Mycoplasma bovis adhesive protein P24 and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a mycoplasma bovis adhesive protein P24 and application thereof. The novel mycoplasma bovis adhesion protein is identified through various biological and immunological methods, the protein can promote combination of mycoplasma bovis and host cells, and further research shows that the protein achieves the adhesion function by being combined with heparin. In addition, the polyclonal antibody of the anti-P24 protein can obviously inhibit the mycoplasma bovis from adhering to host cells, and the adhesion function is closely related to bacterial virulence. And an effective target is provided for research and development of novel vaccines and diagnosis and treatment preparations.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a bovine mycoplasma adhesion protein P24 and an application thereof. Background Art

[0002] Mycoplasma bovis (M. bovis) is an important animal pathogen. Infected cattle may present with a variety of clinical symptoms such as bronchopneumonia, mastitis, arthritis, keratoconjunctivitis and genital diseases. This pathogen can co-infect with a variety of pathogens, such as Pasteurella multocida, Haemophilus somnus, bovine respiratory syncytial virus, bovine herpesvirus type 1, bovine viral diarrhea virus, Histolytica somnus and parainfluenza virus type 3. In addition, M. bovis is also one of the main pathogens of bovine respiratory syndrome. In view of the harmfulness of M. bovis, people have paid more and more attention to this pathogen. However, despite great efforts, the virulence and pathogenic mechanism of M. bovis are still poorly understood.

[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 mycoplasmas. In fact, this process is often very complex and may involve multiple proteins or attachment mechanisms for a single microorganism, and there may even be redundancy in adhesins. The diversity of receptor targeting mechanisms is very important for the success of adhesion and subsequent pathogenesis, and many bacteria can use phase changes to achieve immune evasion through mutations in surface proteins. After continuous efforts by researchers, a variety of M.bovis adhesins have been identified. For example, adhesins with clear host binding targets include NOX, MbfN, FBA, TrmFO, α-enolase, MilA and P27; as well as VpmaX, P26, VSP, Mbov-0503 and 24kDa protein, and these adhesins have not yet identified host target proteins. Unfortunately, although more than a dozen M.bovis adhesins have been identified, none of them can completely block the adhesion of bovine Mycoplasma 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] The binding targets of bacterial adhesins are usually the main components of the host cell surface, including collagen, elastin, fibronectin, plasminogen, laminin, heparin, platelet-derived growth factor, etc. (Li J, Wang J, Shao J, Li Y, Yu Y, Shao G, Feng Z, Xiong Q. 2022. The variable lipoprotein family participates in the interaction of Mycoplasma hyorhinis with host extracellular matrix and plasminogen. Vet Microbiol 265: 109310). At present, 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 Bovis adhesins and their target proteins. Front Immunol 13: 1016641.). Among these adhesin binding targets, heparin is a special target. As a ubiquitous sulfated glycosaminoglycan, heparin sulfate is widely present in all types of tissues and cells at the extracellular and cellular levels. Heparin sulfate chains are synthesized in the Golgi apparatus, and the various binding activities of heparin sulfate are closely related to its extended structural variability. Heparin sulfate chains are diverse, which provides a basis for the binding of various ligands to polysaccharides. It has been reported that a variety of pathogens use heparan 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] The present invention identifies a novel Mycoplasma bovis adhesion protein through multiple biological and immunological methods, which can promote the binding of Mycoplasma bovis to host cells. Further studies show that the protein achieves adhesion function by binding to heparin. This provides an effective target for the development of new vaccines, diagnostic and therapeutic preparations. Based on this, the present invention is completed.

[0006] In a first aspect, the present invention provides a Mycoplasma bovis vaccine containing Mycoplasma bovis P24 protein, wherein the vaccine further contains an immune adjuvant.

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

[0008] Furthermore, the nucleotide sequence of the Mycoplasma bovis 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] In a second aspect, the present invention provides a product for detecting Mycoplasma bovis infection, wherein the product comprises a reagent for detecting Mycoplasma bovis 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 Mycoplasma bovis 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] In a third aspect, the present invention provides a use of a bovine Mycoplasma P24 protein in the preparation of a reagent for detecting bovine Mycoplasma infection.

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

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

[0018] Furthermore, the nucleotide sequence of the Mycoplasma bovis 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] In a fourth aspect, the present invention provides a use of a Mycoplasma bovis P24 protein in the preparation of a medicament for preventing or treating a disease caused by Mycoplasma bovis.

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

[0022] Furthermore, the nucleotide sequence of the Mycoplasma bovis 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] In a fifth aspect, the present invention provides a use of a Mycoplasma bovis P24 protein in the preparation of a Mycoplasma bovis vaccine.

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

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

[0027] Furthermore, the nucleotide sequence of the Mycoplasma bovis 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] In a sixth aspect, the present invention provides a use 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 an antigen-binding fragment or a derivative thereof.

[0031] Furthermore, the anti-bovine Mycoplasma P24 protein antibody includes a monoclonal antibody or a polyclonal antibody.

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

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

[0034] Furthermore, the nucleotide sequence of the Mycoplasma bovis 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 a use of an anti-Mycoplasma bovis P24 protein antibody in the preparation of a medicament for preventing or treating a disease caused by Mycoplasma bovis.

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

[0038] Furthermore, the antibody also includes an 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 a variety of diseases such as bovine pneumonia, mastitis, arthritis, otitis, conjunctivitis and reproductive system diseases.

[0040] Furthermore, the nucleotide sequence of the Mycoplasma bovis 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 the present invention is encoded by the bovine Mycoplasma MBOVJF4278_00820 gene, which proves that the P24 protein has immunogenicity and reactogenicity, and can be used for the detection of bovine Mycoplasma antibodies. It can also be used for the detection of bovine Mycoplasma antigens by preparing anti-P24 protein polyclonal antibodies or monoclonal antibodies.

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

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

[0046] 4. After predicting the tertiary structure of the P24 protein, the present invention found that although the heparin binding region and the serum antibody binding region of natural infection do not overlap, they are closely connected in the tertiary structure. This means that in the natural structure, the serum of infected animals can more effectively block the adhesion of Mycoplasma bovis to host cells through the P24 encoded protein. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0048] Figure 2 It is the identification of the immunogenicity of P24 protein. Note: A: P24 protein reacts with the positive serum of Mycoplasma bovis (lane 7); B: P24 protein has no reaction with the negative serum of Mycoplasma bovis (lane 8).

[0049] Figure 3is the subcellular localization of P24 protein. Bar = 650 μm.

[0050] Figure 4 It is to identify the adhesion ability of P24 protein to EBL cells.

[0051] Figure 5 It is the identification of the adhesion ability of P24 protein to MDBK cells.

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

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

[0054] Figure 8 It is the effect of bovine Mycoplasma positive serum on the interaction of P24 protein and EBL membrane components.

[0055] Fig. 9 It is the effect of bovine mycoplasma positive serum on the interaction between P24 protein and MDBK membrane components.

[0056] Fig.10 Western blot analysis of the direct interaction between different concentrations of P24 protein and heparin.

[0057] Fig.11 ELISA was used to analyze the interaction between different concentrations of P24 protein and heparin.

[0058] Fig.12 Western blot analysis was performed to examine the effects of serum from natural infection with Mycoplasma bovis and mouse anti-P24 protein serum on the reaction of different concentrations of P24 protein with heparin.

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

[0060] Fig.14It is the identification of the sequence of the reaction between P24 protein and 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: the first segment of truncated expression protein of P24 (amino acids 1-40); Lanes 4, 10 and 16: the second segment of truncated expression protein of P24 (amino acids 31-70); Lanes 5, 11 and 17: the third segment of truncated expression protein of P24 (amino acids 61-100); Lanes 6, 12 and 18: the fourth segment of truncated expression protein of P24 (amino acids 91-126). Lanes 1 to 6 are the results of the detection of protein samples by anti-GST antibody; Lanes 7 to 12 are the results of the reaction of protein with heparin; Lanes 13 to 18 are the results of the reaction of protein with positive serum of Mycoplasma bovis.

[0061] Fig.15 The heparin and antibody binding characteristics of P24 protein are analyzed based on the tertiary structure of the protein. DETAILED DESCRIPTION

[0062] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, 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] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0064] Example 1 Bacterial strains, plasmids and culture conditions

[0065] The M. bovis TJ strain was grown to mid-logarithmic growth phase in M. bovis broth (pleuropneumonia-like organism medium containing 20% ​​horse serum, 10% yeast extract, 0.125 mg / ml thallium acetate and 200 IU / ml penicillin).

[0066] E. coli DH5α strain containing the expression vector pET28a(+) and its derivatives were cultured in Luria Bertani broth at 37° C. In order to select transformants, the transformants were screened in solid Luria Bertani medium containing agarose supplemented with kanamycin (50 μg / ml).

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

[0068] Example 2 Gene cloning and protein expression

[0069] The Mycoplasma bovis Tianjin strain (TJ) is a strain isolated in my country and is also a strain preserved in this laboratory. The strain has not been sequenced for the whole genome. The Mycoplasma bovis JF4278 strain is a strain whose whole genome sequence is published in NCBI. Considering that the protein screened by the present invention is highly conservative, the gene sequence of the JF4278 strain is used as a template when designing primers in the present invention, and the MBOVJF4278_00820 gene of the JF4278 strain is cloned using the TJ strain genome as a template.

[0070] The genomic DNA of the Mycoplasma bovis TJ strain was extracted using a cell genomic DNA extraction kit. The DNA sequence of the MBOVJF4278_00820 gene of the Mycoplasma bovis JF4278 strain genome (GenBank: LT578453.1) (nucleotide sequence as SEQ ID NO: 3, amino acid sequence as SEQ ID NO: 4) was used as a template to design primers (primer sequences are shown in Table 1), and the extracted Mycoplasma bovis TJ strain genome was used as a template to amplify the MBOVJF4278_00820 gene of the Mycoplasma bovis TJ strain using the primers.

[0071] Then, the amplified MBOVJF4278_00820 gene was 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 with a double enzyme, and 2 μL of the linearized vector (50 ng / μL), 1 μL of the above-mentioned primer PCR product (30 ng / μL), 4 μL of 5xCE II Buffer, 2 μL of Exnase II and 11 μL of deionized water were mixed, reacted in a metal bath at 37°C for 30 minutes, and then immediately placed on ice for cooling. This mixture is the ligation product (this ligation product contains the constructed recombinant plasmid). The ligation product containing the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, cultured in liquid Luria Bertani medium to the logarithmic growth phase, and then 0.5 mM IPGT was added to induce protein expression 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, followed by SDS-PAGE and Western blot analysis.

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

[0073] Table 1 Primers used to amplify the gene of Mycoplasma bovis TJ strain MBOVJF4278_00820

[0074]

[0075] Table 2 Truncated expressed gene sequences of MBOVJF4278_00820

[0076]

[0077] The expression of the protein was identified by SDS-PAGE. Compared with the pET28a(+) empty vector (lane 1), the MBOVJF4278_00820 recombinant bacteria showed a clear band between 15kDa and 25kDa after induction (lane 2). The expressed protein was mainly present 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 present in a soluble form and could be effectively purified (lane 5). Figure 1 A).

[0078] The purified recombinant protein was identified using an anti-HIS-tagged monoclonal antibody, and a clear band was 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, a Western blot analysis was performed using positive sera from animals naturally infected with Mycoplasma bovis. The purified P24 protein was separated by electrophoresis on SDS-PAGE and then transferred to a nitrocellulose (NC) membrane (PALL). The membrane was blocked with 5% gelatin for 2 hours. After washing 3 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. DAB color development kit (Solarbio) was used for color development.

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

[0082] Example 4 Polyclonal Antibody Production

[0083] Immunize Balb / c mice to prepare immune serum against P24 protein. Immunize Balb / c mice with 50 μg of purified antigen emulsified in Freund's complete adjuvant. After 14 days, boost the mice with 50 μg of antigen mixed with Freund's incomplete adjuvant. After boost, collect blood from the tail of the mice every 14 days to determine the serum antibody titer, and boost until the serum antibody titer reaches 1:5000 or more. Then collect blood to prepare serum, store it at -20°C after aliquoting.

[0084] Example 5 Subcellular localization of P24 protein

[0085] In order to locate the subcellular localization of the protein encoded by P24, Western blot analysis was performed according to the method reported in the literature. The mycoplasma culture in the logarithmic growth phase was centrifuged at 12000g for 15 minutes to collect the precipitate, and the precipitate was washed 3 times with PBS. The membrane protein and cytoplasmic protein of bovine Mycoplasma TJ strain were extracted using a membrane protein extraction kit. The whole protein of bovine Mycoplasma TJ strain was obtained by ultrasonic lysis. @Transmembrane Protein Extraction Kit (Millipore) was used to obtain membrane proteins and cytoplasmic proteins of Mycoplasma bovis TJ strain. The specific method was carried out according to the product manual. Total proteins, membrane proteins and cytoplasmic proteins were separated by 12% SDS-PAGE and transferred to NC membrane. Blocked with 5% gelatin for 2 hours, the NC membrane was then incubated with mouse anti-P24 protein polyclonal antibody (diluted 1:1000 in PBS) for 2 hours. Then it was co-incubated with 5000-fold diluted HRP-labeled goat anti-mouse IgG (H+L) antibody (ZSGB-BIO). Finally, the DAB color development kit (Solarbio) was used for color development.

[0086] Western blot results showed that P24 protein bands were found in whole cell proteins, cell membrane proteins and cytoplasmic proteins, indicating that P24 protein was distributed throughout the entire cell of Mycoplasma bovis ( Figure 3 A).

[0087] Since Western blot analysis cannot completely exclude the possibility of cytoplasmic proteins contaminating membrane proteins, in order to further confirm that the P24 protein is located on the surface of bovine M. bovis, the subcellular localization of the 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. Trm F O, a Fibronectin-Binding Adhesin of Mycoplasma bovis. Int J Mol Sci 18.). Bovine M. bovis grown to the logarithmic growth phase was harvested and washed three times with PBS. The intact bovine M. bovis cells (10 10 CCU) were incubated with mouse anti-P24 protein serum and pre-immune serum respectively. After washing with PBST for 3 times, the samples were incubated with FITC-labeled goat anti-mouse IgG (whole molecule) enzyme-labeled antibody at 37°C for 1 hour. After washing with PBS for 5 times, the samples were observed under a fluorescence microscope and photographed to record the results.

[0088] It was further confirmed that the P24 protein was located on the surface of bovine Mycoplasma. After bovine Mycoplasma reacted with anti-P24 serum, a clear green fluorescence signal could be observed under a fluorescence microscope ( Figure 3 B), while no fluorescence signal was observed in the serum of pre-immune mice (see Figure 3 C). The above experiments all show that the P24 protein is located on the surface of Mycoplasma bovis.

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

[0090] In order to identify whether P24 protein can directly adhere to EBL (fetal bovine lung cells) or MDBK (bovine kidney cells) cells, the adhesion of P24 protein to host cells was analyzed by IFA.

[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. The cells were blocked with 1% BSA at 37°C for 2 hours. Subsequently, the cells were incubated with 1 ml of PBS containing 100 μg of P24 protein. Cells incubated with PBS without P24 protein and pre-immune serum were set as controls. To ensure the activity of the recombinant protein, adhesion verification was performed immediately after purification of the recombinant protein. The bound 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 P24 protein was co-incubated with EBL cells and MDBK cells, anti-P24 protein serum was used to detect the EBL cells ( Figure 4 ) and MDBK cells ( Figure 5 ) were observed to have obvious green fluorescence. However, no specific fluorescence was found in the pre-immune serum group and the non-recombinant protein group. This result indicates that P24 protein can adhere to EBL and MDBK cells.

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

[0094] To further verify the adhesion characteristics of the recombinant protein to the host cells, the membrane proteins of EBL and MDBK cells were extracted 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 on ELISA plates (CORNING) at a concentration of 10 μg / ml with carbonate buffer (18 mM NaHCO3, 27 mM Na2CO3, pH 9.6) and coated at 4°C for 12 hours. Blocked with 5% gelatin at 37°C for 2 hours. The coated membrane proteins were incubated with purified P24 proteins at different concentrations (10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625 μg / ml) at 37°C for 2 hours. Mouse anti-P24 protein polyclonal antibody (1:5000) was used to react with the bound protein. Then, HRP-labeled goat anti-mouse IgG (H+L) enzyme-labeled antibody (1:4000) was added and incubated at 37°C for 1 hour. Then, TMB substrate was used to develop color in the dark for ten minutes. To ensure the rigor of the experiment, the analysis was repeated three times independently.

[0096] For EBL( Figure 6 ) and MDBK( Figure 7 ) cell membrane components, P24 protein binding reached a plateau at a concentration of 2.5μg / ml, and the OD value was close to the background level after 0.3125μg / ml. This result shows that P24 protein can adhere to the membrane extracts of EBL and MDBK cells, and this adhesion effect shows a dose-dependent characteristic.

[0097] Example 8 Inhibitory Effect of Mycoplasma bovis Positive Serum on the Reaction of P24 Protein and Cell Membrane Components

[0098] In order to investigate whether positive sera from animals infected with Mycoplasma bovis affect the binding of P24 protein to host cell membrane components, ELISA was used to analyze the inhibitory capacity.

[0099] The EBL / MDBK cell membrane protein was coated on the ELISA plate at 10 μg / mL and incubated at 4°C for 12 hours. The ELISA plate was then blocked with 5% gelatin at 37°C for 2 hours. A 50-fold diluted bovine Mycoplasma positive serum was incubated with 25 μg / ml of 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 colorimetric solution was added and the OD was measured after color development for 10 minutes in the dark. 450nm Absorbance value. Negative bovine serum was set as the control. Unpaired t-test analysis was performed on different serum treatment groups using GraphPad Prism software. To ensure the rigor of the experiment, the analysis was repeated three times independently.

[0100] Compared with negative serum, 50-fold dilution of bovine Mycoplasma positive serum could significantly inhibit the binding of P24 protein to EBL( Figure 8 ) and MDBK( Fig. 9 )Adhesion of cell membrane components.

[0101] Example 9 Determination of the ability of P24 protein to bind heparin

[0102] Western blot analysis was performed to analyze the binding ability of P24 protein to heparin. P24 protein was transferred to NC membrane and incubated with heparin-biotin sodium salt, and then incubated with HRP-labeled goat anti-biotin antibody. Finally, DAB color development kit was used for color development. The specific method was carried out according to the product manual.

[0103] In order to further verify the binding ability of P24 protein to heparin, the interaction between the two was further analyzed by ELISA. The purified P24 protein (5 μg / ml) was coated on a 96-well plate at 4°C for 12 hours. After the ELISA coating was completed, 5% gelatin was used to block at 37°C for 2 hours. After washing 3 times, the ELISA plate was incubated with different concentrations of heparin-biotin sodium salt (10 2 ,10 1 ,10 -1 ,10 0 ,10 -2 ,10 -3 ,10 -4 μg / ml) at 37°C for 1.5 hours. Wash with PBST 3 times to remove unbound heparin, add HRP-labeled goat anti-biotin antibody, and incubate at 37°C for 1 hour. Add 50 μl of TMB to each well and color at 37°C for 10 minutes in the dark, then stop the reaction with 2M H2SO4 and read the OD 450nm Absorbance value.

[0104] After DAB substrate development, a visible band can be observed at the expected position ( Fig.10 ). This result suggests that the P24 protein can bind to heparin on the cell surface, thereby facilitating pathogen adhesion.

[0105] The results showed that heparin could interact with P24 protein, and the reaction between the two was dose-dependent ( Fig.11 ).

[0106] Example 10 Inhibitory effects of different sera on the interaction between P24 protein and heparin

[0107] In order 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 by Western blot. First, the P24 protein was subjected to SDS-PAGE and then transferred to the NC membrane. The NC membrane was incubated with bovine mycoplasma positive and negative serum (1:100) at room temperature for 2 hours. Then, 50μg / ml heparin-biotin was incubated with the membrane, and then the NC membrane was co-incubated with HRP-labeled goat anti-biotin antibody. Finally, the DAB substrate color development kit was used for color development.

[0108] The results showed that serum from naturally infected cattle could almost completely block the binding of P24 protein to heparin ( Fig.12 ).

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

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

[0111] Example 11 Adhesion inhibition test of Mycoplasma bovis TJ strain

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

[0113] Compared with the pre-immune serum-treated group, the number of colonies in the positive serum-treated group was significantly reduced ( Fig.13 ). This result indicates that anti-P24 protein serum can partially reduce the adhesion of bovine Mycoplasma to host cells.

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

[0115] In order to further analyze the heparin binding properties and antigenicity of the P24 protein, it was truncated and expressed into four segments, namely: the first segment includes amino acid residues from 1 to 40; the second segment includes amino acid residues from 31 to 70; the third segment includes amino acid residues from 61 to 100; the fourth segment includes amino acid residues from 91 to 126. Heparin and positive serum from natural infection were incubated with the segmented expressed protein to determine the reaction area. At the same time, the P24 protein was submitted to the I-TASSER online server (https: / / zhanggroup.org / ) for structural prediction. Based on the tertiary structure, the positions of the heparin reaction area and the naturally infected animal antibody reaction area on the protein were determined and analyzed.

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

[0117] The segmented expression products of P24 protein were transferred to NC membrane and then reacted with positive serum from natural infection of Mycoplasma bovis. The results showed that the fourth segment of P24 protein (amino acids 91-126) reacted with positive serum ( Fig.14 ).

[0118] The heparin-reactive region and the antibody-binding region were analyzed on the tertiary structure of the protein, and the results showed that the two regions did not overlap but were closely connected. From the perspective of protein structure, heparin appears to be more inclined to bind to the α-helical region of the P24 protein, while the antibody is more likely to react with the random coil region ( Fig.15 ).

Claims

1. A Mycoplasma bovis vaccine containing Mycoplasma bovis P24 protein, wherein the vaccine further contains an immune adjuvant.

2. A product for detecting Mycoplasma bovis infection, comprising a reagent for detecting Mycoplasma bovis P24 protein.

3. Application of a bovine Mycoplasma P24 protein in the preparation of a reagent for detecting bovine Mycoplasma infection.

4. The use as claimed in claim 3, wherein the detection reagents include but are not limited to a kit and a test strip.

5. Use of a bovine Mycoplasma P24 protein in the preparation of a drug for preventing or treating a disease caused by bovine Mycoplasma.

6. The use according to claim 5, wherein the diseases caused by Mycoplasma bovis include but are not limited to one or more of a variety of diseases such as bovine pneumonia, mastitis, arthritis, otitis, conjunctivitis and reproductive system diseases.

7. Use of a bovine Mycoplasma P24 protein in the preparation of a bovine Mycoplasma vaccine.

8. Use of an anti-bovine Mycoplasma P24 protein antibody in the preparation of a detection reagent for bovine Mycoplasma antigen.

9. Use of an anti-bovine Mycoplasma P24 protein antibody in the preparation of a drug for preventing or treating a disease caused by bovine Mycoplasma.

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