Chicken mycoplasma synoviae subunit vaccine, preparation method, detection and application thereof

Abstract

The invention provides a chicken mycoplasma synoviae subunit vaccine, a preparation method, detection and application thereof. The chicken mycoplasma synoviae VY930 protein expressed on the basis of an escherichia coli prokaryotic expression system has good immunogenicity, and a subunit vaccine containing the protein is prepared from the chicken mycoplasma synoviae VY930 protein so as to effectively prevent and control chicken mycoplasma synoviae infection. Animal challenge protection tests prove that the subunit vaccine has immune protection potential equivalent to or slightly superior to that of an inactivated vaccine, and clinical symptoms caused by the mycoplasma synoviae can be remarkably relieved. The subunit vaccine disclosed by the invention is simple and convenient in preparation process and low in cost, can adapt to a large-scale production mode, and has the potential of preventing and controlling clinical infection of the mycoplasma synoviae and promoting a population purification process. The invention further provides a monoclonal antibody aiming at the protein, the monoclonal antibody can be used as an index for judging whether immunization is carried out or not, meanwhile, the immune protection evaluation criterion after chicken mycoplasma synoviae foot pad challenge is refined, and a meticulous measurement standard is provided for follow-up vaccine research and development.

Description

Technical Field

[0001] The present invention belongs to the field of protein vaccines, and in particular relates to a Mycoplasma synoviae subunit vaccine, a preparation method, a detection method and an application thereof. Background Art

[0002] Mycoplasma synoviae (MS) is a major avian pathogen. Infection in poultry can cause bursitis and air sacculitis, leading to reduced egg production and quality, lower hatchability, increased chick culling, and negatively impacting broiler growth and development. MS persists in chicken flocks for a long time and, in practice, often co-infects with other pathogens, such as Newcastle disease virus and Escherichia coli. MS is a global epidemic and extremely difficult to eradicate, resulting in significant economic losses for the poultry industry. Therefore, a combination of strict biosecurity controls, rational antimicrobial use, and effective vaccination strategies is crucial for MS prevention and control and achieving population eradication goals.

[0003] Mycoplasma infection spreads rapidly, persists for a long time, can be transmitted both horizontally and vertically, and is prone to developing drug resistance, making it difficult to control and eliminate mycoplasma. Currently, prevention and control of mycoplasma mainly involves three aspects: stock purification, drug control, and vaccination. Stock purification is costly and difficult to implement. Extensive use of antibiotics has led to the rapid development of drug resistance in MS, suggesting that drugs are no longer a viable long-term prevention and control measure for MS. Vaccination remains the primary method of infection control worldwide, especially in areas with large poultry production.

[0004] MS vaccines are primarily categorized into three main categories: inactivated vaccines, attenuated live vaccines, and genetically engineered subunit vaccines. Currently, there are only two registered MS vaccines in my country: the live attenuated vaccine strain MS-H from Australian Bioresources and the YBF-MS1 strain developed by Qingdao Yibang Bioengineering Co., Ltd. Following vaccination, the MS-H strain can colonize the upper respiratory tract, thereby rejecting wild-type infections and inducing a mucosal immune response. However, this vaccine can only be used in negative chicken flocks; vaccination of chickens already infected with MS may actually aggravate clinical symptoms. The large-scale application of live and inactivated vaccines is limited by the difficulty, high cost, and long culture cycles of mycoplasmas. With the advancement of bioinformatics, the design of genetically engineered subunit vaccines has provided new options for MS vaccine development. In recent years, researchers have also developed subunit vaccines for a number of mycoplasma diseases. Bercic et al. (Bercic RL, Slavec B, Lavric M, et al. Identification of major immunogenic proteins of Mycoplasma synoviae isolates [J]. Veterinary Microbiology, 2008, 127(1-2):147-154) attempted to identify MS immunogenic proteins using N-terminal sequencing, but only a limited number of proteins were identified, and the protective effect was not verified. While MS vaccination may not completely prevent pathogen colonization, it has been shown to be an important method for controlling the disease, reducing infection rates, delaying onset, and mitigating vertical transmission. Existing registered vaccines still suffer from weak immune protection. The live attenuated MS-H vaccine strain, after vaccination, can colonize the upper respiratory tract, thereby repelling wild-type infection and eliciting a mucosal immune response. However, this vaccine can only be used in chickens that are negative for MS; vaccination in chickens already infected with MS may actually exacerbate clinical symptoms. Furthermore, the use of attenuated vaccines can affect serological testing in chickens, making false positives more likely. In addition, the high cost and long cultivation cycle of inactivated and live attenuated vaccines limit their large-scale application. Therefore, developing MS vaccines with good protection, high safety, simple operation, and suitable for large-scale immunization applications, and establishing intuitive and simple immune protection efficacy detection methods, are of great significance for the prevention and control of MS in my country. Summary of the Invention

[0005] The purpose of the present invention is to provide a detailed rule for evaluating the effect of a Mycoplasma synoviae vaccine, a preparation method and application of a subunit vaccine for the immune prevention of Mycoplasma synoviae disease.

[0006] The object of the present invention is achieved through the following technical solutions:

[0007] The present invention provides an expression vector for a Mycoplasma synoviae protein, which contains a target gene fragment, including a gene encoding a Mycoplasma synoviae VY930 protein. The sequence of the target gene fragment after codon mutation is as shown in SEQ ID NO.2, and the amino acid sequence is as shown in SEQ ID NO.3.

[0008] Specifically, the present invention employed pan-genomic technology to analyze the core genes of MS, discovering 102 conserved core genes. This was then combined with a reverse vaccinology approach to predict and analyze the MS core genes, resulting in the highly conserved, highly antigenic, and immunogenic VY930 protein. Genetic codon mutagenesis was then performed to select the dominant codons in E. coli. The resulting gene sequence is shown in SEQ ID NO. 2.

[0009] The present invention also provides a Mycoplasma synoviae VY930 protein subunit vaccine, which comprises a preparation method of the VY930 protein and an adjuvant.

[0010] Furthermore, the adjuvant is a water-in-oil emulsion.

[0011] Furthermore, the subunit vaccine is a mixture of an emulsified protein solution and an adjuvant, and the volume ratio of the protein to the adjuvant is 1:1.

[0012] Specifically, the preparation method involves concentrating the purified VY930 protein using a 3kD pore size ultrafiltration tube and resuspending it in PBS buffer. The protein, displaced into PBS buffer, is diluted according to the immunization dose and uniformly mixed with Freund's adjuvant at a 1:1 ratio. The mixture is emulsified in an emulsifier for 1 minute to form a uniform emulsion, which is the VY930 protein subunit vaccine.

[0013] Furthermore, the present invention provides a monoclonal antibody that specifically recognizes the above-mentioned VY930 protein, wherein the monoclonal antibody is 6F4 monoclonal antibody, whose heavy chain variable region sequence is shown in SEQ ID NO.14, wherein the sequences of HCDR1-3 are shown in SEQ ID NOs.16-18, respectively; the light chain variable region sequence is shown in SEQ ID NO.15, wherein the sequences of LCDR1-3 are shown in SEQ ID NOs.19-21, respectively.

[0014] Furthermore, the present invention provides a composition comprising the aforementioned Mycoplasma synoviae VY930 protein subunit vaccine and / or a monoclonal antibody that specifically recognizes the aforementioned VY930 protein.

[0015] Furthermore, the present invention provides a use of the above composition in preparing a medicament for treating or preventing chicken synoviae mycoplasma disease.

[0016] The present invention also provides an evaluation criterion for determining the protective efficacy of the Mycoplasma synoviae vaccine strain, which is used for protective evaluation in the MS vaccine development process.

[0017] Furthermore, the assessment criteria apply to the footpad infection method.

[0018] Furthermore, the assessment criteria are divided into 6 levels (0, 1, 2, 3, 4, 5) according to the presence and severity of the lesion, and points are assigned according to the severity of the lesion. The scores are: 0, 0.2, 0.4, 0.6, 0.8, 1.0. Among them, level 0 means no disease, levels 1-3 mean foot pad symptoms, and levels 4-5 mean that the lesion spreads from the foot pad to the metatarsal bones, tarsal joints and other parts. The score comparison of the degree of lesion is as follows Figure 5 shown.

[0019] Beneficial effects

[0020] The prokaryotic expression vector system provided by the present invention expresses the VY930 protein, and its antigen is easy to obtain, the process is safe and simple, the cost is low, the expression level is high, the immune effect is good, and it has the potential for large-scale production and application. The subunit vaccine of the present invention has a slightly better protective effect than the inactivated vaccine. Compared with traditional attenuated vaccines and inactivated vaccines, it can reduce the detection troubles caused by serological positivity and reduce the cost and time of mycoplasma culture. The present invention provides a new idea for MS prevention and control, which is beneficial to disease prevention and control and population purification, and is a subunit vaccine with great application prospects. The monoclonal antibody provided can effectively recognize the VY930 protein in the present invention, and provide a judgment basis for the cluster immunization strategy. The present invention provides an evaluation rule for determining the protective efficacy of chicken synoviae Mycoplasma vaccine strains. The rules introduce a grade and scoring system, and refine the evaluation method of the vaccine protection effect after footpad infection, thereby effectively reducing the subjectivity of clinical result interpretation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Overlap PCR and PCR identification results of the target gene of the present invention and the construction of the expression vector, where A is the map of the expression vector pCold I used in the present invention, B is the construction process of the VY930 recombinant expression strain, where a is the segmented amplification of the VY930 gene, b is the full-length amplification of the VY930 gene, and c is the PCR identification of the VY930 recombinant expression strain.

[0023] Figure 2 The figures show the SDS-PAGE and immunogenicity verification results of the proteins expressed in the present invention, where A is the SDS-PAGE of the VY930 recombinant protein expressed in the supernatant, which is consistent with the expected size; B is the purification result of the VY930 recombinant protein; C shows that the VY930 recombinant protein can react with the immune serum and is visualized at a molecular weight of 29.0 kDa, demonstrating its good immunogenicity. Figure 3 Specific detection of VY930 protein monoclonal antibody, where M is a marker, 1 is Mycoplasma gallisepticum (MG), 2 is Mycoplasma avium (MI), 3 is Haemophilus paragallinarum, and 4 is Mycoplasma synoviae (MS);

[0024] Figure 4 The subunit vaccine immunization program and humoral immunity level determination of the present invention, wherein A is the subunit vaccine immunization program of the present invention, and B is the humoral immunity level determination result;

[0025] Figure 5 The table description and diagram of the protective effect scoring method based on the foot pad challenge established by the present invention are as follows: A is a table of the protective effect scoring method based on the foot pad challenge established by the present invention, and B is a diagram of the protective effect scoring method based on the foot pad challenge.

[0026] Figure 6 The figures are the dynamic evaluation results of the protective effects of the subunit vaccine of the present invention and the inactivated vaccine. A is the clinical dynamic protective effect after the challenge, and B is the lesion score after the challenge, where a is the average lesion score in the group one week after the challenge, b is the average lesion score in the group two weeks after the challenge, c is the average lesion score in the group three weeks after the challenge, and d is the dynamic change trend of the total lesion score in each group three weeks after the challenge.

[0027] Figure 7 The results of T lymphocyte subset sorting after immunization with the vaccine of the present invention, where A is the CD4 + T cell changes, B is CD8 after immunization + T cell changes, C is CD3 after immunization + T cell changes, D is CD4 after immunization + T / CD8 + T cell changes. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value within any stated value or stated range and any other stated value or intermediate value within the range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Unless otherwise specified, the chemical reagents, biochemical reagents and materials used in the present invention can be obtained from commercial channels.

[0033] Example 1 Construction and identification of expression vector pColdⅠ

[0034] The VY930 gene sequence is shown in SEQ ID NO.1. Based on the codon preference of E. coli and the translation characteristics of mycoplasma amino acids, the codons of the VY930 gene were point mutated and the stop codon TGA was replaced with TGG. The optimized sequence is shown in SEQ ID NO.2 and its amino acid sequence is shown in SEQ ID NO.3. 1853(GenBank accession number CP011096) DNA was used as a template, and the primer sequences shown in Table 1 were used to amplify and recover the four primer pairs. The amplification system was: 2 μL each of upstream and downstream primers, 25 μL of TaKaRa (PrimeSTAR@Max DNA Polymerase), MS WVU 1853 1 μL of whole genomic DNA was added to 50 μL with ddH2O. Amplification conditions were as follows: denaturation at 98°C for 10 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 5 seconds; 35 cycles were performed. Subsequently, the recovered DNA was used as a template for full-length amplification using SEQ ID NO. 4 and SEQ ID NO. 11 to obtain the full-length VY930 gene.

[0035] The VY930 target gene was linked into the expression vector pColdⅠ to form the recombinant expression plasmid pColdⅠ-VY930. Subsequently, the recombinant plasmid was transformed into E. coli DH5α, and a single clone was picked. The bacterial liquid was identified by PCR and sequencing using the pColdⅠ universal primers (sequences shown as SEQ ID NO.12 and SEQ ID NO.13, respectively). The positive clone strain was named E. coli DH5α (pColdⅠ-VY930). The PCR results are shown in Figure 1. Figure 1 shown.

[0036] Table 1 Gene Overlap PCR amplification primer sequences

[0037]

[0038] The pColdⅠ universal primer sequence is as follows:

[0039] pCold-F: 5'-ACGCCATATCGCCGAAAGG-3' (SEQ ID NO.12)

[0040] pCold-R: 5'-GGCAGGGATCTTAGATTCTG-3' (SEQ ID NO. 13).

[0041] Example 2 Induced Expression and Purification of Chicken Mycoplasma Synoviae Protein VY930

[0042] Take freshly cultured E. coli BL21 (pColdI-VY930) bacterial suspension and inoculate it into LB medium containing 100 μg / mL ampicillin at a transfer ratio of 1:100, and culture at 37°C and 180 rpm until the bacterial suspension OD reaches 600nm The expression of the protein was induced at 16 ° C and 110 rpm for 24 h.

[0043] After induction, the cells were collected by centrifugation at 8000 rpm for 10 minutes, resuspended in Binding buffer, and then disrupted using a high-pressure disruptor. Appropriate amounts of protein loading buffer were added to the whole cells before induction, the whole cells after induction, the supernatant after induction, and the precipitate after induction, respectively. After boiling for 15 minutes, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. The results are shown in Figure 2. Figure 2 As shown in A, the VY930 protein was expressed in the supernatant and had a molecular weight of 29.0 kDa, which was consistent with the expected size.

[0044] The supernatant after crushing was used Ni-NTAResin Ni-NTAResin purification of VY930 protein includes column packing, equilibration, sample loading, washing, storage, and verification.

[0045] (1) Column loading: Resuspend the medium and add an appropriate amount of medium to the column according to the amount of protein to be purified, and let it stand.

[0046] (2) Equilibration: Equilibrate the column with 5-10 column volumes of Binding Buffer.

[0047] (3) Sample loading: To avoid clogging the chromatography column, the protein sample was filtered through a 0.45 μm filter and then added to the column bed.

[0048] (4) Washing: After loading, use Washing Buffer to elute impurities, and then use Elution Buffer to perform gradient elution of the protein.

[0049] (5) Storage: Wash the column bed with a large amount of Elution Buffer containing 500 mM imidazole, then wash it twice with deionized water. Finally, add 20 mL of 20% ethanol and store at 4°C after 10 mL has flowed out.

[0050] (6) Verification: Take 40 μL of protein from each washing gradient, add 10 μL of 5× protein loading buffer, boil in boiling water for 15 min, centrifuge briefly, and take 10 μL of sample for SDS-PAGE.

[0051] Main buffer configuration for protein purification:

[0052] (1) Binding Buffer: 50 mM NaH2PO4, 300 mM NaCl, 0 mM imidazole, add 800 mL of deionized water, fully dissolve on a magnetic stirrer, adjust the pH to 8.0, and finally dilute to 1 L and store at room temperature until ready for use.

[0053] (2) Washing Buffer: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, add 800 mL of deionized water, fully dissolve on a magnetic stirrer, adjust the pH to 8.0, and finally dilute to 1 L and store at room temperature until ready for use.

[0054] (3) Elution Buffer: 50mM NaH2PO4, 300mM NaCl, 30 / 50 / 100 / 200 / 300 / 500mM imidazole, add 800mL of deionized water, fully dissolve on a magnetic stirrer, and adjust the pH to 8.0. Finally, adjust the volume to 1L and store at room temperature for use. The results of VY930 protein purification are shown in the figure. Figure 2 As shown in B, the target band is clear and single, with a molecular weight of 29.0 kDa, which is consistent with the expected size, indicating that the protein was successfully purified.

[0055] Example 3 Preparation of VY930 antigen protein as a candidate vaccine antigen

[0056] The purified VY930 protein from Example 2 was concentrated using a 3 kD pore size ultrafiltration tube and resuspended in PBS buffer. The protein, replaced in PBS buffer, was diluted according to the immunization dose and uniformly mixed with Freund's adjuvant at a 1:1 ratio. Emulsification was performed using an emulsifier for 1 minute to form a uniform emulsion, which is the VY930 protein subunit vaccine.

[0057] Example 4 Preparation of rabbit polyclonal antibody serum against VY930 antigen protein

[0058] 1. Animal immunization

[0059] Two-month-old New Zealand white rabbits were immunized with the MS subunit vaccine prepared in Example 3 via multiple subcutaneous injections. For the first immunization, the purified protein was emulsified with an equal volume of Freund's complete adjuvant to prepare a subunit vaccine, with an immunization dose of 500 μg per rabbit. Two weeks later, a second immunization was performed, and two weeks after the second, a third immunization was performed, with a dose of 300 μg per rabbit. Freund's incomplete adjuvant was used in both immunizations.

[0060] 2. Antibody Collection

[0061] One week after the second immunization, blood samples were collected from the rabbits' ear veins. One week after the third immunization, blood samples were collected from the heart. Blood samples were refrigerated at 4°C overnight. The next day, the supernatant was collected by centrifugation at 4000 rpm for 20 minutes to obtain polyclonal antibody serum, which was then stored at -20°C.

[0062] 3. Antibody titer detection

[0063] The collected rabbit serum was tested by ELISA. The VY930 protein was diluted to 5 μg / mL using the coating solution, and 100 μL was added to each well, and the plate was coated overnight at 4°C. After washing with PBST, 200 μL of 5% skim milk blocking solution was added to each well and the plate was incubated in a 37°C constant temperature incubator for 2 hours. After washing with PBST, the rabbit polyclonal antibody serum to be tested was diluted two-fold from 1:100, and the diluted serum was added to the enzyme labeling plate, 100 μL per well, and the plate was incubated in a 37°C constant temperature incubator for 2 hours. After washing with PBST three times, the goat anti-rabbit enzyme-labeled secondary antibody IgG was diluted 1:10000, 100 μL per well, and the plate was incubated in a 37°C constant temperature incubator for 2 hours. After washing with PBST, 100 μL of TMB substrate color development solution was added to each well and the plate was placed in a 37°C constant temperature incubator in the dark for 15 minutes. Finally, 50 μL of stop solution was added to each well to terminate the reaction, and the OD was measured using a multifunctional enzyme reader. 450nm A positive result was determined if the ratio of the test well value to the negative control well value was ≥2.1. The maximum dilution that could be determined as positive was used as the antibody titer of the test serum. The results showed that the antibody titer reached 1:102400.

[0064] 4. Immunogenicity assay

[0065] The purified protein was subjected to SDS-PAGE gel electrophoresis, and the SDS-PAGE electrophoresis product was transferred to NC (nitrocellulose) membrane, blocked with blocking solution at room temperature for 2 hours, washed 3 times with TBST, added with rabbit polyclonal antibody serum and incubated overnight at 4°C, washed 3 times with TBST, added with HRP (horseradish peroxidase) labeled goat anti-rabbit IgG as secondary antibody, incubated at room temperature for 2 hours, and washed 3 times with TBST. After ECL color development, photos were taken, the results were observed, and the immunogenicity of the protein was analyzed. The results are as follows Figure 2 As shown in C, VY930 protein can react with immune serum and is visualized at a molecular weight of 29.0 kDa, demonstrating its good immunogenicity.

[0066] Example 5 Screening of VY930 protein monoclonal antibodies and evaluation of immune effects

[0067] Monoclonal antibodies against the VY930 protein were prepared and screened according to standard procedures in the art. Briefly, four 40-day-old BALB / c female mice were selected, three of which were routinely immunized with the purified VY930 protein obtained in Example 2. The remaining mouse served as a negative control and was not immunized. The serum from the mouse after the fourth immunization was used to establish an indirect ELISA assay for hybridoma screening.

[0068] (1) Dilute the VY930 protein to 0.5 μg / mL using antigen coating solution (0.05 mol / L, pH 9.6±0.2 carbonate buffer), add 100 μL to each well, coat overnight at 4°C (approximately 8 h), and wash three times with PBST.

[0069] (2) Add 200 μL of 5% skim milk solution to each well, block at 37°C for 2 h, and wash three times with PBST.

[0070] (3) Mouse serum samples were diluted in a gradient from 1:200 to 1:25600. Three replicate wells were set for each sample, 100 μL / well, incubated at 37°C for 1 h, and washed three times with PBST.

[0071] (4) Dilute HRP-labeled goat anti-mouse IgG (H+L) antibody at 1:5000 with 2.5% skim milk, add 100 μL to each well, incubate at 37°C for 1 h, and wash three times with PBST.

[0072] (5) Add 100 μL of TMB colorimetric solution to each well and incubate at room temperature for 10 min in the dark.

[0073] (6) Add 50 μL / well of ELISA stop solution (1 M HCl), stop color development, and measure the absorbance at 450 nm within 5 minutes.

[0074] (7) Calculate the P / N value (the ratio of serum absorbance of immunized mice to that of negative control mice). A P / N value > 2 is considered positive (i.e., the immunized mice successfully produced antibodies against the VY930 protein); otherwise, it is considered negative. The mouse with the highest antibody titer is selected for cell fusion experiments to prepare monoclonal antibodies.

[0075] 30 days before fusion, SP2 / 0 cells were revived and subcultured to the logarithmic growth phase. Cells in good condition were collected for cell fusion, and the stable hybridoma cell line 6F4 was screened and amplified.

[0076] The above-mentioned indirect ELISA method was used to detect the hybridoma cell culture supernatant with VY930 protein as the coating antigen. The coating conditions were optimized by array titration method, and the positive / negative absorbance ratio (P / N value) was maximized and the positive serum OD was 450A P / N value close to 1.0 is optimal. A P / N value > 2 is considered positive. Indirect ELISA results showed that the P / N value was highest when coated with 1 μg / mL of VY930 protein. This indirect ELISA method was established to detect the supernatant of monoclonal antibodies secreted by hybridoma cells and screen for positive monoclonal cells. Hybridomas that tested positive in both assays were subcloned by limiting dilution. After 2-4 rounds of cloning, cell lines stably secreting monoclonal antibodies were obtained. Antibodies were purified using the octanoic acid-ammonium sulfate method and identified using the aforementioned ELISA method. The titer of the purified antibodies reached 1:102,400.

[0077] The above 6F4 monoclonal antibody was expanded and sent to a genetic company for sequencing. The results showed:

[0078] The heavy chain variable region sequence is:

[0079] EVQLQHQSGAELRPGASVAKMLSCKATAGYASGFTFASNYGWIHWVKPGHPGAQGLE WVGVPGSGADSFTKFMANQKFKGKTKLTAVSSTAYMELSSLATNEAIYYCVYCARLPYDW AAFDYWGQGTSVTVSS(SEQ IDNO.14);

[0080] Wherein HCDR1 is: GFTFASN (SEQ ID NO.16);

[0081] HCDR2 is: VPGSGADSFTKFMANQKFKG (SEQ ID NO.17);

[0082] HCDR3 is: CARLPYDWAAFDY (SEQ ID NO. 18),

[0083] The light chain variable region sequence is:

[0084] DIVITQSPASLNPATSGESVSISCRSQNIYKAGQNIYLSWFLQQKPPAQSLLIVYWMLASA SGVARFSGGSGSTLAITLEISGVKEDAATVYSCQQSASVEPHYAFGSAGTELIR (SEQ ID NO. 15);

[0085] wherein LCDR1 is: RSQNIYKAGQNI (SEQ ID NO. 19);

[0086] LCDR2 is: WMLASAS (SEQ ID NO. 20);

[0087] LCDR3 is: QQSASVEPHYA (SEQ ID NO. 21).

[0088] Common pathogens found in chicken farms were selected for specificity testing, including Mycoplasma gallisepticum (MG), Mycoplasma fowlii (MI), Mycoplasma synoviae (MS), and Haemophilus paragallinarum. The above-mentioned other pathogen strains were inoculated into 100 mL of liquid culture medium at a ratio of 1% (v / v) and cultured at 37°C. After the culture medium turned yellow, the bacterial precipitate was collected at 10,000 r / min for 30 minutes, resuspended and washed three times with PBS, and resuspended with 1 / 10 volume of PBS in the culture medium and ultrasonically disrupted in an ice bath. The supernatant was collected at 10,000 r / min for 20 minutes, which is the whole bacterial protein of the pathogen. The protein concentration was measured using a BCA protein quantification kit. After quantitative packaging, the samples were stored at -70°C. Western blotting was used to detect the specificity of the prepared monoclonal antibodies against different mycoplasma strains.

[0089] The results show that Figure 3 As shown in the figure, the 6F4 monoclonal antibody had no reaction bands with Mycoplasma gallisepticum (MG), Mycoplasma avium (MI), and Haemophilus paragallinarum, but showed specific reaction bands with the whole bacterial protein of Mycoplasma synoviae (MS). This shows that the obtained monoclonal antibody can specifically bind to Mycoplasma synoviae (MS), can effectively distinguish Mycoplasma synoviae (MS) from other common pathogens, and can be used to detect Mycoplasma synoviae (MS) with high specificity.

[0090] Example 6 Evaluation of the immune protection effect of VY930 antigen protein

[0091] 1. Animal immunization

[0092] Freshly hatched SPF chickens were randomly divided into 4 groups (15 / group), namely, the MS subunit vaccine immunization group, the inactivated vaccine immunization group, the adjuvant immunization control group and the PBS control group. The MS subunit vaccine prepared in Example 3 was immunized in SPF chickens by multiple subcutaneous injections in the neck. The immunization procedure was as follows: Figure 4 As shown in Figure A, the prepared subunit vaccine was administered twice via multiple subcutaneous injections in the neck. The doses were 150 μg / animal and 100 μg / animal at 7 and 21 days of age, respectively. The adjuvant used for the first immunization was Freund's complete adjuvant, and the second immunization was Freund's incomplete adjuvant. The inactivated vaccine group was immunized according to the instructions in the manufacturer's instructions.

[0093] 2. Polyclonal Antibody Collection

[0094] After the first immunization, blood was collected weekly from the sub-wing vein of the chickens for serum isolation. Blood samples were refrigerated at 4°C overnight. The next day, the supernatant was collected by centrifugation at 4000 rpm for 20 minutes to obtain polyclonal antibody serum, which was then stored at -20°C.

[0095] 3. Antibody titer detection

[0096] The collected chicken serum was subjected to ELISA test. The detection method was the same as that shown in Example 4. The enzyme-labeled secondary antibody used was HRP-labeled goat anti-chicken IgY. The results showed that the antibody titer showed a gradual upward trend 7-28 days after immunization, and the titer reached 1:102400 two weeks after the second immunization. The test results are shown in Figure 2. Figure 4 As shown in B.

[0097] 4. MS attack protection test

[0098] Two weeks after the second immunization, the MS subunit vaccine immunization group, the inactivated vaccine immunization group and the challenge control group were challenged with the virus. The strain used was the clinical isolate MS YC1 preserved in our laboratory (Zhao Luru, Qi Jingjing, Zhang Kaiqin, et al. Isolation and identification of Mycoplasma synoviae in some areas of Jiangsu Province and evaluation of pathogenicity and drug resistance [J]. Chinese Journal of Veterinary Science, 2024, 54(12): 1617-1626.), and the footpad challenge dose was 2×10 5 Clinical symptoms were recorded weekly after infection, and pathological changes were observed and scored three weeks later.

[0099] 5. Clinical criteria for MS footpad challenge

[0100] Two weeks after vaccination, the chickens were challenged with footpad virus at a dose of 2×10 5 CCU / mL, each chicken is challenged with one footpad. The present invention uses the left footpad as an example. The observation period after challenge is three weeks. The clinical symptoms of the chickens in the third week are used as the judgment criteria. The evaluation criteria for the protective efficacy of the chicken synoviae vaccine strain are formulated, such as Figure 5As shown. The detailed rules are divided into 6 levels (0, 1, 2, 3, 4, 5) according to the presence or absence and severity of lesions in the chickens in the third week after the infection, among which level 0 means no disease, levels 1-3 mean foot pad symptoms, and levels 4-5 mean lesions spread from the foot pad to the metatarsal bones, tarsal joints and other parts. The scores of each level are: 0, 0.2, 0.4, 0.6, 0.8, 1.0. The specific scoring criteria are as follows: Level 0 is 0, the chicken looks normal and no clinical symptoms occur; Level 1 is 0.2, the foot pad on the infected side is slightly swollen, and needs to be carefully compared with the foot pad on the non-infected side. No clinical symptoms occur in other parts of the chicken; Level 2 is 0.4, the foot pad on the infected side is obviously swollen, but it has not spread to the whole foot. There are no clinical symptoms in other parts; Level 3 is 0.6, the foot pad on the infected side is obviously swollen and spreads to the whole foot , seriously affecting the normal walking of chickens, causing lameness and other phenomena, but it did not spread to the non-infected side, and no lesions were found in the metatarsal bones and tarsal joints; the score of level 4 was 0.8, the foot pad on the infected side was similar to level 3, and some swelling spread to the non-infected foot pad, the metatarsal bones and tarsal joints on the infected side were slightly swollen, and the sick chickens may show symptoms such as air sacitis during autopsy; the score of level 5 was 1.0, the foot pad on the infected or non-infected side was severely swollen, the metatarsal bones and tarsal joints were severely swollen, and the autopsy showed symptoms of air sacitis.

[0101] The present invention establishes a scoring and evaluation method for the immune protection effect of MS vaccine based on the footpad challenge pathway. Compared with the existing air sac evaluation and pathological section evaluation methods, the experimenter can make an evaluation by intuitively interpreting the clinical symptoms of the chicken flock after the challenge, and the scoring method is more intuitive and simple.

[0102] 6. Evaluation of the protective effect of the VY930 subunit vaccine against virus infection

[0103] 14 days after the second immunization, the experimental group was inoculated with about 2×10 5 CCU / mL of MS YC1 culture were observed for 21 days and the clinical manifestations were recorded. Figure 6 Results showed that chickens in the adjuvant-immunized control group had a 100% incidence rate starting in the first week. During the observation period, severe footpad swelling developed, and some chickens experienced severe swelling and deformation of the metatarsals and tarsal joints. Autopsy revealed cheesy material adhering to the surface of the air sacs. Chickens in the PBS control group showed no clinical symptoms. Compared with the challenge group, the VY930 subunit vaccine-immunized and inactivated vaccine-immunized groups showed significantly reduced symptoms. A single-factor ANOVA test using SPSS software revealed that clinical symptoms were significantly reduced in the vaccine-immunized groups compared with the adjuvant-immunized control group. VY930 provided superior protection compared to the inactivated vaccine in the second week. Overall, the lesion scores in the subunit vaccine-immunized groups were lower, confirming the VY930 subunit vaccine's significant protective effect against clinical MS infection.

[0104] 7. T lymphocyte subset sorting

[0105] In order to evaluate the cellular immunity level of chickens induced by VY930 subunit vaccine and inactivated vaccine, the spleen of chickens was aseptically removed before the challenge, and spleen lymphocytes were isolated using a commercial chicken spleen lymphocyte isolation kit. The isolated lymphocytes were diluted to 1×10 6 The cells were incubated with fluorescent dye-conjugated antibodies PE-CD3 (0.3 μg), FITC-CD4 (1 μg) and APC-CD8 (0.1 μg) at 25°C for 30 minutes. After incubation with fluorescent antibodies, the cells were washed twice with PBS by centrifugation and resuspended in 300 μL PBS for flow cytometric analysis. The CD3 in spleen lymphocytes of chickens in the VY930 subunit vaccine immunization group, the inactivated vaccine immunization group and the adjuvant immunization control group were detected by flow cytometry. + T cells (total T cells), CD3 + CD4 + T cells (helper T cells) and CD3 + CD8 + T cell (cytotoxic T cell) level. Figure 7 As shown in the figure, compared with the adjuvant immunization control group, the proportion of CD3+ total T lymphocytes in the chickens after immunization with VY930 and inactivated vaccine increased; + CD4 + T cells (p<0.01) and CD3 + CD8 + T cells (p<0.001) were significantly increased; CD3 + CD4 + T cells (p<0.01) were significantly increased, while CD3 + CD8 + The proportion of T cells did not reach statistical significance (p = ns). This result suggests that there are differences in the immune response patterns induced by the two types of vaccines. Inactivated vaccines mainly activate humoral immune responses based on B cell differentiation and antibody production, while CD8 + The activation effect of T cell-mediated cellular immune response is weak. However, the VY930 subunit vaccine constructed in this study showed unique immune activation characteristics and could effectively induce CD4 + T cell-assisted humoral immune response, and CD8 +T cell-mediated cellular immune response, these two immune response pathways may produce a synergistic effect, providing advantages for preventing MS infection. The results show that the present invention uses pan-genomic technology to analyze the MS whole genome, screens out the core genes, and then combines the reverse vaccinology method to finally screen out the MS conserved protein VY930, and conducts prokaryotic expression vector construction, protein purification, immunogenicity analysis and attack protection test verification. This is the first time that VY930 has been used as a protective antigen to explore the potential of this antigen as a vaccine development, and to verify its protective effect from the perspective of cellular immunity. The results show that VY930 subunit vaccine immunized chickens can play a significant protective role against clinical strong strains of MS, can simultaneously induce humoral immunity and cellular immune response, and can effectively reduce clinical incidence. Compared with inactivated vaccines, its clinical protection effect is slightly better, showing good application potential.

[0106] The above description of the embodiments is intended to facilitate understanding and use of the present invention by those skilled in the art. Those skilled in the art will readily be able to make various modifications to these embodiments and apply the general principles described herein to other embodiments without resorting to creative effort. Therefore, the present invention is not limited to the above-described embodiments. Any improvements or modifications made by those skilled in the art based on the principles of the present invention that do not depart from the scope of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A VY930 antigen protein of a Mycoplasma synoviae subunit vaccine, characterized in that: The gene fragment sequence after codon optimization of the antigen protein is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.

3.

2. A Mycoplasma synoviae vaccine, characterized in that: The preparation method for the Mycoplasma synoviae vaccine is as follows: the purified VY930 protein is concentrated using a 3kD pore size ultrafiltration tube, resuspended in PBS buffer, and the protein concentration is determined using a BCA protein concentration assay kit. The protein is diluted with PBS to the desired concentration according to the immunization dose, uniformly mixed with Freund's adjuvant in a 1:1 ratio, and emulsified in an emulsifier for 1 minute to form a uniform emulsion, which is the VY930 protein subunit vaccine. The specific immunization schedule and dosage are as follows: the prepared VY930 subunit vaccine is administered twice via subcutaneous multiple injections in the neck, at 7 and 21 days of age, with immunization doses of 150 μg and 100 μg per animal, respectively. The adjuvant used for the first immunization is Freund's complete adjuvant, and the second immunization is Freund's incomplete adjuvant.

3. A method for evaluating the protective effect of Mycoplasma synoviae vaccine by footpad challenge, characterized in that: The method comprises the following steps: two weeks after vaccination, the chickens are challenged with the footpad of the chickens with a challenge dose of 2×10 5 CCU / mL. One footpad of each chicken was challenged with the virus. An observation period of three weeks followed the challenge. Clinical symptoms in birds during the third week served as the criterion for evaluating the protective efficacy of the Mycoplasma synoviae vaccine strain. This criterion was divided into six grades (0, 1, 2, 3, 4, 5) based on the presence and severity of lesions in birds during the third week after challenge. Grade 0 indicates no disease, grades 1-3 indicate footpad symptoms, and grades 4-5 indicate lesions extending from the footpad to the metatarsus, tarsal joint, and other areas. The scores for each grade were: 0, 0.2, 0.4, 0.6, 0.8, and 1.0, respectively. The specific scoring criteria are as follows: 0: The chicken is visually normal and has no clinical symptoms; 1: 0.2: The foot pad on the infected side is slightly swollen, which needs to be carefully compared with the foot pad on the uninfected side, and no clinical symptoms are produced in other parts of the chicken; 2: 0.4: The foot pad on the infected side is obviously swollen, but it has not spread to the whole foot, and no clinical symptoms are produced in other parts of the chicken; 3: 0.6: The foot pad on the infected side is obviously swollen and spreads to the whole foot , seriously affecting the normal walking of chickens, causing lameness and other phenomena, but it did not spread to the non-infected side, and no lesions were found in the metatarsal bones and tarsal joints; the score of level 4 was 0.8, the foot pad on the infected side was similar to level 3, and some swelling spread to the non-infected foot pad, the metatarsal bones and tarsal joints on the infected side were slightly swollen, and the sick chickens may show symptoms such as air sacitis during autopsy; the score of level 5 was 1.0, the foot pad on the infected or non-infected side was severely swollen, the metatarsal bones and tarsal joints were severely swollen, and the autopsy showed symptoms of air sacitis.

4. A monoclonal antibody specific for VY930 antigen protein, characterized in that The monoclonal antibody is 6F4 monoclonal antibody, the heavy chain variable region sequence of which is shown in SEQ ID NO.14, wherein the sequences of HCDR1-3 are shown in SEQ ID NOs.16-18, respectively; the light chain variable region sequence is shown in SEQ ID NO.15, wherein the sequences of LCDR1-3 are shown in SEQ ID NOs.19-21, respectively.

5. Use of the VY930 antigen protein according to claim 1 and / or the monoclonal antibody according to claim 5 in preparing a kit for detecting Mycoplasma synoviae.

6. A kit, characterized in that It contains the VY930 antigen protein according to claim 1 and / or the monoclonal antibody according to claim 5.

7. Use of the Mycoplasma synoviae vaccine according to claim 2 in the preparation of a medicament for treating or preventing diseases related to Mycoplasma synoviae infection.

8. A drug for treating or preventing diseases related to Mycoplasma gallinarum infection, comprising an effective amount of the Mycoplasma gallinarum vaccine according to claim 2.

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