Mycoplasma bovis lp586 protein, polyclonal antibody thereof and application

Abstract

The application discloses a Mycoplasma bovum LP586 protein, a polyclonal antibody thereof and application. The amino acid sequence of the LP586 protein is shown as SEQ ID NO. 1, the LP586 protein has high conservation, is positioned on the cell membrane surface of the Mycoplasma bovum, and has multiple biological functions such as specific adhesion to EBL cells, combination with extracellular matrix molecules and activation of plasminogen in the presence of tPA. The application further provides a high-titer specific polyclonal antibody prepared by immunization. The LP586 protein can be used as a key antigen for preparing a Mycoplasma bovum subunit vaccine, a diagnostic reagent or a therapeutic drug; and the polyclonal antibody thereof can be used for preparing a diagnostic reagent or a passive immunotherapy preparation. The application provides a brand-new high-efficiency target and product development basis for prevention and control of the Mycoplasma bovum disease.

Description

Technical Field

[0001] This invention belongs to the field of prevention and control technology of bacterial infectious diseases in herbivores, specifically involving a bovine mycoplasma LP586 protein, a polyclonal antibody that specifically binds to the protein, and its application in the prevention, control and detection of bovine mycoplasma disease. Background Technology

[0002] Mycoplasma bovis is a significant pathogen that seriously threatens the health of cattle. It primarily attacks the respiratory tract, leading to multi-systemic diseases such as pneumonia, arthritis, and otitis media. Infection often synergizes with viruses or other bacteria, exacerbating the condition. This not only causes decreased productivity and increased calf mortality in affected cattle but also easily leads to persistent infections, resulting in substantial economic losses for the cattle industry. Because Mycoplasma bovis lacks a cell wall structure, it is naturally resistant to antibiotics that act on the cell wall, such as β-lactams, and the emergence of drug-resistant strains further increases the difficulty of prevention and treatment, making it a key and challenging area in the field of bovine infectious disease control.

[0003] Vaccination is the core means of preventing bovine mycoplasma disease, but currently there is a shortage of commercially available, highly effective vaccines. Bovine mycoplasma has numerous genotypes, and existing inactivated vaccines suffer from drawbacks such as short duration of immune protection, limited cross-protection against different genotypes, complex antigenic composition, and significant side effects. Subunit vaccines use conserved functional antigens identified through screening as immunogens, avoiding the risks of antigen damage, residual proteins, and pathological exacerbation associated with inactivated vaccines. They offer higher safety and no risk of virulence reversion, and can precisely stimulate humoral and cellular immune responses. Furthermore, they are easier to optimize through genetic engineering for expression and adjuvant formulation, making them an ideal technological direction for the precise control of bovine mycoplasma.

[0004] Bacterial adhesion is a crucial initial step in pathogen colonization and infection. As a pathogenic mycoplasma lacking a cell wall and possessing a simple genome, the adhesion process of *Mycoplasma bovis* is an important prerequisite for pathogenicity and a potential target for vaccine design. Functional surface membrane proteins and lipoproteins are key mediators of *Mycoplasma bovis* adhesion. Among them, the variable surface protein Vsp can evade host immune system recognition by altering antigenic structure, leading to persistent *Mycoplasma bovis* infection. Therefore, screening for highly conserved antigens with good immunogenicity is an important strategy for the development of subunit vaccines against *Mycoplasma bovis*. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a highly conserved bovine mycoplasma LP586 protein with adhesion function and excellent immunogenicity, as well as its specific polyclonal antibody, and to provide applications of both in the prevention and detection of bovine mycoplasma disease, thus providing a new target for the efficient prevention and control of bovine mycoplasma disease.

[0006] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: The present invention provides a bovine mycoplasma LP586 protein, the amino acid sequence of which is shown in SEQ ID NO.1; the sequence encoding the LP586 protein is shown in SEQ ID NO.2.

[0007] The bovine mycoplasma LP586 protein has the following biological characteristics: (1) it is highly conserved among different bovine mycoplasma strains; (2) it is located on the surface of bovine mycoplasma cell membranes; (3) it can specifically adhere to fetal bovine lung epithelial cells (EBL cells), and this adhesion can be specifically inhibited by its antiserum; (4) it can bind to extracellular matrix (ECM) proteins (such as fibronectin, laminin, hydrin, and type IV collagen) in a dose-dependent manner; and (5) it can promote the conversion of plasminogen (Plg) into plasmin under the action of tissue plasminogen activator (tPA).

[0008] This protein can be obtained through recombinant expression in a prokaryotic expression system and purification by nickel column affinity chromatography. The specific preparation method is as follows: the optimized bovine mycoplasma LP586 protein-encoding gene is inserted into the pET-30a vector to construct pET-30a- lp586 The recombinant plasmid was transformed into Escherichia coli BL21(DE3) to obtain the recombinant expression strain. After expression was induced by IPTG, the strain was purified by nickel column affinity chromatography.

[0009] This invention provides a polyclonal antibody that specifically binds to the LP586 protein. This polyclonal antibody is obtained by immunizing mice with the LP586 protein as an immunogen, mixing it with an adjuvant, collecting serum, and purifying the antibody. The ELISA titer of this polyclonal antibody is not less than 1:819200, and it can specifically bind to the LP586 protein, as well as specifically inhibit the adhesion of LP586 protein to EBL cells and the adhesion of Mycoplasma bovis to EBL cells.

[0010] This invention provides the application of the above-mentioned LP586 protein in the preparation of bovine mycoplasma subunit vaccines, bovine mycoplasma infection detection reagents / kits, and drugs for the prevention and treatment of bovine mycoplasma infection; it also provides the application of the above-mentioned polyclonal antibody in the preparation of bovine mycoplasma infection detection reagents / kits and drugs for the prevention and treatment of bovine mycoplasma infection.

[0011] The beneficial effects of this invention are: 1. The bovine mycoplasma LP586 protein of the present invention is located on the membrane surface and is highly conserved in different bovine mycoplasma strains, avoiding the problem of immune escape caused by antigen variation, and providing a basis for broad-spectrum prevention and control of bovine mycoplasma infection; 2. The LP586 protein has good immunogenicity and reactivity, which can accurately stimulate the body's immune response. It can also be efficiently expressed and purified through genetic engineering technology, making it suitable as a candidate target for bovine mycoplasma subunit vaccines, thus making up for the shortcomings of existing vaccines, such as poor immune protection and large side effects. 3. The LP586 protein can specifically adhere to host cells, bind to ECM proteins, and promote plasminogen activation, revealing a new pathogenic mechanism of bovine mycoplasma and providing a novel target for drug development for bovine mycoplasma infection. 4. The polyclonal antibody prepared in this invention can specifically bind to the LP586 protein and effectively inhibit the adhesion of bovine mycoplasma to host cells. It can not only be used for the diagnosis of bovine mycoplasma infection, but also directly reduce the adhesion of pathogens to host cells and reduce cell damage, providing a new technical means for the treatment of bovine mycoplasma infection. 5. The technical solution of this invention is simple to operate and cost-controllable. It can produce LP586 protein and polyclonal antibodies on a large scale through genetic engineering, which is easy to apply in industrial applications and is of great significance to promoting the healthy development of the cattle industry and reducing economic losses. Attached Figure Description

[0012] Figure 1 This is an SDS-PAGE electrophoresis image of purified bovine mycoplasma LP586 protein.

[0013] Figure 2 This is a Western blot image showing the expression of the LP586 protein in different bovine mycoplasma strains.

[0014] Figure 3 This is a Western blot image showing the localization of the LP586 protein on the membrane surface of bovine mycoplasma.

[0015] Figure 4 This is an ELISA titer graph of the LP586 protein polyclonal antibody.

[0016] Figure 5 This is a Western blot image showing the reaction of LP586 protein with its antiserum.

[0017] Figure 6 This is a Western blot image showing the reactivity of the LP586 protein.

[0018] Figure 7 This is an immunofluorescence image showing the adhesion and inhibition of LP586 protein to EBL cells. The cell membrane was stained with a DiI staining kit, and the cell nucleus was stained with DAPI.

[0019] Figure 8(A)-(D): Dot blot analysis of LP586 protein interacting with fibronectin, laminin, hyoidin, and type IV collagen. (E)-(H): ELISA analysis of LP586 protein interacting with fibronectin, laminin, hyoidin, and type IV collagen.

[0020] Figure 9 (A)-(B): Dot blot detection of LP586 protein binding to plasminogen and activation. (C): ELISA detection of LP586 protein binding to Plg and tPA.

[0021] Figure 10 This is a diagram showing the effect of LP586 protein antiserum on the specific inhibition of bovine mycoplasma adhesion to EBL cells. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Unless otherwise specified, the test materials used in the following embodiments are commercially available.

[0023] Example 1: Expression and purification of bovine mycoplasma LP586 protein Sequence alignment was performed on prevalent Mycoplasma bovis strains and reference strains. Based on bioinformatics analysis results such as sequence protection and secondary structure, and according to the codon preference of Escherichia coli, the protein encoding gene of Mycoplasma bovis LP586 was optimized and synthesized (as shown in SEQ ID NO.2).

[0024] The nucleotide sequence encoding the LP586 protein (SEQ ID NO.2):

[0025] The optimized synthesized gene was cloned into the multiple cloning site of the pET-30a(+) expression vector. BamH I and Xho I. Constructing the recombinant plasmid pET-30a- lp586 Bovine mycoplasma pET-30a- lp586 Recombinant plasmids were transformed into *E. coli* BL21(DE3) to obtain recombinant bacteria. The recombinant bacteria were inoculated into 5 mL LB medium containing kanamycin (100 μg / mL) and incubated at 37°C with shaking for 5–8 h. Subsequently, the culture was scaled up to 500 mL at a 1:100 ratio and cultured at 37°C with shaking for 2–3 h until the logarithmic growth phase (OD600 nm = 0.6). IPTG was then added to a final concentration of 0.05 mM and induced at 16°C for 16–24 h. The induced bacterial culture was centrifuged at 9000 r / min at 4°C for 10 min to collect the cells. After washing with PBS, the cells were centrifuged again to collect the cells. The cells were resuspended in 10 mM imidazole and disrupted using a pressure cell disruptor until the culture was clear. After disruption, the culture was centrifuged at 4°C and 11000 r / min for 30 min, and the supernatant was collected. After filtration through a 0.45 μm filter, protein expression was analyzed by SDS-PAGE. Add 10 mL of HighAffinity Ni-charged Resin FF to an empty affinity chromatography column. Wash the nickel column three times with ultrapure water, then add 10 mM imidazole to equilibrate the nickel column. Add the collected supernatant protein to the chromatography column and incubate at 4°C with shaking for 1-2 h. Unscrew the stopper to allow the flow-through to elute, and repeat the eluent three times. Collect the flow-through for SDS-PAGE analysis. Wash away any remaining protein with 10 column volumes of washing buffer. Elute with different concentrations of imidazole (100 mM, 200 mM, 300 mM), eluting three times for each concentration. Collect the eluent from each eluent for subsequent SDS-PAGE analysis. The results showed that high-purity LP586 recombinant protein was obtained. Figure 1 ).

[0026] Example 2: Conservation Analysis of LP586 Protein Whole-cell proteins were extracted from nine strains of Mycoplasma bovis and subjected to SDS-PAGE electrophoresis. The proteins were then transferred to a PVDF membrane, blocked with 5% BSA at room temperature for 2 h, and incubated with 1:10000 diluted anti-LP586 mouse serum or pre-immunized mouse serum at room temperature for 2 h. After incubation, the membrane was incubated with 1:5000 diluted HRP-labeled goat anti-mouse IgG at room temperature for 1 h, followed by ECL color development and exposure.

[0027] The results showed that LP586 protein expression was found in both the bovine mycoplasma reference strain PG45 and clinical isolates (derived from cattle or yaks), indicating that LP586 protein is highly conserved among different bovine mycoplasma strains. Figure 2 ).

[0028] The amino acid sequence of the LP586 protein (SEQ ID NO.1): CQKNDKKVVFQFAQDNYWPLPKMLVPLVKYYNTTFSKDKDFLPIELQFSDKTKTTSE FELIKKVKNDIETNNEAALPNLILGAQSGAYVLNQHKRLMDLSDHGITKSIFNKNIADLHSKLPGQKGNDKIYSIPFDNLDVDAVVYNLEVLDYMFKMIKANGGKVDENAEIVKKATEAGKEGVGSK LPENTIWKALEANSNKAFEGLTVNDETFKSVESIKEFAKKFHEGTKLNESKVNDDTLTGEVISVDYANDLLFKELHSQIDKDKLVYDLEETGNPEKPVTVKYNIVQDQDIKSKFKLLLSSYKEAIKRH AHKVGANNKVFQTINFGNKGENSSINIARFKSAIGFAASVGVNSTMYSSRLKESQGKNDPDFAKKVASYEDVYMDPQLTTIKKGSQKIFTEEGGSSLLVLKSKDSSMNKAVVKFVKWLFEGTNNAYNQ GVEENWKTFAKYSGYIMPLASVVNDKSLNWFETEAEKLKQKRANNSISDEEFRILNFLRSAYVSIKSIRDFEKDSSIIAKNNVQDDKTGQIYNSIKSAAVKWTDHNSPSEIKEDDLIKSIENEVNHK.

[0029] Example 3: Localization of LP586 protein in bovine mycoplasma Using Mem-PER TM The Plus Membrane Protein Extraction Kit was used to extract membrane and cytoplasmic proteins from bovine mycoplasma PG45. The membranes were then incubated with LP586 mouse antiserum diluted 1:20000 at room temperature for 2 h. After washing with TBST, the membranes were incubated with HRP-labeled goat anti-mouse IgG diluted 1:5000 at room temperature for 1 h, followed by ECL color development and exposure.

[0030] The results showed that LP586 protein expression could be detected in whole-cell proteins and membrane surface proteins of Mycoplasma bovis, but almost none in the cytoplasm, indicating that LP586 is mainly present in the cell membrane of Mycoplasma bovis. Figure 3 ).

[0031] Example 4: Preparation and titer determination of LP586 polyclonal antibody Polyclonal antibody preparation: Purified bovine mycoplasma protein LP586 was mixed with QuickAntibody-Mouse 5w adjuvant at a 1:1 volume ratio, and each mouse was immunized with an immunization dose of 20 μg / 50 μL. A second immunization was performed on day 21, and mouse serum was collected on day 35. The titer was determined by ELISA.

[0032] Indirect ELISA determination of mouse polyclonal antibody titer: (1) The purified protein was diluted with CB coating buffer at a rate of 100 ng / well and then added to a 96-well plate at a rate of 100 μL per well.

[0033] (2) After adding the antigen, place it at 4°C overnight to coat the ELISA plate; after coating, a solid antigen is formed, and the liquid is discarded.

[0034] (3) Use 5% skim milk powder to block in a constant temperature incubator at 37℃ for 2 h, 100 μL per well, pour out the liquid, and wash the plate with PBST.

[0035] (4) Add 2-fold serially diluted mouse polyclonal antibody to the well for detection, and use pre-immunized mouse serum as a negative control at the same dilution.

[0036] (5) After incubating at 37°C for 1 h, wash the plate 3 times with PBST. Add goat anti-mouse secondary antibody diluted 1:1000 with 5% skim milk powder, incubate at 37°C for 1 h, and then wash the plate with PBST.

[0037] (6) Add 100 μL of TMB colorimetric solution to each well and incubate at 37°C in the dark for 10 min. Then add 100 μL of 2M H2SO4 to each well to stop the colorimetric process.

[0038] (7) Use a microplate reader (Bio-Rad) to read the OD value at a wavelength of 450 nm.

[0039] (8) Use P / N>2.1 as the critical value to judge the valence result.

[0040] The results show that, as indicated by the ELISA results ( Figure 4 The LP586 titer is as high as 1:819200, indicating that it has good immunogenicity.

[0041] Example 5: Immunogenicity detection of LP586 protein The purified protein was subjected to Western blot analysis with its prepared mouse antiserum, following the procedures described above. The primary antibody incubation conditions were: mouse antiserum diluted 1:10000, incubated at room temperature for 2 h. The secondary antibody incubation conditions were: HRP-labeled goat anti-mouse IgG diluted 1:5000, incubated at room temperature for 1 h. Subsequently, the sample was exposed to light.

[0042] The results showed that a specific band appeared, indicating that the serum of immunized mice could react with LP586 protein. Figure 5 ).

[0043] Example 6: LP586 protein reactivity detection Using the same Western blot method as described above, the LP586 recombinant protein was incubated for 2 h at room temperature with 1:50 diluted serum from bovine mycoplasma-infected bovine serum, bovine mycoplasma-immunized bovine serum, and healthy bovine serum. After washing the membrane with TBST, it was incubated for 1 h at room temperature with 1:5000 diluted HRP-labeled goat anti-bovine IgG secondary antibody. Subsequently, ECL was used for color development and exposure.

[0044] The results showed that LP586 protein specifically reacted with bovine serum immunized with Mycoplasma bovis and bovine serum experimentally infected with Mycoplasma bovis, but did not react with healthy bovine serum. Figure 6 This demonstrates that the LP586 protein exhibits good reactivity.

[0045] Example 7: Assay of the adhesion activity of LP586 protein EBL cells were cultured in DMEM medium containing 10% fetal bovine serum and incubated at 37°C in a 5% CO2 incubator. When the cells reached approximately 80% confluence, they were digested with trypsin for 6-8 min, centrifuged at 1000 rpm for 5 min, and then counted. 1 × 10⁶ cells were seeded per well in a 24-well plate. 5500 μL of EBL cells per well were cultured overnight. LP586 was pre-incubated with its antiserum or negative mouse serum at a 1:10 ratio at 37°C for 1 h. After incubation, 100 μg of LP586 protein diluted in Opti-MEM medium was added to each well of EBL cells, and incubated at 37°C for 1 h. Immobilization was then performed with 4% paraformaldehyde at room temperature for 10 min. Penetration was performed using 0.1% Triton X-100 at room temperature for 10 min, followed by washing with PBS. After blocking with 5% BSA, cells were incubated with anti-LP586 mouse serum diluted 1:100 with antibody dilution at 37°C for 3 h. After washing three times with PBS, cells were incubated with goat anti-mouse IgG (H+L) diluted 1:1000 with 0.2% BSA in Alexa Fluor™ 488 at 37°C for 45 min. After washing three times with PBS, the cell membranes were stained with red fluorescent staining kit (DiI) for 10 min. After washing three times with PBS, the cell nuclei were stained with 1:500 diluted DAPI for 10 min, followed by washing with PBS. A coverslip was then placed upside down on a slide and fixed with nail polish. The slide was then stored in the dark at 4°C. Observation was performed using a Zeiss LSM 980 confocal microscope.

[0046] Results showed that, as observed by laser confocal microscopy, the recombinant LP586 protein specifically adhered to the surface of EBL cell membranes, and its antiserum specifically inhibited this adhesion. No significant fluorescence changes were observed after incubation with pre-immune serum. Figure 7 This demonstrates that the LP586 protein can adhere to EBL cells, and that this adhesion can be specifically inhibited by its antiserum.

[0047] Example 8: LP586 protein binding ECM activity Dot Blot Experiment: LP586 and 6×His peptides were serially diluted 2-fold (1 μg to 0.0625 μg), and the diluted proteins were spotted onto nitrocellulose (NC) membranes. After drying, the membranes were blocked with blocking buffer at room temperature for 1 h. ECM proteins (fibronectin, type IV collagen, laminin, and hypotene) were diluted with blocking buffer and incubated overnight at 4°C. After washing with TBST, the corresponding primary antibodies for the ECM proteins were added, and the membranes were incubated at room temperature for 1 h. After washing with TBST, HRP-labeled goat anti-mouse IgG or goat anti-rabbit IgG secondary antibodies were added, and the membranes were incubated at room temperature for 1 h. After washing with TBST, the membranes were then exposed with ECL exposure buffer.

[0048] ELISA assay: Each well was coated with 200 ng of ECM protein and incubated overnight at 4°C. After washing with PBST, the plates were blocked with 5% skim milk at 37°C for 2 h. After washing, 100 μL of serially diluted LP586 protein (200 ng - 1.56 ng) was added to each well and incubated at 37°C for 1 h. Anti-LP586 mouse serum diluted 1:1000 with 5% skim milk was added and incubated at 37°C for 1 h. After washing with PBST, goat anti-mouse HRP-IgG secondary antibody diluted 1:1000 with 5% skim milk was added and incubated at 37°C for 1 h. After washing, TMB was added for color development, and the OD was read at 450 nm.

[0049] The results showed that Dot Blot and ELISA results indicated that LP586 binds to ECM proteins in a dose-dependent manner. Figure 8 ).

[0050] Example 9: LP586 protein Plg activity Dot Blot Experiment: LP586 and 6×His peptides were serially diluted 2-fold (1 μg to 0.0625 μg). The diluted proteins were spotted onto nitrocellulose (NC) membranes, dried, and then blocked with Beyotime blocking buffer at room temperature for 1 h. Plg and tPA proteins were diluted with blocking buffer and incubated overnight at 4°C. After washing with TBST, the primary antibody corresponding to the ECM protein was added, and the membrane was incubated at room temperature for 1 h. After washing with TBST, HRP-labeled goat anti-rabbit IgG secondary antibody was added, and the membrane was incubated at room temperature for 1 h. After washing with TBST, the membrane was then exposed with ECL exposure buffer.

[0051] Plasminogen activation assay: LP586 protein and plasminogen Plg were incubated at 37°C for 1 h and then added to 96-well plates. Subsequently, tPA or the lysine analog ε-ACA (80 mM) was added, and the mixture was incubated at 37°C for 15 min. After adding 0.5 mM of the specific substrate D-Val-Leu-Lys p-nitroaniline dihydrochloride, the absorbance change at 405 nm was monitored every 15 min using an iMark microplate reader.

[0052] The results showed that LP586 binds to plasminogen and tPA in a dose-dependent manner. Figure 9 A, B). Activation experiments revealed that, using 6×His peptide as a control, the OD value of pores containing LP586 protein was higher than that of the control group, indicating that LP586 can promote the conversion of plasminogen to plasmin. Furthermore, it was found that the lysine analog ε-ACA could inhibit the activation ability of LP586. Figure 9 C).

[0053] Example 10: LP586 antiserum inhibits bovine mycoplasma adhesion to host cells (1) Plating: EBL cells were seeded into 12-well plates at a density of 1 mL per well, resulting in a cell concentration of 2 × 10⁻⁶ cells / well. 5 It was then incubated overnight in a 37°C incubator containing 5% CO2.

[0054] (2) LP586 antiserum was pre-incubated with bovine mycoplasma at 37°C for 1 hour at a ratio of 1:10 and 1:100. Pre-immune negative mouse serum was used as a control and pre-incubated with bovine mycoplasma at a ratio of 1:10.

[0055] (3) Take out the 12-well plate and wash it once with PBS. Add all the incubated serum and bovine mycoplasma to 1 mL of cell culture medium and incubate in a 37°C constant temperature incubator containing 5% CO2 for 1.5 h.

[0056] (4) After washing the cells three times with PBS, add 400 μL of Tryptophan to each well. TM Digest cells with Express, incubate at 37°C for 7-8 min, then terminate digestion with 600 μL PBS, blow off the cells and place them into 2 mL EP tubes.

[0057] (5) Take 100 μL of cells from each well to dilute and spread on an MTA plate, and count the CFU.

[0058] The results showed that, compared with the pre-immune negative serum control group, the adhesion rate of Bovine Mycoplasma to EBL was significantly reduced in the experimental group pretreated with LP586 antiserum (P < 0.001), and the adhesion rate of 1:10 diluted anti-LP586 serum was lower than that of 1:100, which was more significant in inhibiting adhesion. Figure 10 ).

[0059] In summary, the bovine mycoplasma LP586 protein is located on the membrane surface and is highly conserved; it possesses good immunogenicity and reactivity; it can specifically adhere to EBL cells; it binds to ECM proteins (fibronectin, type IV collagen, laminin, and hylocinin) in a dose-dependent manner; it has plasminogen activating activity, which can facilitate the spread of bovine mycoplasma between tissues; and its antiserum can inhibit bovine mycoplasma adhesion to EBL cells, directly reducing its adhesion to host cells, thereby alleviating mycoplasma damage to cells and reducing the risk of pathogen spread. This provides a novel target and approach for the anti-infective treatment of bovine mycoplasma disease. Therefore, this protein can serve as a potential target for subunit vaccines and drugs.

[0060] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A bovine mycoplasma LP586 protein, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

1.

2. The bovine mycoplasma LP586 protein according to claim 1, characterized in that: The nucleotide sequence encoding the bovine mycoplasma LP586 protein is shown in SEQ ID NO.

2.

3. The bovine mycoplasma LP586 protein according to claim 1, characterized in that, The LP586 protein is a recombinant protein that is highly conserved among different bovine mycoplasma strains. It is located on the surface of mycoplasma cell membranes and can specifically adhere to EBL cells, bind to ECM molecules, and promote plasminogen activation in the presence of tPA. Its antiserum can inhibit bovine mycoplasma adhesion to host cells.

4. The bovine mycoplasma LP586 protein according to claim 1, characterized in that, The LP586 protein was prepared by inserting the gene encoding the LP586 protein into the pET-30a vector to construct the pET-30a- lp586 The recombinant plasmid was transformed into Escherichia coli BL21(DE3) to obtain the recombinant expression strain. After expression induced by IPTG, the LP586 protein was purified.

5. A polyclonal antibody that specifically binds to the LP586 protein, characterized in that, It is prepared by immunizing animals with the LP586 protein as described in any one of claims 1-4.

6. The polyclonal antibody according to claim 5, characterized in that, The titer of the polyclonal antibody is not less than 1:819200; Its preparation process includes: mixing LP586 protein with adjuvant and immunizing animals, collecting serum from immunized animals, and purifying it.

7. The use of the LP586 protein according to any one of claims 1-4 in the preparation of bovine mycoplasma subunit vaccines.

8. The use of the LP586 protein according to any one of claims 1-4 in the preparation of diagnostic reagents or kits for detecting bovine mycoplasma infection.

9. The use of the LP586 protein according to any one of claims 1-4 or the polyclonal antibody according to any one of claims 5-6 in the preparation of a medicament for the prevention and / or treatment of bovine mycoplasma infection.

10. The use of the polyclonal antibody according to any one of claims 5-6 in the preparation of diagnostic reagents or kits for detecting bovine mycoplasma infection.

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