CN114045298B

CN114045298B - Chicken bursa mycoplasma subunit vaccine and preparation method and application thereof

Info

Publication number: CN114045298B
Application number: CN202111057100.4A
Authority: CN
Inventors: 金梅林; 黄运福; 康超; 邓明勇; 徐巧霞; 姚蓉; 孙小美
Original assignee: Wuhan Keqian Biological Co ltd
Current assignee: Wuhan Keqian Biological Co ltd; Huazhong Agricultural University
Priority date: 2021-09-09
Filing date: 2021-09-09
Publication date: 2023-10-20
Anticipated expiration: 2041-09-09
Also published as: CN114045298A

Abstract

The invention provides a chicken bursa mycoplasma subunit vaccine, a preparation method and application thereof, wherein the chicken bursa mycoplasma PPHT and FBA-II recombinant proteins expressed by a prokaryotic expression system can be specifically identified by chicken bursa mycoplasma positive serum and have good reactivities, which indicate that the chicken bursa mycoplasma positive serum has excellent immunogenicity, so that a combined vaccine containing the two proteins is prepared, and the infection of chicken bursa mycoplasma is effectively prevented and controlled. After immunization, the chicken is free from pathogenicity, and the incidence rate of chicken bursal mycoplasmosis is obviously reduced. Compared with the traditional inactivated vaccine, the genetic engineering subunit vaccine has the advantages of easy acquisition of antigen, good immune effect, safe and simple process, low cost, easy mass production and the like.

Description

Chicken bursa mycoplasma subunit vaccine and preparation method and application thereof

Technical Field

The invention relates to a chicken bursa mycoplasma subunit vaccine and a preparation method and application thereof, belonging to the technical field of animal vaccines and veterinary biological products.

Background

The mycoplasma synoviae disease is an important disease of poultry, which is caused by mycoplasma synoviae (Mycoplasma Synoviae, MS) and is characterized by bursa and tendon inflammation, and enlargement of joints and parenchymal organs, and can also cause the problems of growth stagnation of young chickens, reduction of egg yield of laying hens, reduction of feed conversion rate of broilers, increase of carcass waste rate and the like, and reduce the production performance of chickens, so that the economic loss caused to poultry farming industry is very serious.

MS is a member of the genus Mycoplasma of the family Mycoplasma, gram-negative, cell wall free, plate culture to form a typical "fried egg" like colony, an important causative agent of avian disease. Antibiotic therapy and vaccination have long been the primary means of controlling this disease. MS is sensitive to certain antibiotics such as tilmicosin, florfenicol, etc., but these antibiotics cannot eradicate the spread of MS in chicken flocks, and the long-term unreasonable use of antibiotics brings many hazards to the environment, livestock and human health. Vaccination is therefore a more effective measure for preventing chicken mycoplasma synoviae.

Currently, the vaccines used for preventing and controlling MS infection mainly comprise commercial attenuated vaccines (MS-H strain) and inactivated vaccines. On one hand, although MS attenuated vaccine can effectively control clinical symptoms caused by MS, the severe preservation condition is unfavorable for detection and purification of MS in chicken flocks, and particularly, the chicken flocks inoculated with MS can cause more serious clinical symptoms, so that the vaccine is not suitable for domestic popularization and application for poultry farming in which most of chicken flocks in China are positive for MS. On the other hand, the MS inactivated vaccine can reduce the infection rate of MS and has a certain protection effect, but the application of the vaccine is hindered by the defects of weak immunity protection, high mycoplasma culture cost and the like. Therefore, the development of a safe and efficient novel vaccine for preventing the transmission of chicken bursa mycoplasma diseases is imperative.

Phosphopyruvate hydratase (phosphopyruvate hydratase, abbreviated as PPHT) and fructose-1,6-bisphosphate aldolase type II (frame II, abbreviated as FBA-II) are key regulatory enzymes involved in the sugar metabolism pathway, and in addition to exerting an enzymatic activity in sugar metabolism, they are presumed to exert similar functions in MS by being distributed on the cell membranes of various pathogenic bacteria as immunogenic membrane proteins and participating in the process of infection as virulence factors. Thus, preparing a combination vaccine comprising the two proteins in an effort to effectively prevent and control MS infection is a significant desire of poultry industry production and scientific researchers.

Disclosure of Invention

The invention aims to solve the technical problem of providing a chicken bursa mycoplasma subunit vaccine which is used for immunoprophylaxis of chicken bursa mycoplasma diseases. The concept of the invention is based on that recombinant proteins of mycoplasma synoviae PPHT and FBA-II expressed by a prokaryotic expression system can be specifically identified by positive serum of mycoplasma synoviae and have good reactivities, so that the recombinant proteins have excellent immunogenicity, and a combined vaccine containing the two proteins is prepared so as to effectively prevent and control infection of mycoplasma synoviae. At present, no commercial subunit vaccine for preventing chicken bursal mycoplasma disease exists, no report of preparing vaccine by taking chicken bursal mycoplasma PPHT and FBA-II as antigens exists, the epidemic of the disease is prevented and controlled by using the subunit vaccine prepared by using recombinant proteins of chicken bursal mycoplasma PPHT and FBA-II expressed by a prokaryotic expression system, and a safe and efficient subunit vaccine is provided for research and development of chicken bursal mycoplasma vaccine.

The invention aims to provide a novel chicken bursa mycoplasma PPHT and FBA-II protein subunit vaccine, and a preparation method and application thereof.

In order to achieve the aim of the invention, the invention provides a recombinant expression vector which contains a target gene fragment, wherein the target gene fragment comprises a chicken bursa mycoplasma PPHT protein coding gene, and the sequence of the target gene fragment is a sequence shown as SEQ ID NO. 1 obtained by codon optimization.

Preferably, the 3 'end of the chicken bursa mycoplasma PPHT protein coding gene is connected with a 6XHis Tag peptide sequence, and the 5' end is sequentially connected with an S-Tag peptide sequence and a TrxA Tag peptide sequence.

Preferably, the recombinant expression vector is a pET-PPHT vector in which a gene fragment of interest is inserted between the T7terminator sequence and the lac operator sequence.

In order to achieve the aim of the invention, the invention provides a recombinant expression vector which contains a target gene fragment, wherein the target gene fragment comprises a mycoplasma synoviae FBA-II protein coding gene, and the sequence of the target gene fragment is a sequence shown as SEQ ID NO. 5 obtained by codon optimization.

Preferably, the 3 'end of the chicken bursa mycoplasma FBA-II protein coding gene is connected with a 6XHis Tag peptide sequence, and the 5' end is sequentially connected with an S-Tag peptide sequence and a TrxA Tag peptide sequence.

Preferably, the recombinant expression vector is a pET-FBA-II vector in which a gene fragment of interest is inserted between the T7terminator sequence and the lac operator sequence.

To achieve the object of the present invention, the present invention provides an immunogenic composition comprising: PPHT protein shown as SEQ ID NO. 3, FBA-II protein shown as SEQ ID NO. 5, and pharmaceutically acceptable carrier.

The invention provides novel chicken bursa mycoplasma PPHT and FBA-II protein subunit vaccines comprising the above immunogenic composition, optionally comprising an adjuvant.

Preferably, the adjuvant is a water-in-oil-in-water adjuvant.

More preferably, the adjuvant is a Summit P168 adjuvant.

The subunit vaccine is a mixed solution formed by emulsifying protein solution and an adjuvant, wherein the mass ratio of PPHT to FBA-II proteins in the protein solution is 1:1, and the volume ratio of the protein solution to the adjuvant is 3:2.

the invention provides a preparation method of a novel chicken bursa mycoplasma PPHT and FBA-II protein subunit vaccine, which comprises the following steps:

(1) The PPHT gene sequence of the mycoplasma gallisepticum shown in the nucleotide sequence SEQ ID NO. 2 and the FBA-II gene sequence of the mycoplasma gallisepticum shown in the nucleotide sequence SEQ ID NO. 6 published on GenBank are referred to, the PPHT and FBA-II target genes are artificially synthesized according to the preference of escherichia coli codons, and the nucleotide sequences of the synthesized target genes are shown as SEQ ID NO. 1 and SEQ ID NO. 5 respectively;

(2) Respectively recombining target genes on pUC57 vectors to obtain recombinant plasmids pUC57-PPHT and pUC 57-FBA-II, and respectively taking pUC57-PPHT and pUC 57-FBA-II recombinant plasmids as templates to carry out PCR amplification reaction, wherein the amplification system is 2X Phanta Max Buffer mu L, dNTP Mix 1 mu L, each of the upstream and downstream primers is 2 mu L, the template is 2 mu L, phanta Max Super-Fidelity DNA Polymerase mu L, and dd H2O is added to supplement 50 mu L; the reaction procedure: pre-denaturing at 95℃for 3min, denaturing at 94℃for 20sec, annealing at 60℃for 20sec, and extending at 72℃for 1min, performing 35 cycles of amplification, and extending at 72℃for 5min; electrophoresis of the amplified product in 1% agarose gel, and gel recovery of target fragment to obtain target gene, and preservation at-20 deg.c; the sequences of the upstream and downstream primers corresponding to the recombinant plasmid pUC57-PPHT are shown as SEQ ID NO. 9 and SEQ ID NO. 10, and the sequences of the upstream and downstream primers corresponding to the recombinant plasmid pUC 57-FBA-II are shown as SEQ ID NO. 11 and SEQ ID NO. 12;

(3) Using a recombinase of ClonExpress MultiS One Step Cloning Kit to respectively connect a target gene PPHT and an FBA-II to a pET-32a vector, transforming the connection products into E.coli DH5 alpha competent cells, coating the E.coli DH5 alpha competent cells on an ampicillin-resistant LB plate, culturing for 12-16 h at 37 ℃, picking single colonies to respectively inoculate LB culture media, and identifying recombinant plasmids by PCR and sequencing, wherein the recombinant plasmids are respectively named pET-PPHT and pET-FBA-II;

(4) And (3) transforming the constructed recombinant plasmid into E.coli BL21, identifying a positive strain by PCR, and inducing the positive strain to express PPHT and FBA-II proteins by using ITPG.

(5) And (3) mixing protein solutions obtained by purifying the recombinant expressed PPHT and FBA-II proteins according to the mass ratio of PPHT to FBA-II proteins of 1:1, wherein the concentration of the PPHT and the concentration of the FBA-II proteins in the obtained mixed protein solution are 167 mug/mL, and adding Summit P168 adjuvant with the volume of 2/3 of that of the mixed protein solution to be fully and uniformly mixed.

The invention provides an application of the novel chicken bursa mycoplasma PPHT and FBA-II protein subunit vaccine: is used for preparing the medicine for treating or preventing chicken mycoplasma synoviae infection.

The method, wherein the step (5) comprises: the positive strains are inoculated in 10mL of LB liquid medium, cultured at 37 ℃ to logarithmic phase, transferred into 1L of LB liquid medium according to the inoculation proportion of 1:100, cultured at 37 ℃ to OD600 value of 0.8, added with IPTG with final concentration of 0.5mmol/L and induced at 20 ℃ for 8-12 h.

After the scheme is adopted, compared with the prior art, the invention has the following outstanding advantages and effects: the inactivated vaccine for preventing chicken bursa mycoplasma disease has the problems of weak immunity protection, possibility of incomplete inactivation in the inactivation process and the like, and the preparation of the inactivated vaccine is hindered by the defects of high cost, complex process, diffusion risk and the like of mycoplasma culture.

The invention adopts a prokaryotic expression system to express PPHT and FBA-II proteins, and has the advantages of easy acquisition of antigen, good immune effect, safe and simple process, low cost, easy mass production and the like. The invention has the advantages that PPHT and FBA-II proteins are expressed by a prokaryotic expression system, the expression level is high, and the invention is suitable for large-scale production. The expression products were specifically recognized by MS positive serum and had good reactogenicity, indicating that they had excellent immunogenicity.

The PPHT and FBA-II proteins expressed by the prokaryotic expression system are used, and the antigenicity, immunogenicity and functions of the expression product are similar to those of the natural protein. The prepared vaccine can generate higher level of antibody, and the antibody has quick excitation and long duration. After immunization, the chicken is free from pathogenicity, and the incidence rate of chicken bursal mycoplasmosis is obviously reduced. Compared with the traditional inactivated vaccine, the genetic engineering subunit vaccine has the advantages of easy acquisition of antigen, good immune effect, safe and simple process, low cost, easy mass production and the like.

Drawings

FIG. 1 shows the results of gel electrophoresis of PPHT gene PCR amplification products; wherein 1 is a negative control; 2 is PPHT gene; m is a molecular weight marker;

FIG. 2 shows the results of gel electrophoresis of PCR amplification products of PPHT gene transformed colony samples; wherein 1 is a negative control; 2-6 are colony sample PCR amplified products of PPHT gene transformation; m is a molecular weight marker;

FIG. 3 shows the result of gel electrophoresis of the PCR amplification product of the FBA-II gene; wherein 1 is a negative control; 2 is FBA-II gene; m is a molecular weight marker;

FIG. 4 shows the result of gel electrophoresis of colony sample PCR amplified products transformed with FBA-II gene; wherein 1 is a negative control; 2-6 are colony sample PCR amplified products of FBA-II gene transformation; m is a molecular weight marker;

FIG. 5 is a diagram of a constructed recombinant prokaryotic expression vector pET-PPHT;

FIG. 6 is a map of the constructed recombinant prokaryotic expression vector pET-FBA-II;

FIG. 7 shows the results of SDS-PAGE vertical electrophoresis of recombinant PPHT proteins expressed in example 3, wherein 1 is a negative control; 2 is the sediment after crushing; 3 is the supernatant after crushing; m is a molecular weight marker;

FIG. 8 shows the result of SDS-PAGE vertical electrophoresis of the recombinant FBA-II protein expressed in example 3, wherein 1 is a negative control; 2 is the sediment after crushing; 3 is the supernatant after crushing; m is a molecular weight marker;

FIG. 9 shows the Western blot results of recombinant PPHT proteins expressed in example 3, wherein 1 is a negative control; 2 is purified recovered PPHT protein; m is a molecular weight marker;

FIG. 10 shows the Western blot results of recombinant FBA-II proteins expressed in example 3, wherein 1 is a negative control; 2 is purified and recovered FBA-II protein; m is a molecular weight marker;

FIG. 11 shows the results of comparing the negative control group after challenge with the vaccine group for paw pad, tarsal joint and liver lesions after challenge with the vaccine of the present invention.

Detailed Description

The invention is further described below in connection with specific embodiments. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the invention, but these changes and substitutions fall within the scope of the present invention.

The terms devised by the present invention are explained first as follows:

the term "pUC57" is a cloning plasmid of Escherichia coli, which has a size of 2710bp and contains an ampicillin antibiotic resistance gene, and can grow on LB medium containing ampicillin, and is a high copy plasmid vector which can be used for screening blue-white spots. pUC57 MCS contains 6 restriction sites with an overhanging 3' end that can tolerate E.coli exonuclease III.

The term'Max Super-Fidelity DNA Polymerase "refers to the" strongest template compatible "new generation of high fidelity DNA polymerase offered by the biological technology Co., ltd. "2X Phanta Max Buffer" is a high-fidelity PCR buffer system provided by Nanjinouzan biotechnology Co., ltd, for matching with PhantaSuper-Fidelity DNA Polymerase (P505) was used. "dNTP Mix" (dNTP mixture) is a premixed solution containing dATP, dCTP, dGTP and dTTP in sodium salt, each at a concentration of 10mM and a total concentration of 40mM (pH 7.5).

The term "DH 5. Alpha. Competent cells": DH 5. Alpha. Strain is the most commonly used competent cell in the laboratory. Deletion of endonuclease (endA) improves yield and quality of plasmid DNA; the recombinase defect type (recA) reduces the homologous recombination probability of the inserted fragment, and ensures the stability of the inserted DNA; the presence of lacZΔM15 allows DH 5. Alpha. To be used for blue and white spot screening.

The term "OD600": refers to the absorbance of a solution at a wavelength of 600 nm. The absorbance is proportional to the concentration of the light absorbing species in the solution and is accordingly inversely proportional to the transmittance T of the sample, which is a logarithmic relationship in value. When the OD600 is 1, the bacteria can be grown in a relatively good period, and in this case, studies such as transformation can be performed, and when the OD600 exceeds 0.8 in the culture of the bacteria, the medium is diluted.

The term "IPTG": isopropyl-beta-D-thiogalactoside is an organic substance, has a chemical formula of C9H18O5S, is an isolactose mimic, and can cause the transcription process of a lactose operon, so that the expression of a gene corresponding to the downstream gene of the lactose operon can be induced.

The term "Western Blot identification" adopts polyacrylamide gel electrophoresis, the detected object is protein, the protein sample separated by the polyacrylamide gel electrophoresis is transferred to a solid phase carrier, and the type of the polypeptide separated by the electrophoresis and the biological activity thereof can be kept unchanged. The protein or polypeptide on the solid carrier is used as antigen, reacts with the corresponding antibody, then reacts with the enzyme or the second antibody marked by the isotope, and the protein component expressed by the specific target gene separated by electrophoresis is detected through substrate color development or autoradiography.

The term "goat anti-chicken polyclonal antibody secondary antibody" is prepared by taking goat immune serum (antiserum) of the obtained high-titer antibody as a raw material and purifying by adopting a chicken IgY immune affinity chromatography method, and the high-purity (more than or equal to 95%) high-activity purified goat anti-chicken IgY antibody is prepared, wherein IgY has high reactivity (sensitivity) to mammal antigens, is commonly existing in birds, reptiles and amphibians, and is functionally equivalent to mammal IgG.

EXAMPLE 1 construction and identification of expression vector pET-PPHT

Amplification and recovery of PPHT Gene the PPHT gene sequence (SEQ ID NO: 2) of the reference Mycoplasma synoviae WVU 1853 strain (GenBank No. CP012096.1) was artificially synthesized by Beijing engine biotechnology Co., ltd. According to the preference of E.coli codons into the gene sequence shown in SEQ ID NO:1 and recombined onto pUC57 vector to obtain pUC57-PPHT plasmid vector. The artificially synthesized sequence SEQ ID NO. 1 has 76.62 percent of consistency with the PPHT gene sequence SEQ ID NO. 2 published by GeneBank; the amino acid sequence translated by SEQ ID NO. 1 is SEQ ID NO. 3, and has 100% of identity with the amino acid sequence translated by SEQ ID NO. 2, SEQ ID NO. 4.

pUC57-PPHT plasmid is used as a template, and PPHT-F, PPHT-R primer (PPHT-F, PPHT-R primer sequence is shown as SEQ ID NO:9 and SEQ ID NO: 10) is used as a template according to Nannunofizan biotechnology Co.LtdMax Super-Fidelity DNA Polymerase was PCR amplified. The amplification system and reaction procedure are shown in table 1:

TABLE 1 PPHT Gene amplification System and reaction procedure

The PCR amplified product is detected by 1% agarose gel electrophoresis, the size of the target gene is shown as figure 1, and a target band appears at the 1356bp position, which indicates that the target gene is amplified successfully, and the target gene is recovered and purified by using a gel recovery and purification kit.

Cleavage and recovery of pET-32a vector the extracted pET-32a plasmid was double digested with BamH I and HindIII restriction enzymes, the digested product was subjected to gel electrophoresis, and recovered and purified using a gel recovery and purification kit.

The cleavage system pET-32a plasmid was 10. Mu.L, 10 XK buffer 5.0. Mu.L, bamH I enzyme 2.0. Mu.L, hind III enzyme 2.0. Mu.L, and add dd H2O to make up to 50. Mu.L.

The reaction conditions were cleaved overnight at 37 ℃.

3. Ligation the recovered purified PPHT gene fragment was ligated with the pET-32a plasmid fragment using the recombinase Exnase of Nanjinopran Biotech Co.Ltd ClonExpress MultiS One Step Cloning Kit.

Ligation system PCR recovery of 1. Mu.L, pET-32a plasmid recovery fragment 1. Mu.L, 5 XCE II buffer 2.0. Mu.L, exnase II 1. Mu.L, add dd H2O to make up to 10. Mu.L.

The reaction conditions were connected at 37℃for 30min.

4. Transformation the ligation product was added to 100. Mu.L DH 5. Alpha. Competent cells, gently flicked, mixed well, ice-bath for 30min, heat-shocked in a water bath at 42℃for 90sec, then rapidly allowed to stand in ice for 5min, 500. Mu.L of non-resistant LB liquid medium (Luria-Bertani medium) was added, and incubated at 37℃for 1h. The bacterial liquid is concentrated to 100 mu L and coated on LB solid medium with ampicillin resistance, and the bacterial liquid is cultured for about 12 to 16 hours at 37 ℃.

And 5. The single colonies on the PCR and sequencing identification picking plates are respectively inoculated with LB liquid culture medium and cultured for 2 hours at 37 ℃. PCR identification is carried out by using bacterial liquid as a template through PPHT-F, PPHT-R primers, PCR amplified products are detected through 1% agarose gel electrophoresis, the size of target genes is shown as figure 2, and samples with bands around 1356bp are positive strains. The positive strain was sequenced by Beijing engine biotechnology Co., ltd, thereby constructing a recombinant vector pET-PPHT. The schematic diagram of the vector is shown in fig. 5, and has the following elements: replicon: ori-replication of the original vector; f1 ori-f 1 phage replicon, displaying sense strand synthesis orientation; rop: encoding the ROP protein such that the plasmid maintains low copy levels; lactose operator element-lacI promoter+laci (which is the inhibitor of the lac operator that initiates expression of the repressor), lac operator (lac operator to which the repressor protein binds, inhibiting expression of its subsequent gene), lactose analogue IPTG may bind to the repressor protein and thereby lose control of the lac operator, and thus allow its subsequent gene to be expressed normally); t7 promter: promoters controlled by T7 RNA polymerase are the mainstay of E.coli expression systems today. The powerful T7 promoter is completely and exclusively controlled by T7 RNA polymerase, and the high-activity T7 RNA polymerase synthesizes mRNA at a speed which is 5 times faster than that of the escherichia coli RNA polymerase, when the two are simultaneously present, the transcription competition of host own genes is not over that of a T7 expression system, and almost all cell resources are used for expressing target proteins; 6xHis (6 xHis affinity tag): encoding 6 consecutive Hiss, serving as tags for purifying the target protein; s-tag: is a short peptide derived from the N-terminus of pancreatic ribonuclease A (RNase A), and S-tag is usually constructed on the N-terminus or C-terminus of the target protein so that the S-tag-labeled protein can be analyzed by immunochemical methods, and this S-tag protein can be detected by a commercial S-tag antibody; trxA: for facilitating soluble expression of recombinant proteins; ampr: hydrolyzing the beta-lactam ring and removing the toxicity of the ampicillin.

EXAMPLE 2 construction and identification of expression vector pET-FBA-II

Amplification and recovery of the FBA-II Gene the FBA-II gene (SEQ ID NO: 6) of the reference Mycoplasma synoviae WVU 1853 strain (GenBank No. CP012096.1) was artificially synthesized by Beijing-QINGSHUO Biotechnology Co., ltd. Into the gene sequence shown in SEQ ID NO:5 according to the preference of E.coli codons and recombined onto pUC57 vector to obtain pUC 57-FBA-II plasmid vector. The artificially synthesized sequence SEQ ID NO. 5 has 78.70 percent of consistency with the FBA-II gene sequence SEQ ID NO. 6 published by GeneBank; the amino acid sequence translated by SEQ ID NO. 5 is SEQ ID NO. 7, and has 100% of identity with the amino acid sequence translated by SEQ ID NO. 6, SEQ ID NO. 8.

The pUC 57-FBA-II plasmid is used as a template, and the primer FBA-II-F, FBA-II-R (the gene sequences of the FBA-II-F, FBA-II-R are shown as SEQ ID NO:11 and SEQ ID NO: 12) is used as a template according to Nanjinopran Biotech Co., ltdMax Super-Fidelity DNA Polymerase was PCR amplified. The amplification system and reaction procedure are shown in table 2:

TABLE 2 FBA-II Gene amplification System and reaction procedure

The PCR amplification product was detected by 1% agarose gel electrophoresis, and the size of the target gene was as shown in FIG. 3, and the target band appeared at the 864kbp position, indicating that the amplification of the target gene was successful, and recovery and purification were performed using the gel recovery and purification kit.

Cleavage and recovery of pET-32a vector the extracted pET-32a plasmid was double digested with BamH I and HindIII restriction enzymes. And (3) performing gel electrophoresis on the enzyme-digested product, and recycling and purifying by using a gel recycling and purifying kit.

The reaction conditions were cleaved overnight at 37 ℃.

3. Ligation the recovered purified FBA-II gene fragment was ligated with the pET-32a plasmid fragment using the recombinase Exnase of Nanjinouzan Biotechnology Co.Ltd ClonExpress MultiS One Step Cloning Kit.

The reaction conditions were connected at 37℃for 30min.

4. Conversion the ligation product was added to 100. Mu.L DH 5. Alpha. Competent cells, flicked and mixed well, ice-bath was performed for 30min, and after heat shock in a water bath at 42℃for 90sec, it was rapidly allowed to stand in ice for 5min, 500. Mu.L of non-resistant LB liquid medium was added, and incubated at 37℃for 1h. The bacterial liquid is concentrated to 100 mu L and coated on LB solid medium with ampicillin resistance, and the bacterial liquid is cultured for about 12 to 16 hours at 37 ℃.

And 5. The single colonies on the PCR and sequencing identification picking plates are respectively inoculated with LB liquid culture medium and cultured for 2 hours at 37 ℃. Bacterial liquid is used as a template, colony PCR identification is carried out through an FBA-II-F, FBA-II-R primer, a PCR amplification product is detected through 1% agarose gel electrophoresis, the size of a target gene is shown as in figure 4, and a sample with a band around 864bp is a positive strain. The positive strain was sequenced by Beijing engine biotechnology Co., ltd, thereby constructing recombinant vector pET-FBA-II. The schematic diagram of the vector is shown in FIG. 6. Has the following elements: replicon: ori-replication of the original vector; f1 ori-f 1 phage replicon, displaying sense strand synthesis orientation; rop: encoding the ROP protein such that the plasmid maintains low copy levels; lactose operator element-lacI promoter+laci (which is the inhibitor of the lac operator that initiates expression of the repressor), lac operator (lac operator to which the repressor protein binds, inhibiting expression of its subsequent gene), lactose analogue IPTG may bind to the repressor protein and thereby lose control of the lac operator, and thus allow its subsequent gene to be expressed normally); t7 promter: promoters controlled by T7 RNA polymerase are the mainstay of E.coli expression systems today. The powerful T7 promoter is completely and exclusively controlled by T7 RNA polymerase, and the high-activity T7 RNA polymerase synthesizes mRNA at a speed which is 5 times faster than that of the escherichia coli RNA polymerase, when the two are simultaneously present, the transcription competition of host own genes is not over that of a T7 expression system, and almost all cell resources are used for expressing target proteins; 6xHis (6 xHis affinity tag): encoding 6 consecutive Hiss, serving as tags for purifying the target protein; s-tag: is a short peptide derived from the N-terminus of pancreatic ribonuclease A (RNase A), and S-tag is usually constructed on the N-terminus or C-terminus of the target protein so that the S-tag-labeled protein can be analyzed by immunochemical methods, and this S-tag protein can be detected by a commercial S-tag antibody; trxA: for facilitating soluble expression of recombinant proteins; ampr: hydrolyzing the beta-lactam ring and removing the toxicity of the ampicillin.

EXAMPLE 3 expression and purification of recombinant proteins PPHT and FBA-II

1. Vector transformation expression Strain the recombinant plasmids constructed in example 1 and example 2 and pET-32a (+) empty vector were transformed into E.coli competent cells E.coli BL21, and positive strains were identified by PCR.

2. Inducible expression of recombinant strains positive strains constructed in example 3 were induced to express PPHT and FBA-ii proteins using ITPG. Inoculating the recombinant positive strains into 10mL of LB liquid medium, culturing at 37 ℃ to logarithmic phase, transferring into 1L of LB liquid medium according to the inoculation proportion of 1:100, culturing at 37 ℃ to OD600 value of 0.8, adding IPTG with final concentration of 0.5mmol/L, and inducing at 20 ℃ for 8-12 h. Isopropyl- β -D-thiogalactoside (IPTG), an isolactose mimetic, is capable of causing the transcription process of the lactose operon, and thus is capable of inducing the expression of the corresponding protein of the gene downstream of the lactose operon. In the induced expression of the exogenous gene of the escherichia coli, the mass expression of the target protein can be realized.

3. Recombinant protein SDS-PAGE is detected, centrifugally collected, resuspended by a proper amount of PBS buffer solution, crushed for 3 times by an ultra-high pressure nano homogenizer at 800-1000 bar pressure, centrifugally collected sediment and supernatant, and crushed sediment and crushed supernatant are obtained. Adding appropriate amount of protein loading buffer solution into induced negative control bacterial solution, induced positive bacterial cell disruption and precipitation and disruption supernatant, boiling for 10min, and performing SDS-PAGE (polyacrylamide gel electrophoresis) vertical electrophoresis. After electrophoresis, taking down the gel, putting the gel into coomassie brilliant blue staining solution for full immersion, staining overnight, pouring out the staining solution, adding coomassie brilliant blue decoloring solution, fully covering the gel, decoloring, and observing whether the protein is expressed or not. As shown in FIGS. 7 and 8, both PPHT and FBA-II recombinant proteins were expressed in the supernatant, with bands at positions with molecular weights of about 68kDa and about 50kDa, respectively, consistent with the expected protein size, and the negative control was not band at the corresponding positions.

4. Western blot identification of recombinant protein Western blot identification is carried out by selecting crushed supernatant sample, transferring SDS-PAGE electrophoresis product onto NC (nitrocellulose) membrane, sealing with 5% skimmed milk powder for 2h, adding chicken-derived anti-MS positive serum, incubating overnight, rinsing, adding HRP (horseradish peroxidase) -labeled goat anti-chicken polyclonal antibody secondary antibody (Abbkine company), incubating for 2h, rinsing, adding enhanced chemiluminescence fluorescent substrate for color development, observing whether target band exists or not, and analyzing immunogenicity of recombinant protein. As shown in FIGS. 9 and 10, both PPHT and FBA-II recombinant proteins reacted with MS positive serum and developed at the positions of about 68kDa and about 50kDa, respectively, demonstrated good reactivity, indicating excellent immunogenicity.

5. Protein purification the crushed supernatant from step 3 of this example was purified using a nickel column and eluted with imidazole to give a recombinant protein solution. Protein concentration was determined by BCA, wherein recombinant protein PPHT concentration was 1654. Mu.g/mL, FBA-II concentration was 1860. Mu.g/mL, and the protein solution was sterile filtered through a 0.22 μm filter membrane and stored at 4 ℃.

Example 4 preparation of vaccine

60.58mL of PPHT protein and 53.87mL of FBA-II protein after filtration and sterilization in the step 5 of the example 3 are taken and mixed, and sterilized water is used for supplementing to 600mL, so that the concentration of each antigen protein is 167 mug/mL, and then the mixed protein liquid and an adjuvant Summit P168 adjuvant are uniformly mixed according to the proportion of 3:2 to prepare the vaccine. The adjuvant Summit P168 is purchased from the biological technology Co., ltd. Of the compound river Sang Mite and is a special water-in-oil-in-water adjuvant for the avian influenza vaccine, and the main components of the adjuvant comprise pharmaceutical grade white oil, a surfactant and an immune promoter; specifically, every 600mL of mixed protein solution is added with 168400mL of adjuvant Summit P, and then the mixture is emulsified for 10min by using an emulsifying instrument at 10000rpm, so that a uniform emulsion is formed between the protein phase and the adjuvant phase.

EXAMPLE 5 immunoassay

The chicken bursa mycoplasma subunit vaccine prepared in example 4 was subcutaneously injected into the neck of 0.5mL for 7-day-old SPF chickens (SPF production chicken fingers were grown in a barrier system or isolator, and no chicken flocks having good growth and reproductive performance were found) for the main infectious pathogens of domestic and foreign chickens, 10 otherwise provided with no immune control. Immunization was performed at the same dose twice 14 days after immunization.

Each group of test chickens was inoculated with 0.2mL of Mycoplasma synoviae culture with a viable count of about 106CCU/mL with a claw-pad 14 days after the secondary immunization, and the disease condition of the test chickens was observed within 14 days. Results control chickens were all (10/10) ill, developing listlessness, growth arrest, swelling of joints, lameness, and even deformation; dissecting the tarsal joints of chickens, and appearing as suppurative exudates; kidney enlargement was seen by dissection (as shown in fig. 11). 1 chicken is immunized, the protection rate is 9/10, and the vaccine immunization efficacy is qualified.

It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

<110> prebiological Co., ltd

<120> chicken bursa mycoplasma subunit vaccine, preparation method and application

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1356

<212> DNA

<213> phosphopyruvate hydratase (phosphopyruvate hydratase)

<400> 1

atgagcgcaa tcaaaaagat ccacgcacgc gaagtgctgg atagccgtgg taaccctacc 60

gtgcaggtgg aagtgtatac agaactgaaa ggttatggta gcgcaatggt gcctagcggt 120

gcaagcaccg gtagccgtga agcactggaa ctgcgtgata aaggtagcaa atttgaaagc 180

aactggtttg gtggtaaagg tgtgatgcag gcagtggaaa acgtgaataa actgatcgca 240

cctgcactga tcggctttga agttacagat cagcgccagg ttgatctggc aatgaaagca 300

ctggatggca caaaaaataa agaaaaactg ggtgcaaatg caattctggg tgttagcctg 360

gcagttgcac gtgcagcagc aaatgaactg gatctgccgc tgtataaata tctgggtggt 420

tttaatgcac ataaactgcc gctgccgatg ctgaatgtta ttaatggtgg tgaacatgca 480

agcaataccc tggattttca ggaatttatg gttatgccgg ttggtgcaaa aagctttcgt 540

gaagcactgc agatggcaaa ttttgttttt cataatctgg caaaactgct gaaaaaacat 600

ggtcatggtg ttcaggttgg tgatgaaggt ggttttgcac cgaattttaa aagccatgaa 660

gaagcactgg attttctggt tgaagcaatt aaactgagcg gttataaacc ggcaattagc 720

ggtgaaaaag cagttgcaat tgcaatggat tgtgcaagca gcgaactgta taaagatggt 780

aaatatacct ttggtaaact gaaaaaagca attgaagaaa aacagccggg ttttgaaaat 840

ctgggtaaaa ccaaactggt ttataccacc gatgaactga ttgattatct ggatcatctg 900

gttagcaaat atccgattgt tagcattgaa gatggtctgg cagaaagcga ttgggcaggt 960

tttgaaaaac tgaccaaacg tctgggtcat aaactgcagg ttgttggtga tgatctgacc 1020

gttaccaata ccgaactgct ggcaaaagca attgaacgta aagcaatgaa tagcattctg 1080

attaaagtta atcagattgg tagcctgacc gaaacctttg aagcaattca gatggcacag 1140

atggcaaata tgaccgcagt tgttagccat cgtagcggtg aaaccgaaga taccaccatt 1200

gcagatattg cagttgcaat gaataccggt cagattaaaa ccggtagcat gagccgtacc 1260

gatcgtattg caaaatataa tcgtctgctg gcaattgaag aagaactgag caaagcaagc 1320

acctttccga aagatgcatt ttataatctg aaaaaa 1356

<210> 2

<211> 1356

<212> DNA

<213> phosphopyruvate hydratase (phosphopyruvate hydratase)

<400> 2

atgtcagcaa ttaaaaaaat ccatgcaaga gaagttttag actcacgtgg taatcccact 60

gttcaagttg aagtgtatac agaactaaaa ggatatggtt ctgcaatggt tccatcagga 120

gcttctactg gatcaagaga agctcttgaa ttaagagata aaggttcaaa atttgaatct 180

aattggtttg gtggaaaagg tgtaatgcaa gcggttgaaa acgttaataa attaatcgct 240

cctgcactta taggttttga agtaaccgac caaagacaag tagatttagc tatgaaggct 300

ttagatggaa ctaaaaacaa agaaaaatta ggagctaacg ctattttagg agtttctctt 360

gctgttgcta gagccgctgc taacgaatta gatcttcctt tatataaata tttaggtgga 420

tttaacgctc acaaacttcc tcttccaatg cttaatgtaa tcaacggtgg agagcacgct 480

tctaatactt tagatttcca agaatttatg gttatgccag ttggagctaa atcatttaga 540

gaagcattgc aaatggcaaa ctttgttttc cataaccttg caaaattact taaaaaacac 600

ggacacggtg ttcaagttgg tgatgaaggt ggatttgcac ctaactttaa atcacacgaa 660

gaagcattag attttttagt agaagctatt aaattatcag gatacaaacc tgcaatcagc 720

ggagaaaaag ctgttgctat tgcaatggac tgtgcatcta gcgagttata taaagatggt 780

aaatatacat tcggtaaact aaaaaaagca atcgaagaaa aacaaccagg atttgaaaac 840

ttaggaaaaa ccaaactagt ttatacaacc gatgaattaa ttgattattt agatcacctt 900

gtatctaaat acccaatcgt atctatcgaa gatggacttg cagaaagcga ctgggctgga 960

tttgaaaaat taaccaaaag acttggacac aaattacaag ttgtagggga cgatttaaca 1020

gttacaaaca cagaacttct tgctaaagct atcgaaagaa aagctatgaa ttcaatttta 1080

attaaagtaa atcaaattgg tagcttaaca gaaacatttg aagctattca aatggcacaa 1140

atggcaaaca tgactgcagt tgtgtcacat agatcaggtg aaactgaaga tacaactatt 1200

gcagatattg cagttgctat gaataccgga caaattaaaa ccggatcaat gtcaagaaca 1260

gatagaattg caaaatacaa tagacttctt gcaattgaag aagaattatc aaaagcttca 1320

acatttccaa aagacgcatt ttacaatctt aaaaaa 1356

<210> 3

<211> 452

<212> PRT

<213> phosphopyruvate hydratase (phosphopyruvate hydratase)

<400> 3

Met Ser Ala Ile Lys Lys Ile His Ala Arg Glu Val Leu Asp Ser Arg

1 5 10 15

Gly Asn Pro Thr Val Gln Val Glu Val Tyr Thr Glu Leu Lys Gly Tyr

20 25 30

Gly Ser Ala Met Val Pro Ser Gly Ala Ser Thr Gly Ser Arg Glu Ala

35 40 45

Leu Glu Leu Arg Asp Lys Gly Ser Lys Phe Glu Ser Asn Trp Phe Gly

50 55 60

Gly Lys Gly Val Met Gln Ala Val Glu Asn Val Asn Lys Leu Ile Ala

65 70 75 80

Pro Ala Leu Ile Gly Phe Glu Val Thr Asp Gln Arg Gln Val Asp Leu

85 90 95

Ala Met Lys Ala Leu Asp Gly Thr Lys Asn Lys Glu Lys Leu Gly Ala

100 105 110

Asn Ala Ile Leu Gly Val Ser Leu Ala Val Ala Arg Ala Ala Ala Asn

115 120 125

Glu Leu Asp Leu Pro Leu Tyr Lys Tyr Leu Gly Gly Phe Asn Ala His

130 135 140

Lys Leu Pro Leu Pro Met Leu Asn Val Ile Asn Gly Gly Glu His Ala

145 150 155 160

Ser Asn Thr Leu Asp Phe Gln Glu Phe Met Val Met Pro Val Gly Ala

165 170 175

Lys Ser Phe Arg Glu Ala Leu Gln Met Ala Asn Phe Val Phe His Asn

180 185 190

Leu Ala Lys Leu Leu Lys Lys His Gly His Gly Val Gln Val Gly Asp

195 200 205

Glu Gly Gly Phe Ala Pro Asn Phe Lys Ser His Glu Glu Ala Leu Asp

210 215 220

Phe Leu Val Glu Ala Ile Lys Leu Ser Gly Tyr Lys Pro Ala Ile Ser

225 230 235 240

Gly Glu Lys Ala Val Ala Ile Ala Met Asp Cys Ala Ser Ser Glu Leu

245 250 255

Tyr Lys Asp Gly Lys Tyr Thr Phe Gly Lys Leu Lys Lys Ala Ile Glu

260 265 270

Glu Lys Gln Pro Gly Phe Glu Asn Leu Gly Lys Thr Lys Leu Val Tyr

275 280 285

Thr Thr Asp Glu Leu Ile Asp Tyr Leu Asp His Leu Val Ser Lys Tyr

290 295 300

Pro Ile Val Ser Ile Glu Asp Gly Leu Ala Glu Ser Asp Trp Ala Gly

305 310 315 320

Phe Glu Lys Leu Thr Lys Arg Leu Gly His Lys Leu Gln Val Val Gly

325 330 335

Asp Asp Leu Thr Val Thr Asn Thr Glu Leu Leu Ala Lys Ala Ile Glu

340 345 350

Arg Lys Ala Met Asn Ser Ile Leu Ile Lys Val Asn Gln Ile Gly Ser

355 360 365

Leu Thr Glu Thr Phe Glu Ala Ile Gln Met Ala Gln Met Ala Asn Met

370 375 380

Thr Ala Val Val Ser His Arg Ser Gly Glu Thr Glu Asp Thr Thr Ile

385 390 395 400

Ala Asp Ile Ala Val Ala Met Asn Thr Gly Gln Ile Lys Thr Gly Ser

405 410 415

Met Ser Arg Thr Asp Arg Ile Ala Lys Tyr Asn Arg Leu Leu Ala Ile

420 425 430

Glu Glu Glu Leu Ser Lys Ala Ser Thr Phe Pro Lys Asp Ala Phe Tyr

435 440 445

Asn Leu Lys Lys

450

<210> 4

<211> 452

<212> PRT

<213> phosphopyruvate hydratase (phosphopyruvate hydratase)

<400> 4

Met Ser Ala Ile Lys Lys Ile His Ala Arg Glu Val Leu Asp Ser Arg

1 5 10 15

Gly Asn Pro Thr Val Gln Val Glu Val Tyr Thr Glu Leu Lys Gly Tyr

20 25 30

Gly Ser Ala Met Val Pro Ser Gly Ala Ser Thr Gly Ser Arg Glu Ala

35 40 45

Leu Glu Leu Arg Asp Lys Gly Ser Lys Phe Glu Ser Asn Trp Phe Gly

50 55 60

Gly Lys Gly Val Met Gln Ala Val Glu Asn Val Asn Lys Leu Ile Ala

65 70 75 80

Pro Ala Leu Ile Gly Phe Glu Val Thr Asp Gln Arg Gln Val Asp Leu

85 90 95

Ala Met Lys Ala Leu Asp Gly Thr Lys Asn Lys Glu Lys Leu Gly Ala

100 105 110

Asn Ala Ile Leu Gly Val Ser Leu Ala Val Ala Arg Ala Ala Ala Asn

115 120 125

Glu Leu Asp Leu Pro Leu Tyr Lys Tyr Leu Gly Gly Phe Asn Ala His

130 135 140

Lys Leu Pro Leu Pro Met Leu Asn Val Ile Asn Gly Gly Glu His Ala

145 150 155 160

Ser Asn Thr Leu Asp Phe Gln Glu Phe Met Val Met Pro Val Gly Ala

165 170 175

Lys Ser Phe Arg Glu Ala Leu Gln Met Ala Asn Phe Val Phe His Asn

180 185 190

Leu Ala Lys Leu Leu Lys Lys His Gly His Gly Val Gln Val Gly Asp

195 200 205

Glu Gly Gly Phe Ala Pro Asn Phe Lys Ser His Glu Glu Ala Leu Asp

210 215 220

Phe Leu Val Glu Ala Ile Lys Leu Ser Gly Tyr Lys Pro Ala Ile Ser

225 230 235 240

Gly Glu Lys Ala Val Ala Ile Ala Met Asp Cys Ala Ser Ser Glu Leu

245 250 255

Tyr Lys Asp Gly Lys Tyr Thr Phe Gly Lys Leu Lys Lys Ala Ile Glu

260 265 270

Glu Lys Gln Pro Gly Phe Glu Asn Leu Gly Lys Thr Lys Leu Val Tyr

275 280 285

Thr Thr Asp Glu Leu Ile Asp Tyr Leu Asp His Leu Val Ser Lys Tyr

290 295 300

Pro Ile Val Ser Ile Glu Asp Gly Leu Ala Glu Ser Asp Trp Ala Gly

305 310 315 320

Phe Glu Lys Leu Thr Lys Arg Leu Gly His Lys Leu Gln Val Val Gly

325 330 335

Asp Asp Leu Thr Val Thr Asn Thr Glu Leu Leu Ala Lys Ala Ile Glu

340 345 350

Arg Lys Ala Met Asn Ser Ile Leu Ile Lys Val Asn Gln Ile Gly Ser

355 360 365

Leu Thr Glu Thr Phe Glu Ala Ile Gln Met Ala Gln Met Ala Asn Met

370 375 380

Thr Ala Val Val Ser His Arg Ser Gly Glu Thr Glu Asp Thr Thr Ile

385 390 395 400

Ala Asp Ile Ala Val Ala Met Asn Thr Gly Gln Ile Lys Thr Gly Ser

405 410 415

Met Ser Arg Thr Asp Arg Ile Ala Lys Tyr Asn Arg Leu Leu Ala Ile

420 425 430

Glu Glu Glu Leu Ser Lys Ala Ser Thr Phe Pro Lys Asp Ala Phe Tyr

435 440 445

Asn Leu Lys Lys

450

<210> 5

<211> 864

<212> DNA

<213> fructose-1,6-bisphosphate aldolase type II (fructose-1, 6-bisphosphate aldolase, class II)

<400> 5

atgccgctga ccaacgccaa agaaatgctg attaaagcaa aagaaggtcg ctatgccgtg 60

ccgcatatta acattaacaa cctggaatgg gccaaagcgg ttctgctggc cgcccaggaa 120

gttaacagcc cgctgattct ggccacaagc gaaggtgcag tgaaatatat gggtggtttt 180

aaaacagtgg caaacatggt taaaggtctg gtgaatgatc tgaatattag tatcccggtt 240

gcactgcatc tggatcatgg tagctatgaa ggtgttaaaa aggcgctgga aacggatggc 300

tatagcagcg tgatgtttga tggtagccat tataaatttg cagaaaatta tgaaaaaacc 360

aaagaactgc tggaactggc aaaaaccgca aattgtagct ttgaagcaga agttggtacc 420

attggtggtg aagaagatgg tattattggt agcggtgaac tggcagatgc aggtgaagca 480

aaacagatgg cagaactggg tattgatgtt ctggcagcag gtattggtaa tgttcatggt 540

ccgtatccgg aaaattggaa aagcctggat tttgataaac tgaaagaaat tgttagcagc 600

gcaaaaattg gtattgttct gcatggtggt agcggtattc cgcagaaaca gattcagaaa 660

gcaattagcc tgggtgttac caaaattaat gttaataccg aactgcagca ggcaaatcat 720

aaagcactgc gtgaatttat tctgagcaat aaagatctgg aaggtaaaaa ttttgatccg 780

cgtaaactgt ataaaccggg ttttgatgca atgcagaaaa ccgttaaaga aaaaattcat 840

gaatttggta gccagaataa agca 864

<210> 6

<211> 864

<212> DNA

<400> 6

atgccattaa caaacgcaaa ggaaatgctt attaaagcca aagaaggtag atacgcggtt 60

ccacatatta atattaacaa tttagaatgg gctaaagctg tgcttcttgc agctcaagaa 120

gtaaattcgc cattaatttt agcaaccagc gaaggtgcag ttaaatacat gggtggattt 180

aagaccgtag ctaatatggt taaagggtta gtaaatgatt taaatattag cattccagta 240

gcgcttcatt tagatcatgg atcatatgaa ggagtgaaaa aagctctaga aaccgatgga 300

tattcatctg ttatgtttga tggaagccat tataaatttg cagaaaacta cgaaaaaacc 360

aaagaattat tagagctagc taaaactgct aattgttcat ttgaagccga ggttggaacc 420

atcggtggtg aagaagatgg aattatcggt tcaggagagc tagccgatgc tggtgaagca 480

aaacaaatgg cagagctagg aattgacgtt cttgcagccg gaatcggaaa cgttcacggg 540

ccatatccag aaaattggaa atctcttgat tttgacaaat taaaagaaat agtatcaagc 600

gctaaaattg gaattgttct tcatggtggt agtggtatcc cacaaaaaca aattcaaaaa 660

gctatatctc taggagttac taaaattaac gtaaacaccg aactccaaca agcaaatcat 720

aaagctttaa gagaatttat tctttcaaat aaagaccttg aaggaaaaaa ttttgatcca 780

agaaaattat ataaaccagg ttttgatgca atgcaaaaaa ccgtaaaaga aaaaattcac 840

gaatttggat cacaaaataa agct 864

<210> 7

<211> 288

<212> PRT

<400> 7

Met Pro Leu Thr Asn Ala Lys Glu Met Leu Ile Lys Ala Lys Glu Gly

1 5 10 15

Arg Tyr Ala Val Pro His Ile Asn Ile Asn Asn Leu Glu Trp Ala Lys

20 25 30

Ala Val Leu Leu Ala Ala Gln Glu Val Asn Ser Pro Leu Ile Leu Ala

35 40 45

Thr Ser Glu Gly Ala Val Lys Tyr Met Gly Gly Phe Lys Thr Val Ala

50 55 60

Asn Met Val Lys Gly Leu Val Asn Asp Leu Asn Ile Ser Ile Pro Val

65 70 75 80

Ala Leu His Leu Asp His Gly Ser Tyr Glu Gly Val Lys Lys Ala Leu

85 90 95

Glu Thr Asp Gly Tyr Ser Ser Val Met Phe Asp Gly Ser His Tyr Lys

100 105 110

Phe Ala Glu Asn Tyr Glu Lys Thr Lys Glu Leu Leu Glu Leu Ala Lys

115 120 125

Thr Ala Asn Cys Ser Phe Glu Ala Glu Val Gly Thr Ile Gly Gly Glu

130 135 140

Glu Asp Gly Ile Ile Gly Ser Gly Glu Leu Ala Asp Ala Gly Glu Ala

145 150 155 160

Lys Gln Met Ala Glu Leu Gly Ile Asp Val Leu Ala Ala Gly Ile Gly

165 170 175

Asn Val His Gly Pro Tyr Pro Glu Asn Trp Lys Ser Leu Asp Phe Asp

180 185 190

Lys Leu Lys Glu Ile Val Ser Ser Ala Lys Ile Gly Ile Val Leu His

195 200 205

Gly Gly Ser Gly Ile Pro Gln Lys Gln Ile Gln Lys Ala Ile Ser Leu

210 215 220

Gly Val Thr Lys Ile Asn Val Asn Thr Glu Leu Gln Gln Ala Asn His

225 230 235 240

Lys Ala Leu Arg Glu Phe Ile Leu Ser Asn Lys Asp Leu Glu Gly Lys

245 250 255

Asn Phe Asp Pro Arg Lys Leu Tyr Lys Pro Gly Phe Asp Ala Met Gln

260 265 270

Lys Thr Val Lys Glu Lys Ile His Glu Phe Gly Ser Gln Asn Lys Ala

275 280 285

<210> 8

<211> 288

<212> PRT

<400> 8

Met Pro Leu Thr Asn Ala Lys Glu Met Leu Ile Lys Ala Lys Glu Gly

1 5 10 15

Arg Tyr Ala Val Pro His Ile Asn Ile Asn Asn Leu Glu Trp Ala Lys

20 25 30

Ala Val Leu Leu Ala Ala Gln Glu Val Asn Ser Pro Leu Ile Leu Ala

35 40 45

Thr Ser Glu Gly Ala Val Lys Tyr Met Gly Gly Phe Lys Thr Val Ala

50 55 60

Asn Met Val Lys Gly Leu Val Asn Asp Leu Asn Ile Ser Ile Pro Val

65 70 75 80

Ala Leu His Leu Asp His Gly Ser Tyr Glu Gly Val Lys Lys Ala Leu

85 90 95

Glu Thr Asp Gly Tyr Ser Ser Val Met Phe Asp Gly Ser His Tyr Lys

100 105 110

Phe Ala Glu Asn Tyr Glu Lys Thr Lys Glu Leu Leu Glu Leu Ala Lys

115 120 125

Thr Ala Asn Cys Ser Phe Glu Ala Glu Val Gly Thr Ile Gly Gly Glu

130 135 140

Glu Asp Gly Ile Ile Gly Ser Gly Glu Leu Ala Asp Ala Gly Glu Ala

145 150 155 160

Lys Gln Met Ala Glu Leu Gly Ile Asp Val Leu Ala Ala Gly Ile Gly

165 170 175

Asn Val His Gly Pro Tyr Pro Glu Asn Trp Lys Ser Leu Asp Phe Asp

180 185 190

Lys Leu Lys Glu Ile Val Ser Ser Ala Lys Ile Gly Ile Val Leu His

195 200 205

Gly Gly Ser Gly Ile Pro Gln Lys Gln Ile Gln Lys Ala Ile Ser Leu

210 215 220

Gly Val Thr Lys Ile Asn Val Asn Thr Glu Leu Gln Gln Ala Asn His

225 230 235 240

Lys Ala Leu Arg Glu Phe Ile Leu Ser Asn Lys Asp Leu Glu Gly Lys

245 250 255

Asn Phe Asp Pro Arg Lys Leu Tyr Lys Pro Gly Phe Asp Ala Met Gln

260 265 270

Lys Thr Val Lys Glu Lys Ile His Glu Phe Gly Ser Gln Asn Lys Ala

275 280 285

<210> 9

<211> 43

<212> DNA

<213> Artificial sequence (primer bind)

<400> 9

gccatggctg atatcggatc catgagcgca atcaaaaaga tcc 43

<210> 10

<211> 49

<212> DNA

<213> Artificial sequence (primer bind)

<400> 10

ctcgagtgcg gccgcaagct tttttttcag attataaaat gcatctttc 49

<210> 11

<211> 39

<212> DNA

<213> Artificial sequence (primer bind)

<400> 11

gccatggctg atatcggatc catgccgctg accaacgcc 39

<210> 12

<211> 45

<212> DNA

<213> Artificial sequence (primer bind)

<400> 12

ctcgagtgcg gccgcaagct ttgctttatt ctggctacca aattc 45

Claims

1. An immunogenic composition comprising: PPHT protein expressed and purified by pET-PPHT vector as shown in SEQ ID NO. 3, and FBA-II protein expressed and purified by pET-FBA-II vector as shown in SEQ ID NO. 7, and pharmaceutically acceptable carrier.

2. A mycoplasma synoviae PPHT and FBA-ii protein subunit vaccine comprising the immunogenic composition of claim 1, optionally comprising an adjuvant.

3. The subunit vaccine of claim 2, wherein the adjuvant is a water-in-oil-in-water adjuvant.

4. A subunit vaccine according to claim 3, wherein the adjuvant is Summit P168 adjuvant, the subunit vaccine is a mixture of a protein solution and an emulsified adjuvant, the mass ratio of PPHT to FBA-ii protein in the protein solution is 1:1, and the volume ratio of the protein solution to the adjuvant is 3:2.

5. The method for preparing chicken bursa mycoplasma PPHT and FBA-ii protein subunit vaccine according to claim 2, comprising the steps of:

(1) The PPHT gene sequence of the mycoplasma gallisepticum shown in the nucleotide sequence SEQ ID NO. 2 and the FBA-II gene sequence of the mycoplasma gallisepticum shown in the nucleotide sequence SEQ ID NO. 6 published on GenBank are referred to, target genes of PPHT and FBA-II are artificially synthesized according to the preference of escherichia coli codons, and the nucleotide sequences of the synthesized target genes are shown as SEQ ID NO. 1 and SEQ ID NO. 5 respectively;

(2) Respectively recombining target genes on pUC57 vectors to obtain recombinant plasmids pUC57-PPHT and pUC 57-FBA-II, and respectively taking pUC57-PPHT and pUC 57-FBA-II recombinant plasmids as templates to carry out PCR amplification reaction, wherein an amplification system is 2X Phanta Max Buffer mu L, dNTP Mix 1 mu L, each of the upper and lower primers is 2 mu L, the template is 2 mu L, phanta Max Super-Fidelity DNA Polymerase 1 mu L, and dd H is added ₂ O is supplemented to 50 mu L; the reaction procedure: 95. pre-denaturing at a temperature of 94 ℃ for 20sec, annealing at a temperature of 60 ℃ for 20sec, extending at a temperature of 72 ℃ for 1min, performing 35-cycle amplification, and extending at a temperature of 72 ℃ for 5min; the amplified product is subjected to agarose gel electrophoresis with concentration of 1%, target fragments are recovered by gel to obtain target genes, the target genes are preserved at the temperature of minus 20 ℃, the sequences of the upstream primer and the downstream primer corresponding to the recombinant plasmid pUC57-PPHT are shown as SEQ ID NO. 9 and SEQ ID NO. 10, and the target genes are heavyThe sequences of the corresponding upstream and downstream primers of the group plasmid pUC 57-FBA-II are shown as SEQ ID NO. 11 and SEQ ID NO. 12;

(3) Connecting target genes PPHT and FBA-II to pET-32a vectors respectively by using a recombinase of ClonExpress MultiS One Step Cloning Kit, transforming DH5 alpha competent cells from the connection products, coating the cells on an ampicillin-resistant LB plate, culturing for 12-16 h at 37 ℃, picking single colonies, inoculating LB culture media respectively, and identifying recombinant plasmids by PCR and sequencing, wherein the recombinant plasmids are named pET-PPHT and pET-FBA-II respectively;

(4) E.coli BL21 is transformed by the constructed recombinant plasmid, positive strains are identified by PCR, the positive strains are respectively inoculated into 10mL LB liquid culture medium, are cultured at 37 ℃ to logarithmic phase, are transferred into 1L LB liquid culture medium according to the inoculation ratio of 1:100, and are cultured at 37 ℃ to OD ₆₀₀ Adding IPTG with the final concentration of 0.5mmol/L at 20 ℃ for induction for 8-12 h, so that the positive strain expresses PPHT and FBA-II proteins;

(5) Purifying the recombinant expressed PPHT and FBA-II proteins, mixing the obtained protein solution according to the mass ratio of PPHT to FBA-II proteins of 1:1, wherein the concentration of the PPHT and the concentration of the FBA-II proteins in the obtained mixed protein solution are 167 mug/mL, and adding Summit P168 adjuvant with the volume of 2/3 of that of the mixed protein solution to be fully and uniformly mixed.

6. Use of an immunogenic composition according to claim 1, or a subunit vaccine according to any one of claims 2 to 4, or a subunit vaccine prepared by a method of preparation according to claim 5, in the manufacture of a medicament for the prevention of infection by mycoplasma synoviae in chickens.