compared with negative control group Aobvious, and IgG1, IgG2a, IgG2b and IgG3 level in immunity brucella rMEP mice serum is right apparently higher than feminine gender According to group and vaccine strain M5-90 group, the brucella rMEP vaccine prepared by explanation can cause stimulates body to produce strong body Liquid immunne response.

(3) mouse spleen lymphocyte separates

Last immunity terminates two weeks after, and cervical dislocation puts to death mice, and aseptic separating mouse spleen after body surface sterilization, by spleen It is placed on steel mesh, adds appropriate 1640 culture medium and be lightly ground spleen, prepare single cell suspension, after 1000r/min is centrifuged 5min, Abandoning supernatant, add 10mL erythrocyte cracked liquid, static 5min, 1000r/min are centrifuged 5min, with 1640 culture medium centrifuge washings Twice of cell, is resuspended in cell in 1640 culture medium, obtains mouse spleen lymphocyte single cell suspension.

(4) cytokine IFN-γ and IL-6 measures

Take above-mentioned mouse spleen lymphocyte single cell suspension, with 1640 cultivations containing 10% hyclone adjust cell concentrations be 2 × 10⁶Individual/mL, joins in 24 orifice plates, every hole 2mL, 37 DEG C, 5%CO₂Overnight incubation, discards culture fluid, and every hole is separately added into 1mg/ ML fusion protein, PBS or 2.5 mg/mL ConA, 37 DEG C, 5%CO2 cultivates 48h, collects supernatant, for the survey of cytokine Fixed.IFN-γ and IL-6 cytokine assay test kit (Peprotech, Rocky Hill, NJ, USA) is used to measure spleen thin The level of IFN-γ and IL-6 in born of the same parents' culture supernatant, concrete operation step is: be coated enzyme mark with the capture antibody in test kit Plate, 100 μ L/ holes, 4 DEG C overnight, and PBST claps after washing three times, with 1%BSA, 300 μ L/ holes, closes room temperature and closes, and every hole is separately added into The standard substance of variable concentrations or spleen cell cultures supernatant, 100 μ L/ holes, incubated at room 2h, PBST claps after washing three times, adds raw The detection antibody of thing element labelling, 100 μ L/ holes, incubated at room 2h, PBST claps after washing three times, the HRP of addition Avidin labelling, and 1: 2000 dilutions, 100 μ L/ holes, incubated at room 0.5h, PBST claps after washing three times, adds ABTS nitrite ion, and 100 μ L/ holes, room temperature is kept away Photoreaction 10 ~ 20 min, adds 2M H₂SO₄, 50 μ L/ holes terminate reaction, and microplate reader reads OD450.Result as shown in Figure 8, is exempted from After epidemic disease brucella rMEP vaccine and vaccine strain M5-90, the IFN-γ produced in mouse boosting cell culture supernatant and IL-6 water Flat significantly raised, and the IFN-γ that produces in immunity brucella rMEP vaccine mouse boosting cell culture supernatant and IL-6 water Flat apparently higher than negative control group and vaccine strain M5-90 group, the brucella rMEP vaccine prepared by same explanation can cause Body is stimulated to produce strong humoral immunoresponse(HI).

(5) CTL killing experiments

Taking above-mentioned steps (3) mouse spleen lymphocyte single cell suspension, adjusting cell concentration is 3 × 10⁶Individual/mL joins 96 holes In Tissue Culture Plate, 37 DEG C, 5%CO₂Cultivating 2h, attached cell is macrophage, washes away the most adherent cell, adds 10 μ g/ The fusion protein of mL, overnight incubation, according to splenocyte: macrophage=25:1 adds splenocyte, arrange simultaneously only have splenocyte and The only matched group of effector lymphocyte, 37 DEG C, 5%CO₂Cultivating 24h, every hole adds 20 μ L MTT, 37 DEG C, 5%CO₂Cultivate 4h, discard Supernatant culture fluid, every hole adds 150 μ L DMSO, and microplate reader reads 0D492.Result is as it is shown in figure 9, illustrate immunity brucella The CTL activity of the mice of rMEP vaccine and vaccine strain M5-90 is all remarkably higher than negative control group, the brucella prepared by explanation The CTL activity of splenocyte can preferably be induced by rMEP vaccine.

(6) T cell hypotype measures

Taking above-mentioned steps (3) mouse spleen lymphocyte single cell suspension, adjusting cell concentration is 1 × 10⁷Individual/mL, takes 100 respectively μ L joins in EP pipe, and 1000r/min is centrifuged 5min, abandons supernatant, with the PBS re-suspended cell containing 2% hyclone, adds FITC The anti-Mus CD4 antibody of labelling, the anti-Mus CD8 antibody of PE labelling, the anti-Mus CD3 antibody of APC-Cy7 labelling, lucifuge, ice bath reacts 15 ~ 20min, 1000r/min are centrifuged 5min, with the PBS washed cell containing 2% hyclone twice, with 350 μ L containing 2% tire cattle The PBS re-suspended cell of serum, uses flow cytometer to detect.Result as shown in Figure 10, illustrates immunity brucella rMEP CD3, CD4 of vaccine mice and cd8 cell ratio are above mice and the negative control of immunity brucella vaccine strain M5-90 Group, the brucella rMEP vaccine prepared by explanation has more preferable immanoprotection action than brucella vaccine strain M5-90.

(7) mice challenge viral dosage

Last immunity terminates to carry out for latter 30 days mice challenge viral dosage, every mouse peritoneal injection 5 × 10⁵CFU B. melitensis 16M, counteracting toxic substances terminate after 2 weeks, execution mice, aseptic take spleen, grind with the PBS containing 0.1% Triton X-100 Mill spleen, lapping liquid is applied to TSA flat board after carrying out doubling dilution, 37 DEG C of cultivations carry out colony counting.Result is as shown in table 2, with PBS compares, and Shandong Salmonella rMEP vaccine has obvious protectiveness, the brucella that its immune protective efficiency is existing frequently-used higher than China Vaccine strain M5-90.Experimental result display brucella rMEP vaccine has immune protective to Infected with Brucella.

Immune protective efficiency^a: log in PBS immune group splenocyte₁₀The meansigma methods of CFU deducts in experiment immunization group splenocyte log₁₀The meansigma methods of CFU.

<110>Jilin University

<120>preparation of brucella Multi-Epitope Fusion Protein vaccine and application

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Claims

1. a brucellosis specific fusion protein antigen, its aminoacid sequence is as shown in sequence table SQ ID No.1.

2. a brucellosis specific fusion protein antigen gene, its base sequence is as shown in sequence table SQ ID No.2.

3. a prokaryotic expression carrier, it is to insert such as sequence in the pronucleus protein expression vector containing T7 promoter Gene shown in list SQ ID No.2.

A kind of prokaryotic expression carrier the most according to claim 3, it is characterised in that: described prokaryotic expression carrier is pET- 28b。

5. a colibacillus engineering, it is the e. coli bl21 (DE3) having transfected above-mentioned prokaryotic expression carrier.

6. the detection kit of a brucellosis, it is characterised in that its diagnostic antigen is special described in claim 1 Property fusion protein antigen, detection kit behaviour brucellosis antibody ELISA test kit.