Mycoplasma bovis adhesion protein P24 and use thereof

Abstract

The application belongs to the field of biotechnology, and particularly relates to a Mycoplasma bovum adhesion protein P24 and application thereof. A novel Mycoplasma bovum adhesion protein is identified by various biological and immunological methods, the protein can promote the combination of Mycoplasma bovum and host cells, and further research shows that the protein realizes the adhesion function by combining with heparin. In addition, the polyclonal antibody against the P24 protein can obviously inhibit the adhesion of Mycoplasma bovum to host cells, and the adhesion function is closely related to bacterial virulence. An effective target is provided for the development of novel vaccines, diagnostic and therapeutic preparations.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a bovine mycoplasma adhesion protein P24 and its applications. Background Technology

[0002] Mycoplasma bovis is an important animal pathogen that can cause various clinical symptoms in cattle, including bronchopneumonia, mastitis, arthritis, keratoconjunctivitis, and genital diseases. This pathogen can cause co-infections with various other pathogens, such as Pasteurella multocida, Haemophilus influenzae, bovine respiratory syncytial virus, bovine herpesvirus type I, bovine viral diarrhea virus, Histobacterium spp., and parainfluenza virus type 3. Furthermore, M. bovis is one of the main pathogens of bovine respiratory syndrome. Given the harmfulness of M. bovis, this pathogen has received increasing attention. However, despite significant efforts, little is known about the virulence and pathogenic mechanisms of Mycoplasma bovis.

[0003] It is well known that adhesion to host tissues and cells is a prerequisite for bacterial colonization and virulence. This is also true for mycoplasma. In fact, this process is often very complex, and for a single microorganism, it may involve multiple proteins or adhesion mechanisms, and even adhesin redundancy may exist. The diversity of receptor targeting mechanisms is crucial for successful adhesion and subsequent pathogenesis; many bacteria can utilize phase transitions to achieve immune evasion through mutations in surface proteins. Through continuous research, several *M. bovis* adhesins have been identified. For example, adhesins with well-defined host-binding targets include NOX, MbfN, FBA, TrmFO, α-enolase, MilA, and P27; and VpmaX, P26, VSP, Mbov-0503, and a 24kDa protein, for which host target proteins have not yet been identified. Unfortunately, although more than ten *M. bovis* adhesins have been identified, none of them can completely block the adhesion of *Mycoplasma bovis* to host cells. This means that the adhesion between *M. bovis* and host cells is the result of the combined action of multiple adhesins.

[0004] Bacterial adhesins typically bind to major components on the surface of host cells, including collagen, elastin, fibronectin, plasminogen, laminin, heparin, and platelet-derived growth factor (Li J, Wang J, Shao J, Li Y, Yu Y, Shao G, Feng Z, Xiong Q. 2022. The variable lipoprotein family participants in the interaction of Mycoplasma hyorhinis with host extracellular matrix and plasminogen. Vet Microbiol 265:109310). Currently, four types of *M. bovis* adhesin-binding components have been identified, including fibronectin (FN), plasminogen (Plg), heparin sulfate (HS), and amyloid precursor-like protein-2 (APLP-2) (Xu QY, Pan Q, Wu Q, Xin JQ. 2022. Mycoplasma Bovisadhesins and their target proteins. Front Immunol 13:1016641.). Among these adhesin-binding targets, heparin is a specific target. As a widely distributed sulfated glycosaminoglycan, heparin sulfate is widely present in all types of tissues and cells at both the extracellular and cellular levels. The heparin sulfate chain is synthesized in the Golgi apparatus, and the diverse binding activities of heparin sulfate are closely related to its extensive structural variability. The diversity of the heparin sulfate chain provides a basis for the binding of various ligands of the polysaccharide. It has been reported that various pathogens use heparin sulfate proteoglycans as infection targets, and the binding of ligands to heparin is closely related to pathogen internalization and pathogenesis (García B, Fernández-Vega I, García-Suárez O, S, Quirós LM. 2014. The role of heparan sulfateproteoglycans inbacterial infections. Journal of Medical Microbiology 3:1-7.). Summary of the Invention

[0005] This invention identifies a novel bovine mycoplasma adhesion protein using various biological and immunological methods. This protein promotes the binding of bovine mycoplasma to host cells, and further research shows that the protein achieves its adhesion function by binding to heparin. This provides an effective target for the development of novel vaccines, diagnostics, and therapeutic agents. Based on this, this invention was completed.

[0006] In a first aspect, the present invention provides a bovine mycoplasma vaccine containing bovine mycoplasma P24 protein, the vaccine further containing an immune adjuvant.

[0007] Furthermore, the vaccines include inactivated vaccines and attenuated live vaccines.

[0008] Furthermore, the nucleotide sequence of the bovine mycoplasma p24 protein is shown in SEQ ID NO: 1.

[0009] Furthermore, the amino acid sequence of the bovine mycoplasma p24 protein is shown in SEQ ID NO: 2.

[0010] Secondly, the present invention provides a product for detecting bovine mycoplasma infection, the product comprising a reagent for detecting bovine mycoplasma p24 protein.

[0011] Furthermore, the reagent is an antibody produced after mice are immunized with the antigen P24 protein.

[0012] Furthermore, the antibodies include monoclonal antibodies and polyclonal antibodies.

[0013] Furthermore, the nucleotide sequence of the bovine mycoplasma p24 protein is shown in SEQ ID NO: 1.

[0014] Furthermore, the amino acid sequence of the bovine mycoplasma p24 protein is shown in SEQ ID NO: 2.

[0015] Thirdly, the present invention provides the application of bovine mycoplasma p24 protein in the preparation of reagents for detecting bovine mycoplasma infection.

[0016] Furthermore, the detection reagents include, but are not limited to, kits and test strips.

[0017] Furthermore, the methods for detecting bovine mycoplasma infection include, but are not limited to, colloidal gold method, ELISA, qPCR, and PCR.

[0018] Furthermore, the nucleotide sequence of the bovine mycoplasma p24 protein is shown in SEQ ID NO: 1.

[0019] Furthermore, the amino acid sequence of the bovine mycoplasma p24 protein is shown in SEQ ID NO: 2.

[0020] Fourthly, the present invention provides the use of bovine mycoplasma p24 protein in the preparation of a medicament for the prevention or treatment of diseases caused by bovine mycoplasma.

[0021] Furthermore, the diseases caused by bovine mycoplasma include, but are not limited to, one or more of the following diseases: bovine pneumonia, mastitis, arthritis, otitis media, conjunctivitis, and reproductive system diseases.

[0022] Furthermore, the nucleotide sequence of the bovine mycoplasma p24 protein is shown in SEQ ID NO: 1.

[0023] Furthermore, the amino acid sequence of the bovine mycoplasma p24 protein is shown in SEQ ID NO: 2.

[0024] Fifthly, the present invention provides the application of bovine mycoplasma p24 protein in the preparation of bovine mycoplasma vaccines.

[0025] Furthermore, the vaccine refers to a vaccine containing the P24 protein or a vaccine containing P24 protein nucleic acid.

[0026] Furthermore, the vaccines include inactivated vaccines and attenuated live vaccines.

[0027] Furthermore, the nucleotide sequence of the bovine mycoplasma p24 protein is shown in SEQ ID NO: 1.

[0028] Furthermore, the amino acid sequence of the bovine mycoplasma p24 protein is shown in SEQ ID NO: 2.

[0029] Sixthly, the present invention provides the application of an anti-Bovine Mycoplasma P24 protein antibody in the preparation of a detection reagent for Bovine Mycoplasma antigen.

[0030] Furthermore, the anti-Bovine Mycoplasma p24 protein antibody also includes its antigen-binding fragment or its derivative.

[0031] Furthermore, the anti-Bovine Mycoplasma p24 protein antibody includes monoclonal antibodies or polyclonal antibodies.

[0032] Furthermore, the reagents for detecting bovine mycoplasma antigen include, but are not limited to, kits and test strips.

[0033] Furthermore, the methods for detecting bovine mycoplasma antigen include, but are not limited to, colloidal gold method, ELISA, qPCR, or PCR.

[0034] Furthermore, the nucleotide sequence of the bovine mycoplasma p24 protein is shown in SEQ ID NO: 1.

[0035] Furthermore, the amino acid sequence of the bovine mycoplasma p24 protein is shown in SEQ ID NO: 2.

[0036] In a seventh aspect, the present invention provides the use of an anti-Bovine Mycoplasma P24 protein antibody in the preparation of a medicament for the prevention or treatment of diseases caused by Bovine Mycoplasma.

[0037] Furthermore, the antibody includes monoclonal antibodies or polyclonal antibodies.

[0038] Furthermore, the antibody also includes its antigen-binding fragment or a derivative thereof.

[0039] Furthermore, the diseases caused by bovine mycoplasma include, but are not limited to, one or more of the following diseases: bovine pneumonia, mastitis, arthritis, otitis media, conjunctivitis, and reproductive system diseases.

[0040] Furthermore, the nucleotide sequence of the bovine mycoplasma p24 protein is shown in SEQ ID NO: 1.

[0041] Furthermore, the amino acid sequence of the bovine mycoplasma p24 protein is shown in SEQ ID NO: 2.

[0042] Beneficial effects

[0043] 1. The P24 protein in this invention is encoded by the bovine mycoplasma MBOVJF4278_00820 gene, confirming that the P24 protein has immunogenicity and reactivity, and can be used for the detection of bovine mycoplasma antibodies. Furthermore, it can be used for the detection of bovine mycoplasma antigens by preparing polyclonal or monoclonal antibodies against the P24 protein.

[0044] 2. The P24 protein in this invention can bind not only to EBL cells but also to MDBK cells, indicating that this protein may play a role in the colonization of bovine mycoplasma in different cells.

[0045] 3. This invention found that naturally infected bovine serum can significantly block the binding of P24 protein to heparin, indicating that this protein is a potential subunit vaccine component.

[0046] 4. After predicting the tertiary structure of the P24 protein, this invention found that although the heparin-binding region and the antibody-binding region of naturally infected serum do not overlap, they are closely connected in the tertiary structure. This means that in the native structure, the serum of infected animals can more effectively block the adhesion of Mycoplasma bovis to host cells through the P24-encoded protein. Attached Figure Description

[0047] Figure 1 This is for the identification of protein expression. Note: A: SDS-PAGE for the identification of P24 protein expression; B: Western blot for the identification of purified P24 protein expression; lane M is the protein marker, the same below.

[0048] Figure 2 This section describes the identification of the immunogenicity of the P24 protein. Note: A: P24 protein reacts with bovine mycoplasma-positive serum (lane 7); B: P24 protein does not react with bovine mycoplasma-negative serum (lane 8).

[0049] Figure 3This indicates the subcellular localization of the P24 protein. Strip size = 650 μm.

[0050] Figure 4 This study identifies the adhesion ability of P24 protein to EBL cells.

[0051] Figure 5 This study identifies the adhesion ability of P24 protein to MDBK cells.

[0052] Figure 6 This is an identification of the interaction between the P24 protein and EBL cell membrane components.

[0053] Figure 7 This is an identification of the interaction between P24 protein and MDBK cell membrane components.

[0054] Figure 8 This study investigated the effect of bovine mycoplasma-positive serum on the interaction of P24 protein with EBL membrane components.

[0055] Figure 9 This study investigated the effect of bovine mycoplasma-positive serum on the interaction of P24 protein with MDBK membrane components.

[0056] Figure 10 The method involves Western blot analysis of the direct interaction between different concentrations of P24 protein and heparin.

[0057] Figure 11 It is an ELISA analysis of the interaction between different concentrations of P24 protein and heparin.

[0058] Figure 12 The study used Western blot analysis to examine the effects of bovine mycoplasma naturally infected serum and mouse anti-P24 protein serum on the response of different concentrations of P24 protein to heparin.

[0059] Figure 13 The adhesion inhibition assay was used to analyze the effect of mouse anti-P24 protein serum on the adhesion of bovine mycoplasma to host cells.

[0060] Figure 14This section describes the identification of the P24 protein reaction sequence with heparin and positive serum. Note: Lanes 1, 7, and 13: GST-tagged protein; Lanes 2, 8, and 14: P24 protein; Lanes 3, 9, and 15: First truncated expression of P24 (amino acids 1-40); Lanes 4, 10, and 16: Second truncated expression of P24 (amino acids 31-70); Lanes 5, 11, and 17: Third truncated expression of P24 (amino acids 61-100); Lanes 6, 12, and 18: Fourth truncated expression of P24 (amino acids 91-126). Lanes 1-6 show the results of anti-GST antibody detection of the protein sample; Lanes 7-12 show the results of the protein reaction with heparin; Lanes 13-18 show the results of the protein reaction with bovine mycoplasma positive serum.

[0061] Figure 15 The analysis of heparin and antibody binding characteristics of the P24 protein is based on the tertiary structure of the protein. Detailed Implementation

[0062] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0063] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0064] Example 1: Bacterial strain, plasmid, and culture conditions

[0065] The bovine mycoplasma TJ strain was cultured in bovine mycoplasma broth (a medium containing 20% ​​horse serum, 10% yeast extract, 0.125 mg / ml thallium acetate and 200 IU / ml penicillin for pleuropneumonia-like organisms) to mid-log growth.

[0066] Escherichia coli DH5α strain containing expression vector pET28a(+) and its derivatives was cultured in Luria Bertani broth at 37°C. Transformants were selected by adding kanamycin (50 μg / ml) to solid Luria Bertani medium containing agarose for screening.

[0067] Embryonic bovine lung cells (EBL) and bovine kidney cells (MDBK) were cultured in modified DMEM medium containing 10% inactivated fetal bovine serum (containing 10% fetal bovine serum, 100 U / ml penicillin, and 0.1 mg / ml streptomycin).

[0068] Example 2: Gene Cloning and Protein Expression

[0069] Mycoplasma bovis Tianjin strain (TJ) is an isolate from my country and is also a strain preserved in our laboratory. This strain has not undergone whole-genome sequencing. Mycoplasma bovis strain JF4278 is a strain whose whole-genome sequence is published in NCBI. Considering the extremely high conservation of the proteins screened in this invention, the gene sequence of strain JF4278 was used as a template in primer design, and the MBOVJF4278_00820 gene of strain JF4278 was cloned using the genome of strain TJ as a template.

[0070] Genomic DNA was extracted from the bovine mycoplasma TJ strain using a cellular genomic DNA extraction kit. Primers were designed using the DNA sequence (nucleotide sequence as shown in SEQ ID NO: 3, amino acid sequence as shown in SEQ ID NO: 4) of the bovine mycoplasma JF4278 strain genome (GenBank: LT578453.1) MBOVJF4278_00820 gene as a template (primer sequences are shown in Table 1). The extracted bovine mycoplasma TJ strain genome was used as a template to amplify the bovine mycoplasma TJ strain MBOVJF4278_00820 gene using these primers.

[0071] The amplified MBOVJF4278_00820 gene was then cloned into the pET-28a vector using the ClonExpress II one-step cloning kit to construct a recombinant plasmid. The pET-28a vector was linearized using two enzymes. A mixture of 2 μL linearized vector (50 ng / μL), 1 μL of the primer PCR product (30 ng / μL), 4 μL 5xCE II Buffer, 2 μL Exnase II, and 11 μL deionized water was incubated at 37°C for 30 minutes, then immediately cooled on ice. This mixture was the ligation product (containing the constructed recombinant plasmid). The ligation product containing the recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells and cultured in liquid Luria Bertani medium until the logarithmic growth phase. Then, 0.5 mM IPGT was added, and protein expression was induced at 16°C for 24 hours. The expressed recombinant protein was purified using a Ni column, and the concentration of the purified protein was determined using a BCA protein quantification kit. Then, SDS-PAGE and Western blot analysis were performed.

[0072] The cloned gene sequence was divided into four segments to synthesize a truncated expression gene of MBOVJF4278_00820 (gene sequence shown in Table 2). After synthesis, the gene was directly cloned into the pGEX-4T-1 vector and transformed into BL21(DE3) competent cells. After being cultured to the logarithmic growth phase, the cells were induced with 0.5 mM IPTG at 16°C for 24 hours. The truncated expression protein was purified using a GST resin column to obtain the bovine mycoplasma protein described in this invention, named P24 protein (nucleotide sequence as shown in SEQ ID NO: 1, amino acid sequence as shown in SEQ ID NO: 2).

[0073] Table 1 Primers used for amplifying the MBOVJF4278_00820 gene of Bovine Mycoplasma TJ strain.

[0074]

[0075] Table 2. Truncated gene sequences of MBOVJF4278_00820

[0076]

[0077] The expression of the protein was identified using SDS-PAGE. Compared with the pET28a(+) empty vector (lane 1), the recombinant MBOVJF4278_00820 strain showed a clear band between 15 kDa and 25 kDa after induction (lane 2). The expressed protein was mainly in a soluble form (lane 3), and only a small amount of the target protein was present in the cell lysis precipitate (lane 4). The expressed protein could be effectively purified (lane 5). This indicates that the target protein was effectively expressed under the above conditions, and the expressed protein was mainly in a soluble form and could be effectively purified. Figure 1 A).

[0078] The purified recombinant protein was identified using an anti-HIS-labeled monoclonal antibody, and clear bands were observed between 15 kDa and 25 kDa. Figure 1 B, Lane 6).

[0079] Example 3 Antigenicity Analysis

[0080] To identify the immunogenicity of the P24 protein, Western blot analysis was performed using positive serum from animals naturally infected with Mycoplasma bovis. The purified P24 protein was separated by SDS-PAGE and then transferred to a nitrocellulose (NC) membrane (PALL). The membrane was blocked with 5% gelatin for 2 hours. After washing three times with PBST (PBS containing 0.05% Tween-20), the NC membrane was incubated with bovine serum (diluted 1:100 in PBS) for 2 hours. Subsequently, it was incubated with HRP-labeled rabbit anti-bovine IgG (diluted 1:8000) for 1 hour. Color development was performed using a DAB chromogenic kit (Solarbio).

[0081] After colorimetric reaction, it was found that bovine mycoplasma-positive serum could react with p24 protein. Figure 2 A), while negative serum cannot recognize this protein ( Figure 2 B). This result indicates that cattle infected with Mycoplasma bovis can produce antibodies against the P24 protein, which means that this protein is one of the antigenic components of Mycoplasma bovis.

[0082] Example 4: Production of Polyclonal Antibodies

[0083] Anti-P24 protein immune serum was prepared by immunizing Balb / c mice. Balb / c mice were immunized with 50 μg of purified antigen emulsified in Freund's complete adjuvant. Fourteen days later, mice were boosted with 50 μg of antigen mixed with Freund's incomplete adjuvant. Following booster immunization, blood was collected from the tail of the mice every 14 days to determine serum antibody titers, and booster immunizations were performed until the serum antibody titer reached ≥1:5000. Serum was then collected, aliquoted, and stored at -20°C.

[0084] Example 5: Subcellular localization of the p24 protein

[0085] To determine the subcellular localization of the p24-encoded protein, Western blot analysis was performed according to reported methods. Mycoplasma cultures in logarithmic growth phase were centrifuged at 12000g for 15 min, and the precipitate was collected and washed three times with PBS. Membrane and cytoplasmic proteins of *Mycoplasma bovis* strain TJ were extracted using a membrane protein extraction kit. The total protein of *Mycoplasma bovis* strain TJ was obtained using sonication lysis. ProteoExtract was used to... @Membrane and cytoplasmic proteins of *Mycoplasma bovis* strain TJ were obtained using the Transmembrane Protein Extraction Kit (Millipore), following the instructions provided in the product manual. Total protein, membrane proteins, and cytoplasmic proteins were separated by 12% SDS-PAGE and transferred to an intracellular (NC) membrane. The membrane was blocked with 5% gelatin for 2 hours, and then incubated for 2 hours with mouse anti-P24 protein polyclonal antibody (diluted 1:1000 in PBS). It was then co-incubated with a 5000-fold dilution of HRP-labeled goat anti-mouse IgG (H+L) antibody (ZSGB-BIO). Finally, the membrane was developed using a DAB chromogenic kit (Solarbio).

[0086] Western blot results showed that P24 protein bands were present in whole-cell proteins, cell membrane proteins, and cytoplasmic proteins, indicating that P24 protein is distributed throughout the entire bovine mycoplasma cell. Figure 3 A).

[0087] Since Western blot analysis could not completely rule out the possibility of cytoplasmic protein contamination of membrane proteins, to further confirm that the P24 protein is located on the surface of bovine mycoplasma, the subcellular localization of this protein was re-analyzed using the IFA method reported in the literature (Guo Y, Zhu H, Wang J, Huang J, Khan FA, Zhang J, Guo A, Chen X. 2017. TrmFO, a Fibronectin-Binding Adhesin of Mycoplasma bovis. Int J Mol Sci 18.). Bovine mycoplasma cells grown to the logarithmic growth phase were harvested and washed three times with PBS. Intact bovine mycoplasma cells (10 10 CCU) was incubated with mouse anti-P24 protein serum and pre-immunization serum, respectively. After washing three times with PBST, the samples were incubated with FITC-labeled goat anti-mouse IgG (full molecular weight) enzyme-labeled antibody at 37°C for 1 hour. After washing five times with PBS, the results were observed and photographed under a fluorescence microscope.

[0088] This further confirmed that the P24 protein is located on the surface of Bovine Mycoplasma. After Bovine Mycoplasma reacted with anti-P24 serum, a clear green fluorescent signal could be observed under a fluorescence microscope. Figure 3 B), while no fluorescent signal was observed in the serum of mice before immunization (see B). Figure 3 C). All the above experiments show that the P24 protein is located on the surface of bovine mycoplasma.

[0089] Example 6: Identification of the binding of P24 protein to EBL and MDBK cells

[0090] To determine whether the P24 protein can directly adhere to EBL (fetal bovine lung cells) or MDBK (bovine kidney cells) cells, IFA was used to analyze the adhesion of the P24 protein to host cells.

[0091] EBL / MDBK cells were cultured for 24 hours, washed three times with PBS, and then fixed with 4% paraformaldehyde at 4°C for 30 minutes. Cells were then blocked with 1% BSA at 37°C for 2 hours. Subsequently, the cells were co-incubated with 1 ml of PBS containing 100 μg of P24 protein. Cells incubated with PBS without P24 protein and pre-immunization serum served as controls. To ensure the activity of the recombinant protein, adhesion verification was performed immediately after purification. The binding protein was stained with mouse anti-P24 protein polyclonal antibody and FITC-labeled goat anti-mouse IgG antibody at 37°C for 1 hour, and the cell nuclei were stained with DAPI. Immunofluorescence was detected by confocal microscopy, and the results were recorded by photography.

[0092] The results showed that after co-incubation of P24 protein with EBL cells and MDBK cells, the levels of P24 protein in EBL cells (…) were significantly lower than those in MDBK cells (…). Figure 4 ) and MDBK cells ( Figure 5 A distinct green fluorescence was observed in all samples. However, no specific fluorescence was found in the pre-immunization serum group and the non-recombinant protein group. This result indicates that the P24 protein can adhere to EBL and MDBK cells.

[0093] Example 7: Identification of the binding ability of P24 protein to host cell membrane components

[0094] To further verify the adhesion characteristics of recombinant proteins to host cells, membrane proteins were extracted from EBL and MDBK cells using a membrane protein extraction kit, and the concentration of the extracted membrane proteins was determined using a BCA protein quantification kit.

[0095] The extracted membrane proteins were coated onto ELISA plates (corning) at a concentration of 10 μg / ml using carbonate buffer (18 mM NaHCO3, 27 mM Na2CO3, pH 9.6) and incubated at 4°C for 12 hours. The plates were then blocked with 5% gelatin at 37°C for 2 hours. The coated membrane proteins were incubated with purified P24 protein at different concentrations (10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625 μg / ml) at 37°C for 2 hours. The bound proteins were reacted with a mouse anti-P24 protein polyclonal antibody (1:5000). Then, HRP-labeled goat anti-mouse IgG (H+L) enzyme-labeled antibody (1:4000) was added, and the plates were incubated at 37°C for 1 hour. Finally, TMB substrate was used for color development in the dark for 10 minutes. To ensure experimental rigor, the analysis was performed in three independent replicates.

[0096] For EBL ( Figure 6 ) and MDBK ( Figure 7 The binding of P24 protein, a membrane component of cells, reached a plateau at a concentration of 2.5 μg / ml, while the OD value approached background levels after 0.3125 μg / ml. This result indicates that P24 protein can adhere to membrane extracts from EBL and MDBK cells, and that this adhesion exhibits a dose-dependent characteristic.

[0097] Example 8: Inhibitory effect of bovine mycoplasma-positive serum on the reaction of P24 protein and cell membrane components.

[0098] To investigate whether positive serum from animals infected with Mycoplasma bovis affects the binding of P24 protein to host cell membrane components, ELISA was used to analyze its inhibitory capacity.

[0099] EBL / MDBK cell membrane protein was coated onto an ELISA plate at 10 μg / mL and incubated at 4°C for 12 hours. The plate was then blocked with 5% gelatin at 37°C for 2 hours. A 50-fold diluted bovine mycoplasma-positive serum solution was incubated with 25 μg / mL P24 protein at 37°C for 1 hour. The mixture was added to the ELISA plate and incubated at 37°C for 2 hours. After washing three times with PBST, mouse anti-HIS antibody (1:2000) was added and incubated at 37°C for 1 hour. HRP-labeled goat anti-mouse IgG enzyme-labeled antibody was then added. Finally, TMB substrate chromogenic solution was added, and the plate was incubated in the dark for 10 minutes before measuring the OD. 450nm Absorbance values. Negative bovine serum was used as a control. Unpaired t-tests were performed on the different serum treatment groups using GraphPad Prism software. To ensure the rigor of the experiment, the analysis was performed three independent replicates.

[0100] Compared with negative serum, a 50-fold dilution of bovine mycoplasma positive serum significantly inhibited the interaction between p24 protein and EBL (…). Figure 8 ) and MDBK ( Figure 9 Adhesion of cell membrane components.

[0101] Example 9: Determination of the binding capacity of P24 protein to heparin

[0102] To analyze the binding affinity of p24 protein to heparin, Western blot analysis was performed. P24 protein was transferred to an NC membrane and incubated with heparin-biotin sodium salt, followed by incubation with HRP-labeled goat anti-biotin antibody. Finally, color development was performed using a DAB colorimetric kit, following the manufacturer's instructions.

[0103] To further verify the binding ability of P24 protein to heparin, the interaction between the two was further analyzed using ELISA. Purified P24 protein (5 μg / ml) was coated onto 96-well plates at 4°C for 12 hours. After ELISA coating, the plates were blocked with 5% gelatin at 37°C for 2 hours. After washing three times, the ELISA plates were coated with different concentrations of heparin-biotin sodium salt (10 μg / ml). 2 10 1 10 -1 10 0 10 -2 10 -3 10 -4 The mixture (μg / ml) was incubated at 37°C for 1.5 hours. Unbound heparin was removed by washing three times with PBST, and HRP-labeled goat anti-biotin antibody was added. The mixture was incubated at 37°C for 1 hour. 50 μl of TMB was added to each well, and the mixture was incubated at 37°C in the dark for 10 minutes. The reaction was then stopped with 2M H₂SO₄, and the OD was read. 450nm Absorbance value.

[0104] After developing the DAB substrate, a visible band can be observed at the expected location. Figure 10 This result indicates that the P24 protein can bind to heparin on the cell surface, thereby facilitating pathogen adhesion.

[0105] The results showed that heparin can interact with the P24 protein, and the response between the two is dose-dependent. Figure 11 ).

[0106] Example 10: Inhibitory effect of different serums on the interaction between p24 protein and heparin

[0107] To analyze the effect of bovine mycoplasma-positive serum on the reaction of P24 protein and heparin, the binding of different serum inhibitory proteins to heparin was analyzed using Western blot. First, P24 protein was SDS-PAGEed and transferred to an intracellular matrix (NC) membrane. The NC membrane was then incubated with bovine mycoplasma-positive and negative sera (1:100) at room temperature for 2 hours. Next, the membrane was incubated with 50 μg / ml heparin-biotin, followed by co-incubation with HRP-labeled goat anti-biotin antibody. Finally, the membrane was developed using a DAB substrate chromogenic kit.

[0108] The results showed that serum from naturally infected cattle could almost completely block the binding of p24 protein to heparin. Figure 12 ).

[0109] In addition, the effect of mouse anti-P24 protein immune serum on the recombinant protein and heparin response was analyzed using the same method as bovine mycoplasma positive serum, with mouse serum before immunization serving as a negative control.

[0110] The results showed that the anti-P24 protein antibody could also inhibit the binding of this protein to heparin, but its inhibitory ability was weaker than that of bovine mycoplasma-positive serum. Figure 12 ).

[0111] Example 11 Adhesion inhibition test of bovine mycoplasma TJ strain

[0112] MDBK cells were seeded into 12-well cell culture plates (2 x 10⁻⁶ cells / well). 5 Cells were grown in a monolayer (cell / well). Bovine Mycoplasma TJ strain was pretreated with 20-fold diluted mouse anti-P24 protein serum or pre-immunization serum at 37°C for 1 hour, followed by infection of MDBK cells with the treated Bovine Mycoplasma. Samples were washed five times with PBST to remove unbound Bovine Mycoplasma. Cells were harvested after digestion with 0.25% trypsin in MEM. CFU of bound Bovine Mycoplasma were detected by colony counting on modified PPLO agar plates. Unpaired t-tests were performed on CFU from different groups using GraphPad Prism software. Three independent experiments were conducted.

[0113] Compared with the pre-immunization serum treatment group, the number of colonies in the positive serum treatment group was significantly reduced. Figure 13 This result indicates that anti-P24 protein serum can partially reduce the adhesion of Mycoplasma bovis to host cells.

[0114] Example 12: Analysis of heparin binding and positive serum reaction sequences of P24 protein

[0115] To further analyze the heparin-binding properties and antigenicity of the P24 protein, it was truncated and expressed in four segments: the first segment includes amino acid residues 1 to 40; the second segment includes amino acid residues 31 to 70; the third segment includes amino acid residues 61 to 100; and the fourth segment includes amino acid residues 91 to 126. The segmented protein was incubated with heparin and naturally infected positive serum to determine the reactive regions. Simultaneously, the P24 protein was submitted to the I-TASSER online server (https: / / zhanggroup.org / ) for structural prediction. Based on the tertiary structure, the locations of the heparin reactive region and the antibody reactive region from naturally infected animals were determined and analyzed on the protein.

[0116] After incubation with HRP-labeled goat anti-biotin antibody, color development was performed using DAB substrate. Figure 14 The results showed that the first segment (amino acids 1-40) and the second segment (amino acids 31-70) of the P24 protein reacted with heparin, while the other parts did not, indicating that the 1-70 amino acid region of the P24 protein is the heparin-reactive region.

[0117] The fragmented expression product of P24 protein was transferred to an NC membrane and then reacted with positive serum from natural Mycoplasma bovis infection. The results showed that the fourth fragment of P24 protein (amino acids 91-126) reacted with the positive serum. Figure 14 ).

[0118] Analysis of the heparin-reacting region and antibody-binding region at the tertiary structure of the protein revealed that these two regions do not overlap but are tightly connected. From a protein structure perspective, heparin appears to bind more readily to the α-helix region of the P24 protein, while the antibody is more likely to react with the randomly coiled region. Figure 15 ).

Claims

1. A Mycoplasma bovis vaccine comprising a P24 protein of Mycoplasma bovis, characterized in that, The vaccine also contains an immune adjuvant; the nucleotide sequence of the Mycoplasma bovis P24 protein is shown as SEQ ID NO:1, and the amino acid sequence is shown as SEQ ID NO:

2.

2. Use of a Mycoplasma bovis P24 protein in the preparation of a medicament for the prevention or treatment of a disease caused by Mycoplasma bovis, characterized in that, The nucleotide sequence of the Mycoplasma bovis P24 protein is shown as SEQ ID NO:1, and the amino acid sequence is shown as SEQ ID NO:

2.

3. The use according to claim 2, wherein the disease caused by Mycoplasma bovis includes bovine pneumonia, mastitis, arthritis, otitis, conjunctivitis and reproductive system disease.

4. Use of a Mycoplasma bovis P24 protein in the preparation of a Mycoplasma bovis vaccine, characterized in that, The nucleotide sequence of the Mycoplasma bovis P24 protein is shown as SEQ ID NO:1, and the amino acid sequence is shown as SEQ ID NO:

2.

5. Use of an antibody against Mycoplasma bovis P24 protein in the preparation of a detection reagent for Mycoplasma bovis antigens, characterized in that, The antibody is a polyclonal antibody; the nucleotide sequence of the Mycoplasma bovis P24 protein is shown as SEQ ID NO:1, and the amino acid sequence is shown as SEQ ID NO:2; and the polyclonal antibody is prepared by the following method: Balb / c mice are immunized to prepare immune serum against P24 protein, and the mice are immunized with 50 μg of purified antigen emulsified in Freund's complete adjuvant; 14 days later, the mice are boosted with 50 μg of antigen mixed with Freund's incomplete adjuvant; after boosting, blood is collected from the tail of the mice every 14 days to determine the serum antibody titer, and boosting is performed until the serum antibody titer reaches more than 1:5000, and then blood is collected to prepare serum to obtain the polyclonal antibody.

6. Use of an antibody against Mycoplasma bovis P24 protein in the preparation of a medicament for the prevention or treatment of a disease caused by Mycoplasma bovis, characterized in that, The antibody is a polyclonal antibody; the nucleotide sequence of the Mycoplasma bovis P24 protein is shown as SEQ ID NO:1, and the amino acid sequence is shown as SEQ ID NO:2; and the polyclonal antibody is prepared by the following method: Balb / c mice are immunized to prepare immune serum against P24 protein, and the mice are immunized with 50 μg of purified antigen emulsified in Freund's complete adjuvant; 14 days later, the mice are boosted with 50 μg of antigen mixed with Freund's incomplete adjuvant; after boosting, blood is collected from the tail of the mice every 14 days to determine the serum antibody titer, and boosting is performed until the serum antibody titer reaches more than 1:5000, and then blood is collected to prepare serum to obtain the polyclonal antibody.

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