CN107129527A

CN107129527A - A kind of Malian drainage protective antigens HP0623 and preparation method thereof

Info

Publication number: CN107129527A
Application number: CN201710395446.2A
Authority: CN
Inventors: 魏子贡; 赵鹏鹏; 梁辉煌; 唐斌
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2017-05-26
Filing date: 2017-05-26
Publication date: 2017-09-05
Anticipated expiration: 2037-05-26
Also published as: CN107129527B

Abstract

The present invention relates to a kind of Malian drainage protective antigens HP0623 and preparation method thereof, the Malian drainage protective antigens HP0623 is SEZ HP0623 recombinant proteins, is made up of 429 amino acid residues, molecular weight is 47.04kDa；The preparation method includes gene cloning, digestion enzyme company, induced expression, protein purification；The HP0623 can be used for the preparation of vaccine.Malian drainage protective antigens HP0623 of the present invention shows good immune response originality in mouse experiment; the antibody of generation can provide stronger protection; the sterilizing ability of induced high levels; Malian drainage protective antigens HP0623 is located at SEZ surfaces, and rHP0623 can suppress adhesions of the SEZ to the cells of Hep 2.

Description

A kind of Malian drainage protective antigens HP0623 and preparation method thereof

Technical field

The present invention relates to streptococcus protective antigens, specially a kind of Malian drainage protective antigens HP0623 And preparation method thereof.

Background technology

Streptococcosis is a kind of extremely common animal pathogen, and 20 can be classified as according to group specific antigen Group, from A~V, lacks I and J.Animal pathogen is with B group streptococcus (Group B Streptococci, GBS) and C group streptococcus (Group C Streptococci, GCS) is more, and the pathogenic bacteria of people are generally A group streptococcus (GroupA Streptococci,GAS)。

Malian drainage (Streptococcus zooepidemicus, SEZ) belongs to C group's chains of Lan Shi points of groups Coccus, no special preferendum of host, easily morphs, and different strain antigens of originating may be different.The bacterium can be through alimentary canal, hand Art, wound, vaccinate with the approach infection animal such as wound, so as to cause the septicemia of animal, arthritis, mammitis, meninx The animal epidemics such as scorching and endocarditis.People then may be by drawing with infected animal close contact or the food of edible bacterium pollution Send out the sick infection.The antibiotic such as penicillin and Amoxicillin is used for prevention and treatment always, and this is sick, but due to antibiotic Largely use, cause the bacterium drug resistance generation and antibiotic in animal product residual the problems such as.Therefore, prevented using vaccine These problems can fundamentally be solved by controlling the disease.But it is due to that the whole-bacterial-vaccine of inactivation can often cause the pairs such as immune complex Effect, it is therefore desirable to research and develop new subunit protein vaccine to prevent the generation of such disease.The sugar knot of cell surface grappling Hop protein (HP0623) is a kind of newfound protective antigens of Malian drainage cell surface, and it is mainly acted on Host cell, therefore it is expected to be developed to preventing and treating SEZ subunit vaccine.

The content of the invention

In order to solve the above problems, the invention provides a kind of Malian drainage protective antigens HP0623 and its A kind of preparation method, Malian drainage protective antigens HP0623 can be sub- effective for preventing and treating streptococcus equi epizootic disease Kind.

The technical scheme is that：

A kind of Malian drainage protective antigens HP0623, the HP0623 recombinant proteins are SEZ HP0623 weights Histone (rHP0623), by sez_0623 gene codes, is made up of, molecular weight is 47.04kDa, ammonia 429 amino acid residues Base acid sequence is as shown in SEQ ID NO.2.

A kind of Malian drainage protective antigens HP0623 preparation method, is comprised the steps of：

1) PCR is expanded：The use of Malian drainage SEZ C55138 pnca genes group is template, performing PCR is entered with primer Amplification；

2) it is connected with carrier：PCR primer connects after digestion with restriction enzyme with the pET-32a carriers through same enzyme digestion Connect；Described restriction enzyme is BamH I and Hind III；

3) convert and induce：The above-mentioned carrier conversion connected is entered after Escherichia coli (E.coli BL21), 37 are placed in DEG C shaking table culture, when its OD value is 0.6~1.0, adds IPTG inductions 3-5h；

4) purify：Thalline is collected by centrifugation, after being resuspended through PBS, supernatant is collected by centrifugation after high pressure is broken, by the purpose of supernatant Albumen carries out the HP0623 recombinant proteins that purifying obtains purifying by Ni-NTA chromatographic columns.

The primer includes forward primer and reverse primer, and the forward primer and reverse primer respectively have 1 restriction enzyme site.

Further, the primer is：

Forward primer (SEQ ID NO.3)：5 '-CCCGGATCCGCTTGCCTGCTAGTG-3 ', dashed part is BamH I Restriction enzyme site；

Reverse primer (SEQ ID NO.4)：5 '-CTCAAGCTTGAGGGGAAGATCGTATC-3 ', dashed part is Hind III digestion site；

Applications of the Malian drainage protective antigens HP0623 in Malian drainage vaccine is prepared.

The side of Malian drainage vaccine is prepared using the Malian drainage protective antigens HP0623 Method is：Recombinant protein by successful expression and Jing Guo purification process, i.e. Malian drainage protective antigens HP0623, with As vaccine after Montanide Gel 01PR (SEPPIC, France) adjuvant emulsion, recombinant protein concentration described in vaccine is 100 μ g/mL, the concentration is the minimum effective concentration of recombinant protein in vaccine.

The present invention technique effect be：

Compared with prior art, the streptococcus equi beast that the present invention is purified by steps such as gene cloning, expression, purifying Epidemic disease subspecies protective antigens HP0623 recombinant proteins.The present invention obtains target gene by round pcr first, is connected by digestion enzyme Method obtain recombinant plasmid, be transferred in Escherichia coli progress induced expression HP0623 recombinant proteins, printed through protein immunization Note experiment, shows that the recombinant protein has good immunogenicity.Mouse is immunized after adjuvant emulsion in HP0623 recombinant proteins can To provide higher Vaccine effectiveness for it, two exempt from mice serum can provide preferable passive immune protection for mouse.Through Mice serum after rHP0623 recombinant proteins are immune determines its antibody titer through ELISA, shows that recombinant protein can induce high level Antibody titer, and immune response type is based on Th2.HP0623 recombinant proteins antibody can induce stronger sterilizing ability, After SEZ infecting mouses, the transcriptional level of the SEZ extracted in its spleen tissue sez_0623 genes is higher than external training Foster transcriptional level, rHP0623 can suppress SEZ to the adhesion of Hep-2 cells and infect.

Brief description of the drawings

Fig. 1 represent the SDS-PAGE and immunoblotting analysis figure of HP0623 albumen.

In Fig. 1, swimming lane 1~3 is respectively before recombination bacillus coli is induced, after induction and HP0623 albumen after purification；Swimming Road M is albumen pre-dyed Marker.

Swimming lane 4 is the Westernblot testing results that rHP0623 and SEZ infects rehabilitation Swine serum.

Fig. 2, Fig. 3 are that immune response ELISA testing result figures caused by Mice Body are immunized after mouse in recombinant protein.

Antibody level in Fig. 2 for immune group and negative control group compares；The antibody response class induced in Fig. 3 for HP0623 Type compares.

Fig. 4 represents the mouse of immune group, negative control group and positive controls, after attacking poison through SEZ, the survival rate pair of mouse Than figure.

Fig. 5 represents that rHP0623 bis- exempts from serum (experimental group), SEZ inactivated vaccines two and exempts from serum (positive controls) and normal Serum (negative control group) provides the situation comparison diagram of passive immune protection for mouse.

Fig. 6 show the analysis result figure of quantitative PCR.

It is respectively three mouse each bacteria gene expression situation in spleen from left to right in Fig. 6.

Fig. 7, Fig. 8 are surface distributed figures of the flow cytometries HP0623 in SEZ.

Fig. 9 represent the lethal use comparative result figure of Mouse whole blood.

In Fig. 9,1 it is experimental group, 2 is that positive controls and 3 are negative control group.

Adhesion testing result figures of Figure 10 .SEZ to Hep-2 cells.

Embodiment

In order to be better understood from the present invention, it is described further below in conjunction with embodiment and accompanying drawing.

Normal experiment method and condition are used in the embodiment of the present invention, condition is not those skilled in the art in the present invention Known to member, those skilled in the art can combine prior art and realize the present invention.

Embodiment one, bacterial strain and growth conditions

Bacterial strain is SEZ C55138 bacterial strains；Condition of culture is the TSB culture mediums or TSA culture mediums for adding 5% hyclone； 0.6~1.0 is reached in 37 DEG C of concussion and cultivate about 8h, or OD600.Carrier is Escherichia coli pET-32a, and competent cell is large intestine Bacillus BL21.

The SEZ C55138 bacterial strains, Escherichia coli pET-32a, hyclone and e. coli bl21 competent cell are equal For commercially available prod.

Embodiment two, preparation method

HP0623 albumen is by sez_0623 gene codes, and the HP0623 gene forward primers have 1 BamH I digestions position Point, reverse primer has 1 Hind III digestions site.The forward and reverse primer is designed by SEZ genome sequences, by primer Synesis Company synthesizes.

Malian drainage protective antigens HP0623 preparation method, is comprised the steps of：

1) PCR is expanded：Using the genome of C55138 bacterial strains as template, enter performing PCR using following primer and expand：

Forward primer (SEQ ID NO.3)：5’-CCCGGATCCGCTTGCCTGCTAGTG-3 ', dashed part is BamH I Restriction enzyme site；

Reverse primer (SEQ ID NO.4)：5’-CTCAAGCTTGAGGGGAAGATCGTATC-3 ', dashed part is Hind III digestion site；

2) it is connected with carrier：PCR primer use digestion with restriction enzyme after with the pET-32a carriers through same enzyme digestion Connection；

3) convert and induce：After the above-mentioned carrier conversion competent escherichia coli cell BL21 connected, in 37 DEG C of perseverances Treat that it, to exponential phase of growth, adds final concentration of 1mM IPTG, induce 3h in warm shaking table；Do non-induced check experiment simultaneously.

4) purify：Thalline after induction is collected by centrifugation, it is broken through high pressure after being resuspended with sterile PBS, recombinant protein is collected by centrifugation Supernatant, the recombinant protein for the purifying that the destination protein in supernatant is obtained after Ni-NTA column chromatographies.

Immunoblotting analysis is analyzed：Recombinant protein after purification, primary antibody, goat are used as using the rehabilitation Swine serum for infecting SEZ Anti- pig IgG (H+L)-HRP carries out immunoblotting assay as secondary antibody.

The genome of described C55138 bacterial strains is extracted from C55138 bacterial strains by conventional method and obtained.

Primer is synthesized by Wuhan Jin Kairui bioengineering Co., Ltd, and the restriction enzyme is BamH I and Hind III。

IPTG is isopropyl-β-D-thiogalactoside, is the activity inducement material of beta galactosidase.It is special based on this Property, when being converted with lacZ gene carrier DNA by host of lacZ deletion cells, it, which can be used as, has lac or tac etc. The induced expression thing of the expression vector of promoter is used.

Experimental result

PCR obtains sez_0623 gene order (SEQ ID NO.1), by SEZ C55138 bacterial strain sez_0623 genes gram After grand, the protein (SEQ ID NO.2) of 429 amino acid compositions is given expression to, molecular weight is 47.04kDa.Compare induction and The bacterium of non-induced culture, can prove successful expression destination protein, and the stripe size and the size of prediction of destination protein Unanimously.The recombinant protein of purifying is obtained by way of Ni-NTA affinity chromatographys, by immunoassay experiment show, the albumen with Infection SEZ convalescence Swine serum shows good immunoreactivity, as shown in figure 1, HP0623 albumen size and prediction It is in the same size, and it has good immune response originality.

Embodiment three, antibody titer are determined

By enzyme linked immunosorbent assay mice serum IgG antibody, by the recombinant protein HP0623 of purifying in 4 DEG C of mistakes After night coating, the two mouse blood samplings after immune 10 days are separated after serum, gradient doubling dilution, all dilution factors take 100 μ L adds elisa plate, and the mountain sheep anti-mouse igg of horseradish enzyme mark is added after 37 DEG C of reactions, terminating reaction after the completion of developing the color is waited, uses enzyme mark Instrument reads OD values, takes OD values to be more than the serum maximum dilution multiple conduct of+3 times of variance SD (standard deviation) of control serum mean OD value Serum antibody.In order to infer immunization type, further detect IgG1 and IgG2a to determine Th2 and Th1 immune response.

Experimental result：

The level of the IgG titres of immune group is significantly higher than the antibody level of negative control group, immune group as shown in Figure 2 it is anti- Body titre is apparently higher than negative control group.

As a result show that HP0623 can cause significant Th1/Th2 immune responses, compared with the IgG2 antibody of generation, IgG1 Antibody titer is occupied an leading position, as shown in Figure 3 in the antibody response type that HP0623 is induced, and Th2 reactions are occupied an leading position.

Example IV, mice immunisation and challenge test

1st, 30 6~8 week old KM female mices are taken, 3 groups, every group 10 are randomly divided into；

2nd, experimental group：RHP0623 Montanide Gel 01PR (SEPPIC, the France) adjuvant emulsions of 50 μ g purifying Afterwards, it is injected intraperitoneally and first group of mouse is immunized, interval carries out secondary immunity with dosage to mouse in the same way after 14 days；

3rd, positive controls：By C55138 plants of inactivations of SEZ, helped through Montanide Gel 01PR (SEPPIC, France) After agent emulsification, with 1 × 10⁹Second group of mouse is immunized in CFU/mL, by the way of intraperitoneal injection, and interval is after 14 days, same side Formula carries out secondary immunity；

4th, negative control group：3rd group of 10 mouse be injected intraperitoneally through Montanide Gel 01PR (SEPPIC, France) PBS of adjuvant emulsion is compareed；

5th, all mouse secondary immunities are after 10 days, and serum is collected by centrifugation in tail vein blood, and then all mouse attack malicious SEZ C55138 bacterial strains (2 × 10⁵CFU/mL)；

6th, observe and record the growing state of all mouse.

Experimental result：

1) 10 mouse of experimental group, attack and start within the 3rd day after poison death occur, the mouse of survival shows slight face Bed symptom, such as state of mind is poor, becomes thin, and was that full recovery is normal in 6 days, finally there is 4 dead mouses；

2) mouse of positive controls does not totally show obvious clinical symptoms, only has during entirely attacking poison 1 small Mouse occurs dead；

3) mouse of negative control group starts death for second day after poison is attacked, and the mouse of survival shows obvious clinic Symptom, such as hair are messy, and stimulate the reaction is blunt to external world, not active etc., and gradually dead in 6 days after poison is attacked, and finally only 1 Only survive.

As shown in figure 4, positive controls and immune group can provide higher immunoprotection, and positive controls for mouse It is higher than experimental group.As a result show that restructuring rHP0623 albumen can provide immunoprotection for mouse, resist the sense of SEZ velogen strains Dye.

Embodiment five, passive protection test analysis

It is to give the credit to the special immune response stimulatings of HP0623 to confirm to protect, exempts from serum with rHP0623 bis- and passively exempt from Epidemic disease mouse, then carries out challenge test again.

1st, 30 6 week old KM female mices, are randomly divided into 3 groups, every group 10；

2nd, positive controls：Exempt from first group of mouse of serum intravenous immune with SEZ inactivated vaccines two；

3rd, negative control group：With second group of mouse of Normal Mouse Serum intravenous immune；

4th, experimental group：Exempt from the 3rd group of mouse of serum intravenous immune with rHP0623 bis-；

5th, it is immunized after 24h, all mouse carry out challenge test (2 × 10 with SEZ C55138 bacterial strains⁵CFU/mL)；

6th, observe and record the growing state of all mouse.

Experimental result：

1) positive controls mouse does not show clinical symptoms in challenge test, there is 1 mouse during entirely attacking poison It is final dead；

2) negative control group mouse is mainly shown as One's spirits are drooping after poison is attacked, and stimulate the reaction is slow to external world, hair is thick The symptom such as random, and it is dead successively in 3 days；

3) experimental mice is mainly shown as One's spirits are drooping, slow, thick unrest of hair of stimulate the reaction etc. to external world after poison is attacked Returned to normal after symptom, but 3 days, the final survival rate of mouse is 70%.

As shown in figure 5, as a result showing, experimental group and positive controls are significantly stronger than feminine gender to the passive protection ability of mouse Control group.

Embodiment six, quantitative fluorescent PCR analysis

It is fixed by fluorescent quantitative PCR technique in order to assess expression of the SEZ sez_0623 genes inside and outside Mice Body Transcriptional level inside and outside amount detection sez_0623 genosomes.

1st, propagation bacterium in vivo is obtained out of infection SEZ mouse spleen, sterile collection spleen tissue is milled after 4 DEG C, 850 × g centrifuges 5min, takes supernatant liquor in 4 DEG C, 15500 × g centrifugation 5min precipitate the bacterium in as spleen tissue；In vitro Precipitation is collected by centrifugation after it grows into Midexponential in the bacterium of culture；

2nd, bacterium total serum IgE is extracted with sour phenol method；

3rd, it is cDNAs by RNA reverse transcriptions, quantitative PCR reaction is carried out by template of cDNA samples, all reactions carries out three Repeat；

In real-time fluorescence quantitative PCR, quantitative analysis is carried out to sez_0623 genes using following primer：

Forward primer (SEQ ID NO.5)：5’-CGGTATAGTATAGCCAAGC-3’；

Reverse primer (SEQ ID NO.6)：5’-GCATTTCTCCCAACGA-3’；

In real-time fluorescence quantitative PCR, quantitative analysis is carried out to 16S rRNA genes using following primer：

Forward primer (SEQ ID NO.5)：5’-ATCCGAACTGAGATTGGC-3’；

Reverse primer (SEQ ID NO.6)：5’-CCCTTATGACCTGGGCTA-3’；

Experimental result：

As shown in fig. 6, as a result showing that the transcriptional level of the SEZ extracted in mouse spleen sez_0623 genes will obvious height In the SEZ of in vitro culture transcriptional level.

Embodiment seven, flow cytometry

1st, bacterium is resuspended with PBS/BSA (PBS containing 1%BSA), takes the μ L of bacterium solution 200；

2nd, experimental group adds the μ L of mice serum 100 being immunized through rHP0623, and control group adds the mouse blood being immunized through PBS Clear 100 μ L, 4 DEG C of incubation 15min, are then incubated at room temperature 30min；

3rd, 5000rpm centrifuges 3min, collects bacterium；

4th, washed with PBS/BSA 3 times, add FITC- mountains sheep anti mouse secondary antibody, 45min is incubated at room temperature；

5th, washed with PBS/BSA 3 times, be resuspended with 500 μ L PBS, be added in flow cytometer and detected.

Experimental result：

As shown in Figure 7, Figure 8, relative to blank control group, SEZ HP0623 antibody can cause stronger on SEZ surfaces Fluorescence signal, control group bacterium average fluorescent strength is weaker, and the average fluorescent strength of experimental group bacterium has larger lifting, Show that HP0623 albumen is distributed in SEZ surface.

Embodiment eight, the analysis of whole blood lethal dose

To determine after the bactericidal activity of HP0623 antibody, mouse blood test tube of hepari, serum is exempted from or right using rHP0623 bis- It is estimated after being incubated according to serum with the SEZ survived in blood.

1st, treating, SEZ C55138 strain growths, to Midexponential, are collected by centrifugation thalline, bacterial concentration are adjusted with sterile PBS For 10⁴CFU/mL；

2nd, positive controls：Take 900 μ L healthy mices through anticoagulant heparin whole blood, serum is exempted from 100 μ LSEZ inactivated vaccines two Mixing；

3rd, negative control group：Take 900 μ L healthy mices through anticoagulant heparin whole blood, mixed with 100 μ L Normal Mouse Serums；

4th, experimental group：Take 900 μ L healthy mices through anticoagulant heparin whole blood, exempt from serum with 100 μ L restructuring rHP0623 bis- and mix Close；

5th, 100 μ L SEZ bacterium solutions are added into mixed liquor respectively, 37 DEG C of constant-temperature table concussion and cultivate 90min are placed in；

6th, every group takes 100 μ L samples respectively, is coated in TSA flat boards, carries out 3 repetitions, calculates the front and rear survival of hatching Amount of bacteria；

7th, survival rate is calculated as remaining amount of bacteria relative to the percentage for starting amount of bacteria.

Experimental result：

Negative control group sterilizing rate is 9.21 ± 1.29%；Positive controls sterilizing rate is 91.6 ± 1.35%；Experimental group Sterilizing rate is 61.08 ± 1.60%.

As shown in figure 9, as a result showing that anti-rHP0623 antibody can be with the sterilizing ability of induced high levels, anti- HP0623 antibody can have stronger killing ability to SEZ.

Embodiment nine, adherence inhibition test

The influence of Hep-2 cells is adhered to SEZ for detection HP0623, is detected by the adherence test of Hep-2 cells, Key step is as follows：

1st, the DMEM culture Hep-2 cells containing 10% hyclone are added in 24 orifice plates, 37 DEG C of cell trainings are placed on Support in case and cultivate, treat its length to 80% or so；

2nd, cell is washed 3 times using the PBS/BSA of precooling；

3rd, the rSec_205 that 200 μ g/ml purifying is added per hole is incubated 2h, and identical cell adds 200 μ g/ml BSA It is incubated as a control group；

4th, after the completion of being incubated, washed with PBS/BSA 3 times, 1ml 5 × 10 is then added into each hole⁷CFU/ml SEZ C55138 plants in 37 DEG C, 5%CO₂Under the conditions of be incubated 2h；

5th, nonadherent bacterium is washed away with PBS/BSA, then will be thin with 1ml PBS (the Triton X-100 for containing 0.2%) Born of the same parents are dispelled, and dilution rear plate is counted；

6th, HP0623 is counted to the SEZ inhibiting rates for adhering to Hep-2 cells by equation below：

Inhibiting rate=[1- (bacterial population of the cell of the bacterial population of the cell of HP0623 processing/BSA processing)] × 100%.

Experimental result：RHP0623 can suppress adhesions of the SEZ to Hep-2 cells, as shown in Figure 10, compared to control group, Inhibiting rate is 14.9%.

From above-described embodiment as can be seen that Malian drainage protective antigens HP0623 of the present invention preparation Method, including target gene clone, digestion enzyme company, induced expression, protein purification.HP0623 albumen is recombinated through protein immunization imprinting Analysis, shows that it has good immune response originality.In mice serum after recombinant protein is immune antibody titer apparently higher than In negative control group mouse, and the Antibody types of immune mouse generation, IgG1 titres are higher than IgG2a.Mouse is immunized in rHP0623 Stronger Vaccine effectiveness can be provided for mouse afterwards, anti-rHP0623 mouse two, which exempt from serum, obvious passive protection to mouse Power, anti-rHP0623 antibody can be with the sterilizing ability of induced high levels.Sez_0623 genes are in the Mice Body that SEZ infects Transcriptional level is higher than the transcriptional level of in vitro culture, and Flow cytometry result shows, HP0623 is located at SEZ surfaces, RHP0623 can suppress adhesions of the SEZ to Hep-2 cells, and inhibiting rate is 14.9%.

Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously Therefore the limitation of the scope of the claims of the present invention can not be interpreted as.For the person of ordinary skill of the art, this is not being departed from On the premise of inventive concept, some denaturation and improvement can also be made, these belong to protection scope of the present invention.Therefore, originally The protection domain of patent of invention should be determined by the appended claims.

SEQUENCE LISTING

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<120>A kind of Malian drainage protective antigens HP0623 and preparation method thereof

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Claims

1. a kind of Malian drainage protective antigens HP0623, it is characterised in that：For SEZ HP0623 recombinant proteins, by Sez_0623 gene codes, are made up of 429 amino acid residues, and molecular weight is 47.04kDa, amino acid sequence such as SEQ ID Shown in NO.2.

2. a kind of Malian drainage protective antigens HP0623 preparation method, is divided into following steps：

1) PCR is expanded：The use of Malian drainage SEZ C55138 genomes is template, entering performing PCR with primer expands；

2) connection of carrier：PCR primer is connected after digestion with restriction enzyme with PET-32a carriers；

3) convert and induce：By step 2) carrier that connects converts to Escherichia coli, cultivates to OD values to 0.6~1.0, plus Enter IPTG Fiber differentiations 3-5h；

4) purify：Step 3 is collected by centrifugation) in cultured thalline, with sterile PBS be resuspended after after crushing, albumen is collected by centrifugation Supernatant, takes supernatant through Ni-NTA column chromatographies, obtains the recombinant protein of purifying, as Malian drainage protectiveness Antigen HP0623.

3. Malian drainage protective antigens HP0623 according to claim 2 preparation method, its feature exists In：The primer is：

Forward primer：5’-CCCGGATCCGCTTGCCTGCTAGTG-3’；

Reverse primer：5’-CTCAAGCTTGAGGGGAAGATCGTATC-3’。

4. Malian drainage protective antigens HP0623 according to claim 3 preparation method, its feature exists In：The forward primer and reverse primer have 1 restriction enzyme site respectively.

5. Malian drainage protective antigens HP0623 according to claim 4 preparation method, its feature exists In：The restriction enzyme site of the forward primer is BamH I restriction enzyme sites；Reverse primer restriction enzyme site is Hind III digestions site.

6. Malian drainage protective antigens HP0623 is preparing Malian drainage according to claim 1 Application in vaccine.

7. a kind of method that Malian drainage protective antigens HP0623 prepares Malian drainage vaccine, it is special Levy and be：By step 4 described in any claim in claim 2-5) purification process obtained recombinant protein and Montanide It is Malian drainage vaccine after the PR adjuvant emulsions of Gel 01, the concentration of the recombinant protein is 100 μ g/mL.