A mycoplasma synoviae of chicken subunit vaccine, preparation method, detection and application thereof

Abstract

The application provides a Mycoplasma synoviae subunit vaccine, a preparation method, detection and application thereof. The Mycoplasma synoviae VY930 protein expressed based on an Escherichia coli prokaryotic expression system has good immunogenicity, and a subunit vaccine containing the protein is prepared, so as to effectively prevent and control Mycoplasma synoviae infection. It is proved by animal challenge protection test that the subunit vaccine has equivalent or slightly better immunoprotective potential than the inactivated vaccine, and can significantly reduce the clinical symptoms caused by Mycoplasma synoviae. The subunit vaccine has the potential of preventing and controlling Mycoplasma synoviae clinical infection and promoting the purification process of the population. The application also provides a monoclonal antibody for the above protein, which can be used as an index for judging whether immunization is performed or not, and further refines the immune protection evaluation criteria after footpad challenge of Mycoplasma synoviae, so as to provide a detailed measurement standard for subsequent vaccine research and development.

Description

Technical Field

[0001] This invention belongs to the field of protein vaccines, specifically relating to a chicken synoviae subunit vaccine, its preparation method, detection method, and its application. Background Technology

[0002] Mycoplasma synoviae (MS) is a major avian pathogen that causes synocasitis and air sacculitis in poultry, leading to decreased egg production and quality in laying hens, reduced hatchability of hatching eggs, increased culling rates in chicks, and negatively impacting the growth and development of broilers. MS is a persistent presence in chicken flocks and, in practice, is often co-infected with other pathogens such as Newcastle disease virus and Escherichia coli. Its infection is a global epidemic, extremely difficult to eradicate, and causes significant economic losses to the poultry industry. Therefore, a combination of strict biosecurity measures, appropriate antibiotic use, and effective vaccination strategies is crucial for the control of MS and the achievement of population eradication goals.

[0003] MS infection spreads rapidly, has a long carriage time in the body, can be transmitted horizontally and vertically, and easily develops drug resistance, posing challenges to the control and eradication of mycoplasma. Currently, MS control mainly focuses on three aspects: population eradication, drug treatment, and vaccination. Population eradication is costly and impractical. The widespread use of antibiotics has led to rapid development of drug resistance in MS, indicating that drugs are no longer suitable for long-term MS control. Vaccination remains the primary method of infection control worldwide, especially in areas with concentrated poultry farming.

[0004] MS vaccines are mainly classified into three categories: inactivated vaccines, live attenuated vaccines, and genetically engineered subunit vaccines. Currently, only two MS vaccines are registered in my country: the MS-H live attenuated vaccine strain from Australian Bioresources and the YBF-MS1 strain developed by Qingdao Yibang Biotechnology Co., Ltd. The MS-H strain can colonize the upper respiratory tract after inoculation, thus resisting wild-type virus infection and inducing a mucosal immune response. However, this vaccine can only be used in MS-negative flocks; inoculating MS-infected flocks may worsen their clinical symptoms. The difficulty, high cost, and long culture cycle of mycoplasma cultivation limit the large-scale application of live and inactivated vaccines. With the development of bioinformatics technology, 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 some mycoplasmal 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 immunoprotective effect was not verified. MS vaccination may not completely prevent pathogen colonization, but it has been proven to be an important way to control the disease, reduce infection rates, delay the onset of the disease, and mitigate vertical transmission. Existing registered vaccines still have the problem of weak correlation with immunoprotective efficacy. After vaccination with the MS-H attenuated live vaccine strain, it can colonize the upper respiratory tract, thereby rejecting wild-type virus infection and inducing mucosal immune responses. However, this vaccine can only be applied to MS-negative flocks; vaccination of MS-infected flocks may worsen their clinical symptoms. Furthermore, the application of attenuated vaccines can affect serological testing of flocks, easily leading to false positives. Furthermore, the high cost and long cultivation cycle of inactivated and live attenuated vaccines limit their large-scale application. Therefore, developing MS vaccines with good protective efficacy, high safety, simple operation, and suitability for large-scale immunization, and establishing intuitive and convenient methods for detecting immunoprotective effects, is of great significance for the prevention and control of MS in my country. Summary of the Invention

[0005] The purpose of this invention is to provide a detailed guideline for evaluating the efficacy of a chicken synoviae vaccine, a method for preparing a subunit vaccine, and its application for the immunization prevention of chicken synoviae disease.

[0006] The objective of this invention is achieved through the following technical solution:

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

[0008] Specifically, this invention uses pan-genome technology to analyze the core genes of MS, discovering that MS has 102 conserved core genes. Then, combined with reverse vaccinology methods, the core genes of MS are predicted and analyzed to obtain the VY930 protein, which has good conservation, high antigenicity, and good immunogenicity. Furthermore, through genetic codon mutation, dominant codons of *E. coli* are selected, and the gene sequence after codon mutation is shown in SEQ ID NO.2.

[0009] The present invention also provides a chicken synoviae VY930 protein subunit vaccine, which includes a method for preparing VY930 protein and an adjuvant.

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

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

[0012] Specifically, the preparation method is as follows: Purified VY930 protein is concentrated using a 3kD ultrafiltration tube and resuspended in PBS buffer. The protein displaced in the PBS buffer is diluted according to the immunization dose, and then uniformly mixed with Freund's adjuvant at a 1:1 ratio. The mixture is emulsified for 1 minute using an emulsifier to form a homogeneous 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. The monoclonal antibody is a 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 NO.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 NO.19-21, respectively.

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

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

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

[0017] Furthermore, these assessment guidelines apply to infection modes of footpad infection.

[0018] Furthermore, the assessment criteria are divided into six levels (0, 1, 2, 3, 4, 5) based on the presence and severity of the lesions, with scores assigned according to the severity of the lesions. The scores are 0, 0.2, 0.4, 0.6, 0.8, and 1.0, respectively. Level 0 represents no symptoms, levels 1-3 represent the presence of footpad symptoms, and levels 4-5 represent the spread of the lesion from the footpad to the metatarsals, tarsal joints, and other areas. The score comparison for lesion severity is as follows: Figure 5 As shown.

[0019] Beneficial effects

[0020] The prokaryotic expression vector system provided by this invention expresses the VY930 protein. Its antigen acquisition is simple, the process is safe and straightforward, the cost is low, the expression level is high, and the immunization effect is good, showing potential for large-scale production and application. Compared with inactivated vaccines, the subunit vaccine of this invention has slightly better protective efficacy. Compared with traditional attenuated and inactivated vaccines, it can reduce the detection difficulties caused by serological positivity and reduce the cost and time of mycoplasma culture. This invention provides a new approach to MS control, which is beneficial for disease control and population purification, and is a subunit vaccine with great application prospects. The provided monoclonal antibody can effectively recognize the VY930 protein in this invention, providing a basis for judging cluster immunization strategies. This invention provides a detailed evaluation guideline for determining the protective efficacy of chicken synoviposition mycoplasma vaccine strains. This guideline introduces a grading and scoring system, refining the evaluation method of vaccine protective efficacy after footpad challenge, thereby effectively reducing the subjectivity of clinical result interpretation. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The images show the results of overlap PCR of the target gene and PCR identification of the expression vector construction of this invention. In the images, A is the pCold I expression vector used in this 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 results of SDS-PAGE and immunogenicity verification of the protein expressed in this invention are shown below. 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 immune serum and is visualized at a molecular weight of 29.0 kDa, proving that it has good immunogenicity. Figure 3 The assay is for the specific detection of monoclonal antibodies against VY930 protein, where M is the marker, 1 is Mycoplasma gallisepticum (MG), 2 is Mycoplasma avianis (MI), 3 is Haemophilus paragallinarum, and 4 is Mycoplasma synoviae (MS).

[0024] Figure 4 The present invention provides a subunit vaccine immunization schedule and a humoral immunity level measurement, wherein A is the subunit vaccine immunization schedule of the present invention and B is the humoral immunity level measurement result.

[0025] Figure 5 The table and illustrations are for the protection effect scoring method based on foot pad attack established by the present invention. A is a table for the protection effect scoring method based on foot pad attack established by the present invention, and B is an illustration of the protection effect scoring method based on foot pad attack.

[0026] Figure 6 The results of the dynamic evaluation of the protective effect of the subunit vaccine and the inactivated vaccine of this invention are shown in Figure A, which represents the dynamic clinical protective effect after challenge, and Figure B represents the lesion score after challenge. Among them, a represents the mean lesion score within the group one week after challenge, b represents the mean lesion score within the group two weeks after challenge, c represents the mean lesion score within the group three weeks after challenge, and d represents the dynamic trend of the total lesion score within the group three weeks after challenge.

[0027] Figure 7 This is the result of T lymphocyte subset sorting after immunization with the vaccine of this invention, where A represents CD4+ after immunization. + T cell changes, B represents CD8+ after immunization. + T cell changes, C represents CD3+ after immunization. + T cell changes, D represents CD4+ after immunization. + T / CD8 + Changes in T cells. Detailed Implementation

[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 invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, regarding numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in the invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0032] Unless otherwise specified, all chemical reagents, biochemical reagents and materials used in this invention are commercially available.

[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, point mutations were performed on the codons of the VY930 gene, replacing the stop codon TGA with TGG. The optimized sequence is shown in SEQ ID NO.2, and its amino acid sequence is shown in SEQ ID NO.3. The VY930 gene sequence was amplified using MS WVU. 1853Using DNA (GenBank accession number CP011096) as a template, the primer sequences shown in Table 1 were used to amplify and recover each of the four primer pairs. The amplification system consisted of 2 μL each of forward and reverse primers, 25 μL of TaKaRa (PrimeSTAR@Max DNA Polymerase), and MS WVU. 1853 One μL of whole-genome DNA was collected and brought to a final volume of 50 μL with ddH2O. Amplification conditions were: denaturation at 98°C for 10 s, annealing at 55°C for 15 s, and extension at 72°C for 5 s; 35 cycles. Subsequently, using the recovered DNA as a template, full-length amplification was performed using SEQ ID NO.4 and SEQ ID NO.11 to obtain the full-length VY930 gene.

[0035] The VY930 target gene was ligated into the expression vector pColdⅠ to form the recombinant expression plasmid pColdⅠ-VY930. Subsequently, the recombinant plasmid was transformed into *E. coli* DH5α, and single clones were picked. Colony PCR and sequencing were performed using the universal primers for pColdⅠ (sequences shown in SEQ ID NO. 12 and SEQ ID NO. 13, respectively). The correctly identified positive clones were named *E. coli* DH5α (pColdⅠ-VY930). PCR results are shown below. Figure 1 As shown.

[0036] Table 1 Primer sequences for gene overlap PCR amplification

[0037]

[0038] The universal primer sequence for pColdⅠ 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: Induction and purification of Mycoplasma synoviae protein VY930

[0042] Freshly cultured E. coli BL21 (pColdI-VY930) bacterial suspension was inoculated into LB medium containing 100 μg / mL ampicillin at a 1:100 transfer ratio and cultured at 37°C with shaking at 180 rpm until the bacterial suspension reached OD. 600nm The concentration was 0.6, and IPTG was added to a final concentration of 0.05 μg / μL. Expression was induced at 16℃ and 110 rpm for 24 h.

[0043] After induction, the bacterial cells were collected by centrifugation at 8000 rpm for 10 min, resuspended in binding buffer, and then lysed using a high-pressure homogenizer. The whole cells before induction, the whole cells after induction, the supernatant after induction, and the lysed precipitate after induction were added to an appropriate amount of protein loading buffer, boiled for 15 min, and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results are as follows: Figure 2 As shown in Figure A, the VY930 protein was expressed in the supernatant with a molecular weight of 29.0 kDa, consistent with the expected size.

[0044] Use the supernatant after crushing Ni-NTAResin is used to purify VY930 protein. The process includes: column packing, equilibration, sample loading, washing, storage, and validation.

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

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

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

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

[0049] (5) Preservation: After washing the column bed with a large amount of Elution Buffer containing 500mM imidazole, wash it a second time with deionized water. Finally, add 20mL of 20% ethanol, and after 10mL has eluted, store at 4℃.

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

[0051] Preparation of the main buffer for protein purification:

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

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

[0054] (3) Elution Buffer: 50mM NaH2PO4, 300mM NaCl, 30 / 50 / 100 / 200 / 300 / 500mM imidazole, add 800mL deionized water, dissolve thoroughly on a magnetic stirrer, and adjust the pH to 8.0. Finally, bring the volume to 1L and store at room temperature for later use. The VY930 protein purification results are as follows... Figure 2 As shown in Figure 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 purification was successful.

[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 3kD ultrafiltration tube and resuspended in PBS buffer. The protein displaced in the PBS buffer was diluted according to the immunization dose and mixed uniformly with Freund's adjuvant at a 1:1 ratio. The mixture was emulsified for 1 minute using an emulsifier to form a homogeneous 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] The MS subunit vaccine prepared in Example 3 was used to immunize two-month-old New Zealand white rabbits via subcutaneous multi-site injection. For the first immunization, the purified protein was emulsified with an equal volume of Freund's complete adjuvant to prepare the subunit vaccine, with an immunization dose of 500 μg / rabbit. A second immunization was performed two weeks later, followed by a third immunization two weeks after the second immunization, with an immunization dose of 300 μg / rabbit for each immunization, using Freund's incomplete adjuvant for all immunizations.

[0060] 2. Antibody collection

[0061] One week after the second immunization, blood samples were collected via the marginal ear vein of rabbits. One week after the third immunization, blood samples were collected via cardiac sampling. The blood samples were stored overnight at 4°C. 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 testing

[0063] Rabbit serum was analyzed by ELISA. VY930 protein was diluted to 5 μg / mL using coating buffer, and 100 μL was added to each well. Coating was performed overnight at 4°C. After washing with PBST, 200 μL of 5% skim milk blocking buffer was added to each well, and the plates were incubated at 37°C for 2 hours. After washing with PBST, the rabbit polyclonal antibody serum was serially diluted 1:100, and 100 μL of the diluted serum was added to each well. The plates were incubated at 37°C for 2 hours. After washing three times with PBST, goat anti-rabbit enzyme-labeled secondary antibody IgG was diluted 1:10000, and 100 μL was added to each well. The plates were incubated at 37°C for 2 hours. After washing with PBST, 100 μL of TMB substrate chromogenic solution was added to each well, and the plates were incubated at 37°C 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 multi-mode microplate reader. 450nm A positive result was defined as a ratio of ≥2.1 between the antibody titer of the test well and the negative control well. The highest dilution that could be considered positive was used as the antibody titer of the serum being tested. 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. The SDS-PAGE products were transferred onto an NC (nitrocellulose) membrane, blocked at room temperature for 2 h, washed three times with TBST, incubated overnight at 4°C with rabbit polyclonal antibody serum, washed three times with TBST, and then incubated with HRP (horseradish peroxidase)-labeled goat anti-rabbit IgG as a secondary antibody for 2 h at room temperature, followed by three washes with TBST. After ECL staining, the results were photographed and analyzed to assess the immunogenicity of the protein. Results are as follows: Figure 2 As shown in Figure C, the 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 and evaluation of immunogenicity of VY930 protein monoclonal antibodies

[0067] Monoclonal antibodies for screening VY930 protein were prepared according to standard procedures in the art, briefly described as follows: Four 40-day-old female BALB / c mice were selected, three of which were routinely immunized with the purified VY930 protein obtained in Example 2, and the remaining mouse served as a negative control and was not immunized. An indirect ELISA method for screening hybridoma cells was established using the serum of the mice after the fourth immunization.

[0068] (1) Dilute VY930 protein to 0.5 μg / mL with antigen coating buffer (0.05 mol / L, pH 9.6 ± 0.2 carbonate buffer), add 100 μL to each well, coat overnight at 4°C (about 8 h), and wash 3 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 3 times with PBST.

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

[0071] (4) Dilute the HRP-labeled goat anti-mouse IgG (H+L) antibody with 2.5% skim milk at a ratio of 1:5000, add 100 μL to each well, incubate at 37°C for 1 h, and wash 3 times with PBST.

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

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

[0074] (7) Calculate the P / N value (the ratio of serum absorbance of the immunized group mice to that of the negative control mice). A P / N value greater than 2 is considered positive (meaning that the immunized group mice have successfully produced antibodies against the VY930 protein), otherwise it is considered negative. Select mice with the highest antibody titer for cell fusion experiments to prepare monoclonal antibodies.

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

[0076] Using the aforementioned indirect ELISA method, hybridoma cell culture supernatant was detected with VY930 protein as the coating antigen. Coating conditions were optimized using a checkerboard titration method, aiming for the highest positive / negative absorbance ratio (P / N value) and the highest OD value in positive serum. 450A P / N ratio close to 1.0 is considered optimal. A P / N ratio > 2 is considered positive. Indirect ELISA results showed that the P / N ratio was highest when coated with 1 μg / mL VY930 protein. Based on this, an indirect ELISA method was established to detect the monoclonal antibody supernatant secreted by hybridoma cells and screen for positive monoclonal cells. Hybridoma cells that were positive in both tests were subjected to limiting dilution subcloning. After 2-4 cloning processes, cell lines stably secreting monoclonal antibodies were obtained. The antibodies were purified using the caprylic acid-ammonium sulfate method and identified using the above ELISA method. The purified antibody titer reached 1:102400.

[0077] After the 6F4 monoclonal antibody was expanded and cultured, it was sent to a gene company for sequencing. The results showed that:

[0078] The heavy chain variable region sequence is as follows:

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

[0080] 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 as follows:

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

[0085] 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 specific assays, including Mycoplasma gallisepticum (MG), Mycoplasma avianis (MI), Mycoplasma synoviae (MS), and Haemophilus paragallinarum. Other pathogen strains were inoculated at a ratio of 1% (v / v) into 100 mL of liquid culture medium and incubated at 37°C. After the medium turned yellow, the bacterial pellet was collected at 10,000 rpm for 30 min, resuspended and washed three times with PBS, and then resuspended in 1 / 10 volume of PBS. The pellet was then sonicated on ice. The supernatant was collected at 10,000 rpm for 20 min, yielding the complete bacterial protein of the pathogen. Protein concentration was measured using a BCA protein quantification kit. The quantified protein was aliquoted and stored at -70°C. Western blotting was used to detect the specificity of the prepared monoclonal antibodies against different mycoplasma strains.

[0089] The results showed that, Figure 3 As shown, the 6F4 monoclonal antibody did not show any reaction bands with Mycoplasma gallisepticum (MG), Mycoplasma avianis (MI), or Haemophilus paragallinarum, but it showed a specific reaction band with the whole bacterial protein of Mycoplasma synoviae (MS). This indicates that the obtained monoclonal antibody can specifically bind to Mycoplasma synoviae (MS) and can effectively distinguish Mycoplasma synoviae (MS) from other common pathogens. It can be used to detect Mycoplasma synoviae (MS) and has high specificity.

[0090] Example 6: Evaluation of the immunoprotective effect of VY930 antigen protein

[0091] 1. Animal immunization

[0092] Newly hatched SPF chickens were randomly divided into four groups (15 chickens per group): an MS subunit vaccine immunization group, an inactivated vaccine immunization group, an adjuvant immunization control group, and a PBS control group. The MS subunit vaccine prepared in Example 3 was administered to the SPF chickens via subcutaneous injection at multiple sites in the neck. The immunization schedule is as follows: Figure 4 As shown in Figure A, the prepared subunit vaccine was administered twice via subcutaneous injection at multiple sites in the neck, at ages 7 and 21, with doses of 150 μg / animal and 100 μg / animal, respectively. The first immunization used Freund's complete adjuvant, and the second immunization used Freund's incomplete adjuvant. The inactivated vaccine immunization group was administered according to the instructions.

[0093] 2. Collection of polyclonal antibodies

[0094] Following the first immunization, blood was collected weekly via the subwing vein of chickens, and serum was separated. Blood samples were stored overnight at 4°C. The following 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 testing

[0096] The collected chicken serum was analyzed by ELISA. The detection method was the same as shown in Example 4. The enzyme-labeled secondary antibody used was HRP-labeled goat anti-chicken IgY. The results showed that the antibody titer gradually increased from 7 to 28 days after immunization, reaching 1:102400 two weeks after the second immunization. The detection results are as follows. Figure 4 As shown in B.

[0097] 4. MS virus 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 MS YC1, a clinical isolate preserved in our laboratory (Zhao Luru, Qi Jingjing, Zhang Kaiqin, et al. Isolation, identification, pathogenicity and drug resistance evaluation of Mycoplasma synoviae in chickens from some areas of Jiangsu Province [J]. Chinese Journal of Veterinary Science, 2024, 54(12):1617-1626.). The footpad challenge dose was 2×10⁻⁶. 5 CCU / mL. Clinical symptoms were recorded weekly after challenge, and pathological changes were observed by autopsy after three weeks, with clinical symptoms scored and assessed.

[0099] 5. Clinical criteria for evaluating MS foot pad infection

[0100] Two weeks after vaccination, the flock was challenged with the virus through the footpads at a dose of 2×10⁻⁶. 5 CCU / mL, one footpad of each chicken was challenged with the virus; this invention uses the left footpad as an example. The observation period after challenge was three weeks, with the clinical symptoms observed in the chickens at the third week as the criterion for judgment. Detailed rules for evaluating the protective efficacy of the Mycoplasma synoviae vaccine strain were established, such as... Figure 5As shown. This detailed rule classifies chickens into six levels (0, 1, 2, 3, 4, 5) based on the presence and severity of lesions in the chickens three weeks after infection. Level 0 indicates no disease; levels 1-3 indicate footpad symptoms; and levels 4-5 indicate lesions spreading from the footpad to the metatarsals, tarsal joints, and other areas. The scores for each level are: 0, 0.2, 0.4, 0.6, 0.8, and 1.0, respectively. The specific scoring criteria are as follows: Level 0: 0 points. The chicken appears completely normal, with no clinical symptoms. Level 1: 0.2 points. The footpad on the infected side is slightly swollen, requiring careful comparison with the footpad on the uninfected side; no clinical symptoms appear in other parts of the chicken. Level 2: 0.4 points. The footpad on the infected side shows significant swelling, but it has not spread to severe swelling of the entire foot; no clinical symptoms appear in other parts of the chicken. Level 3: 0.6 points. The footpad on the infected side is significantly swollen and has spread to the entire foot. The symptoms of 4-level disease are: 1. Severely affecting the normal walking of chickens, causing lameness, but not spreading to the non-infected side, and no lesions appear on the metatarsals and tarsal joints; 2. Level 4 has a score of 0.8, the footpad on the infected side is similar to that of level 3, and some swelling spreads to the footpad on the non-infected side, and the metatarsals and tarsal joints on the infected side show slight swelling, and necropsy may show symptoms such as air sacculitis; 3. Level 5 has a score of 1.0, the footpad on the infected or non-infected side is severely swollen, the metatarsals and tarsal joints are severely swollen, and necropsy shows symptoms of air sacculitis.

[0101] This invention establishes a scoring and evaluation method for the immunoprotective effect of MS vaccine based on the footpad challenge route. Compared with existing air sac evaluation and pathological section evaluation methods, the experimenter can conduct the evaluation by intuitively interpreting the clinical symptoms of the flock after challenge, making the scoring method more intuitive and convenient.

[0102] 6. Evaluation of the protective effect of VY930 subunit vaccine against viral challenge

[0103] Fourteen days after the second immunization, the experimental group was inoculated with approximately 2 × 10⁻⁶ live bacteria in the footpads. 5 MS YC1 culture at CCU / mL was observed for 21 days, and clinical manifestations were recorded. The dynamic clinical protective effect and lesion score after challenge were as follows: Figure 6 As shown in the results, the morbidity rate in the adjuvant-immunized control group reached 100% from the first week. During the observation period, severe footpad swelling was observed, and some chickens showed severe swelling and deformation of the metatarsals and tarsal joints. Necropsy revealed caseous material adhering to the surface of the air sacs. No clinical symptoms were observed in the PBS control group. Compared with the challenge group, the VY930 subunit vaccine immunization group and the inactivated vaccine immunization group showed significantly reduced symptoms. Univariate ANOVA analysis using SPSS software showed that, compared with the adjuvant-immunized control group, the vaccine immunization group showed significantly reduced clinical symptoms. The protective effect of VY930 in the second week was superior to that of the inactivated vaccine, and overall, the lesion score of the lesion subunit vaccine immunization group was lower, confirming that the VY930 subunit vaccine has a significant protective effect against clinical MS infection.

[0104] 7. T lymphocyte subset sorting

[0105] To assess the cellular immunity levels induced in chickens by the VY930 subunit vaccine and the inactivated vaccine, chicken spleens were aseptically removed before challenge, and spleen lymphocytes were isolated using a commercially available chicken spleen lymphocyte isolation kit. The isolated lymphocytes were diluted to 1×10⁻⁶. 6 Cells were incubated at 25°C for 30 minutes with fluorescently conjugated antibodies PE-CD3 (0.3 μg), FITC-CD4 (1 μg), and APC-CD8 (0.1 μg) at a concentration of 1 / mL. After incubation with the fluorescent antibodies, the cells were washed twice with PBS by centrifugation and resuspended in 300 μL PBS for flow cytometry analysis. CD3+ levels in spleen lymphocytes from chickens immunized with VY930 subunit vaccine, inactivated vaccine, and adjuvant-immunized control groups 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) levels. Results as follows Figure 7 As shown, compared with the adjuvant-immunized control group, the proportion of CD3+ total T lymphocytes in chicken flocks increased after immunization with VY930 and inactivated vaccines; among them, the CD3+ total T lymphocytes in the VY930 immunized group increased. + CD4 + T cells (p<0.01) and CD3 + CD8 + T cells (p<0.001) were significantly increased in all groups; CD3 count in the inactivated vaccine immunization group was significantly increased. + CD4 + T cells (p<0.01) were significantly increased, while CD3 cells were significantly increased. + CD8 + The proportion of T cells did not show a statistically significant change (p = ns). This result suggests that the two types of vaccines induce different immune response patterns. Inactivated vaccines primarily activate humoral immune responses dominated by B cell differentiation and antibody production, while CD8+ responses are less affected. + T cell-mediated cellular immune responses have a relatively weak activation effect. However, the VY930 subunit vaccine constructed in this study exhibits unique immune activation properties, effectively inducing T cell-mediated cellular immune responses. + T-cell-assisted humoral immune responses, and CD8-mediated humoral immune responses. +T-cell-mediated cellular immune responses and these two immune response pathways may have a synergistic effect, providing an advantage in preventing MS infection. Results showed that this invention utilized pan-genomic technology to analyze the entire MS genome, screened core genes, and then combined this with reverse vaccinology methods to ultimately screen for the MS conserved protein VY930. A prokaryotic expression vector was constructed, the protein was purified, immunogenicity was analyzed, and challenge protection experiments were conducted to verify its effectiveness. This invention is the first to use VY930 as a protective antigen, exploring its potential as a vaccine and verifying its protective effect from the perspective of cellular immunity. Results showed that VY930 subunit vaccine immunization of chicken flocks significantly protected against clinically virulent MS strains, simultaneously inducing humoral and cellular immune responses and effectively reducing clinical morbidity. Compared with inactivated vaccines, its clinical protective effect was slightly better, showing good application potential.

[0106] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A monoclonal antibody specifically targeting the VY930 antigen protein, characterized in that... The monoclonal antibody is a 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 NO.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 NO.19-21 respectively; and the amino acid sequence of the VY930 antigen protein is shown in SEQ ID NO.

3.

2. The application of the monoclonal antibody as described in claim 1 in the preparation of a chicken synoviocyte mycoplasma detection kit.

3. A reagent kit, characterized in that... It contains the monoclonal antibody as described in claim 1.

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