Mycoplasmic adhesion protein ftsz of bovine mycoplasma and application thereof

Abstract

The application discloses a Mycoplasma bovum adhesion protein FtsZ and application thereof. The nucleic acid sequence of the Mycoplasma bovum adhesion protein FtsZ is shown as SEQ ID NO. 1. The application also discloses a recombinant plasmid pET-30a-ftsZ and an E. coli containing the recombinant plasmid pET-30a-ftsZ. ftsZ The recombinant protein rFtsZ has the advantages of being capable of specifically combining with EBL cell membrane protein, being capable of combining with extracellular matrix components (fibronectin, fibronectin, laminin and type IV collagen), having the direct adhesion host cell effect, having good antigenicity, being capable of producing high-level antibodies, being capable of providing good immune protection effect, and providing a new target and thought for elucidating the pathogenic mechanism of the Mycoplasma bovum and developing a new vaccine and medicine.

Description

Technical Field

[0001] This invention relates to a protein and its applications. Specifically, this invention relates to an adhesion protein of bovine mycoplasma, FtsZ, and its applications. Background Technology

[0002] Bovine mycoplasmosis is a serious animal disease that threatens the global cattle industry. The disease is caused by Mycoplasma bovis (… Mycoplasma bovis , M. bovis Mycoplasma bovis causes diseases primarily manifesting as calf pneumonia, arthritis, and mastitis in dairy cows. This disease is currently prevalent worldwide, causing significant economic losses. To date, there is still a lack of effective vaccines to prevent diseases caused by Mycoplasma bovis, and drug-resistant strains are constantly increasing. Decreased sensitivity of Mycoplasma bovis to antibiotics leads to poor clinical treatment outcomes. Therefore, developing vaccines and therapeutic drugs to combat diseases caused by Mycoplasma bovis remains an ongoing challenge, requiring a deeper understanding of the functional genes and pathogenic mechanisms of Mycoplasma bovis.

[0003] Adhesion is not only the first step in pathogen infection of the host, but also crucial for successful infection. Because *Mycoplasma bovis* lacks the cell wall of conventional bacteria, its cell membrane becomes the direct mediator in the attachment process to host cells. The virulence-related genes are closely associated with proteins on the surface of the *Mycoplasma bovis* cell membrane. Other mycoplasma proteins involved in adhesion have been identified, including glycoproteins that promote adhesion by binding extracellular components such as fibronectin and heparin; and outer membrane lipoproteins that anchor to host cells by forming specific adsorption structures that bind to host cell receptors. Furthermore, membrane lipoproteins are highly variable and can evade the host's immune response. Many cell membrane receptors are known to be targets for pathogen adhesion. The extracellular matrix (ECM), a complex network of macromolecules secreted by cells, mainly includes fibronectin and glycosaminoglycans, which often serve as substrates for pathogen adhesion. Screening and identification of adhesion-related proteins on the surface of the *Mycoplasma bovis* cell membrane will be beneficial for the screening of *Mycoplasma bovis* vaccines and drug targets. Summary of the Invention

[0004] This invention provides a novel bovine mycoplasma FtsZ protein with adhesive function and its application in vaccine antigen preparation. The technical problem to be solved is to overcome the shortcomings of existing bovine mycoplasma vaccine antigens, such as lack of antigen and unclear pathogenic mechanism. The nucleic acid sequence of the bovine mycoplasma adhesion protein FtsZ of the present invention is shown in SEQ ID NO.1.

[0005] This invention also discloses a recombinant plasmid pET-30a-ftsZ, which is created by cloning the bovine mycoplasma FtsZ protein-coding gene into the restriction enzyme site of the prokaryotic expression vector pET30a(+).BamH I and Xho I obtained it.

[0006] This invention discloses a plasmid containing recombinant plasmid pET-30a- ftsZ Escherichia coli.

[0007] The bovine mycoplasma adhesion protein FtsZ of the present invention can be used in the preparation of drugs for the prevention and treatment of bovine mycoplasma, and can also be used in the preparation of vaccines for the prevention and treatment of bovine mycoplasma.

[0008] This invention first optimizes and synthesizes bovine mycoplasma using genetic engineering methods. ftsZ Genes were successfully generated, and the recombinant expression plasmid pET-30a- was constructed. ftsZ The recombinant protein rFtsZ was obtained by transforming *E. coli* BL21(DE3) and inducing expression followed by purification. FtsZ is highly conserved in bacteria and is generally considered a mitotic protein in most bacteria, such as *E. coli*, *Staphylococcus aureus*, and *Bacillus subtilis*. During bacterial division, FtsZ first accumulates at the mitotic site, forming a highly dynamic Z-loop, and recruits other interacting proteins to cooperate in completing the division activity. The recombinant protein rFtsZ of this invention has been verified to possess the following functional activities: 1. It can specifically bind to EBL cell membrane proteins. 2. It can bind to extracellular matrix components (glassin, fibronectin, laminin, and type IV collagen). 3. FtsZ protein has a direct adhesion effect to host cells. 4. FtsZ protein has good antigenicity. 5. FtsZ protein stimulates the production of high levels of antibodies, providing good immunoprotection. This invention has the following advantages: 1. The bovine mycoplasma FtsZ protein of the present invention is a novel adhesin with multiple binding functions screened and identified by the inventors.

[0009] 2. Relevant experiments have demonstrated that the bovine mycoplasma recombinant protein rFtsZ of the present invention has the activity of binding host cell membrane proteins and extracellular matrix.

[0010] 3. The FtsZ protein has good immunogenicity and reactivity, is highly conserved, and has good antigenicity, making it a suitable vaccine target.

[0011] 4. Related experiments show that the FtsZ protein of the present invention stimulates the body to produce high levels of antibodies and has a good immune protection effect.

[0012] Therefore, the FtsZ protein of this invention provides a new target and approach for elucidating the pathogenic mechanism of bovine mycoplasma and developing novel vaccines and drugs. Attached Figure Description

[0013] Figure 1This is an SDS-PAGE electrophoresis image of the purified bovine mycoplasma recombinant protein rFtsZ from this invention.

[0014] Figure 2 This is an immunofluorescence assay of rFtsZ and EBL cells according to the present invention. The cell membrane was stained with Dil, and the cell nucleus was stained with DAPI. The white arrow points to the rFtsZ protein attached to the EBL cell membrane.

[0015] Figure 3 This is an ELISA detection image of the binding of rFtsZ to EBL cell membrane protein according to the present invention.

[0016] Figure 4 This is a Dot Blot and ELISA assay of rFtsZ binding to extracellular matrix (ECM) components according to the present invention. (Figure label explanation:) Figure 4 The binding of A:rFtsZ to fibronectin; Figure 4 B:rFtsZ binds to laminin; Figure 4 The binding of C:rFtsZ to hyalin; Figure 4 The binding of D:rFtsZ to type IV collagen.

[0017] Figure 5 This is a graph showing the ELISA titer detection of the FtsZ protein polyclonal antibody of this invention.

[0018] Figure 6 This is a Western blot image of the reactivity of the FtsZ protein in this invention.

[0019] Figure 7 This is a diagram illustrating the inhibitory effect of the FtsZ protein antiserum of this invention on bovine mycoplasma-infected host EBL cells. Detailed Implementation

[0020] The following embodiments are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available. Example

[0021] 1. Optimized synthesis of the FtsZ protein-coding gene from bovine mycoplasma. Sequence alignment was performed on 44 Mycoplasma bovis strains, including the Mycoplasma bovis reference strain PG45 (ATCC 25523). Based on the bioinformatics analysis results, including sequence protection and secondary structure, and according to the codon preference of Escherichia coli, the FtsZ protein encoding gene of Mycoplasma bovis was optimized and synthesized.

[0022] The optimized sequence of the gene encoding the FtsZ protein of Mycoplasma bovis, SEQ ID No. 1, is as follows: 2. Expression and purification of recombinant bovine mycoplasma protein rFtsZ 1) The FtsZ protein-coding gene of bovine mycoplasma was cloned into the restriction enzyme site of the prokaryotic expression vector pET30a(+). BamH I and Xho I. Obtain the recombinant plasmid pET-30a- ftsZ Recombinant E. coli was obtained by transforming it into E. coli BL21(DE3) expression bacteria. The recombinant E. coli was cultured in LB liquid medium containing 100 μg / mL kanamycin until OD = 0.6. One mL of the culture was taken as a pre-induction control, and isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.05 mM. The culture was incubated at 16℃ and 120 r / min for 16 h. Five mL of the culture was then used for the next step: the culture was centrifuged at 9000 r / min for 10 min, the supernatant was discarded, and the culture was resuspended in 3 mL of 0.01M phosphate-buffered saline (PBS) (Solepro) solution. The culture was then centrifuged again under the same conditions, and the supernatant was discarded. This process was repeated once more. Then, 1 mL of PBS was added for resuspending. 20 μL of the resuspended culture was mixed with 5 μL of 5×Sample Buffer (GenScript). The mixture was boiled in 100℃ boiling water for 10 min. After SDS-PAGE gel electrophoresis, it was confirmed that the recombinant FtsZ protein (rFtsZ protein) was mostly expressed in the supernatant. 500 mL of the expression bacterial culture was centrifuged at 8000 r / min for 10 min, the supernatant was discarded, and the culture was washed once with 20 mL of PBS, followed by centrifugation at 8000 r / min for 10 min. This washing was repeated once more with 20 mL of PBS. After discarding the supernatant, 15 mL of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH=8.0) was added for resuspending, and the culture was disrupted using a pressure lysing device. After disruption, the culture was centrifuged at 10000 r / min for 30 min, the supernatant was collected, and the precipitate was resuspended in PBS. The specific purification steps for rFtsZ protein are as follows: (1) Add 3 mL of high-affinity nickel-charged resin (purchased from GenScript) to the affinity chromatography column. (2) Add 15 mL of ddH2O to the affinity chromatography column for washing; (3) Add 15 mL of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH=8.0) and equilibrate the column at room temperature for 10 min. (4) Add protein expression supernatant and bind the column by inverting it at 4°C for 2 h.

[0023] (5) After the binding is complete, collect all the filtered liquid, add the affinity chromatography column, and continue to collect all the filtered liquid, which is labeled as "flow-through liquid".

[0024] (6) Add 200 mL of 20 mM washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH=8.0) to wash away impurities; (7) Add 50 mL of 60 mM washing buffer (50 mM NaH2PO4, 300 mM NaCl, 60 mM imidazole, pH=8.0) and collect the filtrate; (8) Add 50 mL of 80 mM washing buffer (50 mM NaH2PO4, 300 mM NaCl, 80 mM imidazole, pH=8.0) and collect the filtrate; (9) Add 15 mL of 100 mM elution buffer (50 mM NaH2PO4, 300 mM NaCl, 100 mM imidazole, pH=8.0) to elute the target protein and collect the filtrate; (10) Add 15 mL of 200 mM elution buffer (50 mM NaH2PO4, 300 mM NaCl, 200 mM imidazole, pH=8.0) to elute the target protein and collect all the filtrate; (11) Add 15 mL of 300 mM elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole, pH=8.0) to elute the target protein and collect all the filtrate; (12) Take 20 uL of each of the collected filtrates and mix them with 5 uL of 5×Sample Buffer, and boil them in boiling water for 10 min. (13) Prepare an SDS-PAGE gel. Add the purified sample to each well (10 μL / well). After electrophoresis, remove the gel and stain with Coomassie Brilliant Blue at room temperature for 1 h. Then destain to obtain the purified recombinant protein rFtsZ. Figure 1 ).

[0025] 3. Detection of adhesion activity of bovine mycoplasma recombinant protein rFtsZ EBL cell culture: EBL cells were cultured in DMEM complete medium (DMEM medium containing 10% fetal bovine serum) at 37°C and 5% CO2. When the cells reached 80% confluent monolayer, they were digested with 1× trypsin at 37°C for 8 min, and then immediately replaced with DMEM complete medium to stop the digestion. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell pellet was resuspended in an appropriate volume of DMEM complete medium. After mixing, 10 μL was transferred to a cell counting chamber and the cells were counted using a cell counter. The counted EBL cells were then cultured at 1×10⁻⁶ cells / mL. 5 Cells / wells were added to 24-well cell culture plates containing cell spreaders and cultured overnight at 37°C and 5% CO2 to allow EBL cells to adhere.

[0026] Immunofluorescence assay: rFtsZ was diluted with Opti-MEM, and the recombinant protein was pre-incubated with pre-immunized mouse serum and anti-FtsZ mouse serum at 37°C at a concentration of 1:10. After the incubation, the protein was added to a 24-well plate containing EBL cells at a concentration of 150 μg / well and cultured at 37°C and 5% CO2 for 40 min. After culture, unbound proteins were discarded, and cells were washed three times with PBS, 3 min each time. 500 μL of 4% paraformaldehyde was added to each well, and cells were fixed at room temperature for 30 min. After washing away the fixative, 500 μL of 0.1% Triton X-100 was added to each well, and cells were permeabilized at room temperature for 10 min. After washing away the permeabilization buffer, 500 μL of 5% BSA was added to each well, and cells were blocked at 37°C for 2 h. After blocking, 1:100 anti-FtsZ mouse serum was used as the primary antibody, and cells were incubated at 37°C for 3 h. Then, 1:200 goat anti-mouse IgG (H+L) Alexa Fluor™ 488 was used as the secondary antibody, and cells were incubated at 37°C in the dark for 45 min. Cell membranes were stained using DIL, with the staining solution prepared according to the Shanghai Beyotime DIL kit. 500 μL of staining solution was added to each well containing EBL cells, and cells were stained at room temperature in the dark for 10 min. DAPI was diluted to 1:500 with PBS, and 200 μL of DAPI was added to each well. EBL cell nuclei were stained with μL of DAPI staining solution at room temperature in the dark for 10 min. After staining, the cell-containing slides were placed on a glass slide containing an anti-fluorescence quencher, mounted with nail polish, and stored in the dark. Observation was performed using a Zeiss LSM980 laser confocal microscope.

[0027] Extraction of EBL cell membrane proteins: Cells were scraped from 75T cell culture flasks using a cell scraper, PBS was added, and the resulting cell suspension was centrifuged at 1000 rpm for 5 min, discarding the supernatant. The pellet was washed with 3 mL of cell washing buffer (from a cell membrane extraction kit), and the cell suspension was centrifuged at 1000 rpm for 5 min, discarding the supernatant. The cells were resuspended in 1.5 mL of cell washing buffer and transferred to a 2 mL centrifuge tube, centrifuged at 1000 rpm for 5 min, discarding the supernatant. 0.75 mL of permeabilization buffer (from a cell membrane extraction kit) was added, the pellet was resuspended, and incubated at 4°C for 10 min under continuous mixing. The permeabilized cells were centrifuged at 16000 g for 15 min, and the cytoplasmic supernatant was collected. 0.5 mL of enrichment solution was added to the pellet, and the pellet was resuspended, and incubated at 4°C for 30 min under continuous mixing. After incubation, the pellet was centrifuged at 16000 g for 15 min at 4°C. min, collect the supernatant containing soluble membrane proteins, aliquot and store at -80℃.

[0028] ELISA assay of rFtsZ binding to EBL cell membrane protein: EBL cell membrane protein was diluted to 400 ng / well using carbonate coating buffer and coated onto 96-well plates at 4°C. After overnight coating, the liquid was discarded, and the plates were washed three times with PBST (0.01 M PBS, 0.05% Tween-20) on a microplate shaker for 3 min each time. The plates were then dried, and 200 μL of 5% skim milk powder was added to each well. The plates were blocked at 37°C for 2 h. After washing away the blocking solution, rFtsZ diluted 400 ng to 1.56 ng was added to the microplate containing EBL cell membrane protein, and the plates were incubated at 37°C for 1 h. After washing away the protein solution, anti-FtsZ mouse serum was diluted 1:1000 with 5% skim milk powder as the primary antibody and incubated at 37°C for 1 h. After washing away the primary antibody dilution, 1:1000 Beyotime goat anti-mouse HRP-IgG was used as the secondary antibody and incubated at 37°C for 1 h. After washing away the secondary antibody dilution, 100 μL of rFtsZ was added to each well. TMB was incubated at 37°C in the dark for 10 min. After incubation, 50 μL of LELISA stop solution was added to each well to stop the incubation. The OD value was measured at 450 nm using a microplate reader.

[0029] Results Interpretation: Laser confocal microscopy revealed that rFtsZ significantly adhered to EBL cells. Figure 2 (Indicated by the white arrow). When the recombinant protein was pre-incubated with anti-FtsZ serum, the adhesion of rFtsZ to EBL cells was significantly reduced, while mouse negative serum did not show a significant inhibitory effect. Figure 2In ELISA results, compared to the BSA control, rFtsZ could bind to cell membrane proteins in a dose-dependent manner ranging from 25 ng to 400 ng. Figure 3 ).

[0030] 4. Bovine Mycoplasma recombinant protein rFtsZ binding ECM activity Dot Blot experiment of rFtsZ binding to ECM: rFtsZ and 6×His peptide were diluted to 0.4 μg / μL, and then serially diluted five times and spotted onto an NC membrane. Each concentration gradient was 5 μL (2 μg-0.125 μg). After spotting, the membrane was dried at room temperature for 1 h. The membrane was then sealed with plastic film and blocked with Beyotime blocking buffer at room temperature for 1 h. After blocking, the blocking buffer was discarded, and ECM protein (fibronectin, laminin, and type IV collagen) dilution buffer was added. The membrane was incubated overnight at 4°C with an ECM protein concentration of 10 μg / 3 mL. After incubation, the membrane was washed five times with TBST for 10 min each time. The ECM protein antibody was diluted 1:500 with Beyotime primary antibody dilution buffer, added to the plastic film, and incubated at room temperature for 1 h. h; After incubation, the membrane was washed 5 times with TBST, and the goat anti-mouse HRP-IgG was diluted to 1:1000 with Beyotime secondary antibody dilution buffer and added to the plastic seal film. The membrane was incubated at room temperature for 1 h; After incubation, the membrane was washed with TBST, developed with ECL, and then imaged using the ChemiDoc XR+ imaging system.

[0031] (1) ELISA experiment of rFtsZ binding ECM: ECM proteins (glassin, fibronectin, laminin and type IV collagen) were diluted to 200 ng / well using carbonate coating buffer and coated on 96-well plates at 4°C. After overnight coating, the liquid was discarded, and the plates were washed three times with PBST using a microplate shaker, 3 min each time. The plates were then dried, and 200 μL of 5% skim milk powder was added to each well. The plates were blocked at 37°C for 2 h. After washing away the blocking solution, rFtsZ diluted 400 ng to 1.56 ng was added to the microplate containing EBL cell membrane protein, and the plates were incubated at 37°C for 1 h. After washing away the protein solution, anti-FtsZ mouse serum was diluted 1:1000 with 5% skim milk powder as the primary antibody, and the plates were incubated at 37°C for 1 h. After washing away the primary antibody dilution, 1:1000 Beyotime goat anti-mouse HRP-IgG was used as the secondary antibody, and the plates were incubated at 37°C for 1 h. After washing away the secondary antibody dilution, 100 μL of TMB was added to each well, and the plates were developed in the dark at 37°C for 10 min. After development, 50 μL of TMB was added to each well. The color development was stopped using μLELISA stop solution, and the OD value was measured at 405 nm in an ELISA reader.

[0032] (2) Result Interpretation: The results of Dot Blot and ELISA experiments show that rFtsZ can bind to fibronectin, collagen IV, hysterin and laminin in a dose-dependent manner. Figure 4 ).

[0033] 5. Preparation and titer detection of polyclonal antibodies against recombinant bovine mycoplasma protein FtsZ. SPF-grade female BALB / c mice aged 6-8 weeks were immunized three times via intramuscular injection in the leg, with each mouse receiving 20 μg of rFtsZ mixed with QuickAntibodymouse and 5W adjuvant (KX0210041, Biodragon, China) (volume 1:1). The intervals between the first and second immunizations were 3 weeks, and between the second and third immunizations were 2 weeks. Two weeks after the third immunization, orbital venous blood was collected from the mice, and serum was collected. Antibody titers were detected by ELISA. The rFtsZ recombinant protein was diluted to 100 ng / well using carbonate coating buffer and coated onto 96-well plates at 4°C. After overnight coating, the liquid was discarded, and the plate was washed three times with PBST on a microplate shaker, 3 min each time. The plate was dried, and 200 μL of 5% skim milk powder was added to each well. The plate was blocked at 37°C for 2 h. After washing away the blocking solution, anti-FtsZ mouse serum was serially diluted to 1:3200~1:13107200 using 5% skim milk powder as a diluent and incubated at 37°C for 1 h. Pre-immunization mouse serum was used as a control. After washing away the primary antibody diluent, 1:1000 Beyotime goat anti-mouse HRP-IgG was used as a secondary antibody and incubated at 37°C for 1 h. After washing away the secondary antibody diluent, 100 μL of TMB was added to each well and the plate was incubated at 37°C in the dark for 10 min. After the color development was completed, 50 μL of LELISA stop solution was added to each well to stop the color development, and the OD value was measured at 450 nm using a microplate reader. Using P / N > 2.1 as the cutoff value to determine the titer, the ELISA titer of the recombinant FtsZ protein polyclonal antibody reached as high as 1:3276800. Figure 5 The preparation results showed that the FtsZ protein has good immunogenicity.

[0034] 6. FtsZ protein reactivity assay Recombinant rFtsZ protein was isolated using 10% SDS-PAGE and transferred to PVDF. The membrane was blocked with 5% BSA for 2 h at room temperature, then reacted with 1:50 healthy bovine serum, experimentally infected bovine mycoplasma serum, and post-immunization bovine serum for 2 h at room temperature. After washing with TBST, the membrane was incubated with 1:5000 HRP-labeled goat anti-bovine IgG (H+L) for 1 h at room temperature. Images were detected using ECL and the ChemiDoc XR+image System. Results showed that compared to healthy bovine serum, FtsZ reacted with both experimentally infected bovine mycoplasma serum and post-immunization bovine serum. Figure 6 The results indicate that the bovine mycoplasma rFtsZ recombinant protein can react with the specific antibodies produced by its stimulation, demonstrating good reactivity. The combined immunogenicity results show that the FtsZ protein has good antigenicity and is highly conserved, making it a suitable vaccine target.

[0035] 7. Inhibitory effect of FtsZ protein antiserum on bovine mycoplasma infection of host cells. (1) Culture and counting of EBL cells: EBL cells were cultured in DMEM complete medium (DMEM medium containing 10% fetal bovine serum) at 37°C and 5% CO2. When the cells reached 80% confluent monolayer, they were digested with 1× trypsin at 37°C for 8 min, and then DMEM complete medium was added immediately to stop the digestion. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell pellet was resuspended in an appropriate volume of DMEM complete medium. After mixing, 10 μL was transferred to a cell counting chamber and counted using a cell counter. The counted EBL cells were then cultured at 2×10⁻⁶ cells / mL. 5 Add cells / wells to a 12-well cell culture plate, or at a concentration of 1×10⁻⁶ cells / well. 5 Cells / wells were added to 24-well cell culture plates containing cell spreaders and cultured overnight at 37°C and 5% CO2 to allow EBL cells to adhere.

[0036] (2) Culture and counting of bovine mycoplasma: Bovine mycoplasma reference strain PG45 was inoculated into MTB medium at a ratio of 1:10 and incubated statically at 37℃ in a 5% CO2 incubator for 24 h before CFU counting. Alternatively, the cultured bacterial suspension was serially diluted 10-fold, and 100 μL of the appropriately diluted suspension was spread onto MTA medium and incubated statically at 37℃ in a 5% CO2 incubator for 3-7 days before colony counting. The colony count was calculated as CFU / mL = colony count × dilution × 10.

[0037] (3) Plate counting experiment: EBL cells in 12-well plates were infected with Mycoplasma bovis at MOI=1000 and cultured at 37℃ and 5% CO2 for 1.5 h. After culture, unbound Mycoplasma bovis were discarded, and the cells were washed three times with PBS preheated to 37℃, 3 min each time. 400 μL of trypsin was added to each well and digested in a 37℃ and 5% CO2 incubator for 8 min. After digestion, 600 μL of PBS was added to stop digestion. Bacterial and cell fluids were transferred to MTB, diluted to an appropriate concentration, and spread on MTA medium. After static culture in a 37℃ and 5% CO2 incubator for 3-7 days, colony counting was performed. The colony count was calculated as CFU / mL = colony count × dilution × 10. For the adhesion inhibition experiment, Mycoplasma bovis PG45 was pre-incubated with 1:10 and 1:20 pre-immunized mouse serum and anti-FtsZ mouse serum at 37℃ before infection.

[0038] (4) Results Interpretation: After treatment with FtsZ antiserum, compared with untreated and pre-immune serum treatment, the adhesion ability of Mycoplasma bovis PG45 strain to EBL cells was significantly reduced, and this adhesion inhibition was dose-dependent. The 1:10 dilution of anti-FtsZ serum showed a higher level of adhesion inhibition than the 1:20 dilution. Figure 7 The results indicate that the FtsZ protein antiserum has a significant adhesion inhibition effect, which can significantly reduce the adhesion of bovine mycoplasma to host cells and reduce the number of viable bacteria, thus showing potential for treating bovine mycoplasma.

[0039] 8. Immunological and immunoprotective effects of recombinant protein FtsZ 400 μg rFtsZ was mixed with ISA 201 VG adjuvant at a 1:1 ratio, and a final concentration of 1 mg / ml saponin was added. After emulsification, the mixture was stored at 4°C. Ten healthy New Zealand white rabbits (Experimental Animal Center, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were divided into an FtsZ group and a PBS control group, with five rabbits in each group. Each rabbit was subcutaneously injected with 2 ml of the PBS. Fourteen days after the first immunization, a second immunization was administered. Fourteen days after the second immunization, ten rabbits were injected with the PBS. 9CFU (Chronic Fumarate) was administered via standard bovine mycoplasma challenge, with intranasal instillation for 5 consecutive days. Seven days after challenge, necropsy was performed. Results showed significant reduction in clinical symptoms in the FtsZ group. In the PBS control group, 100% (5 / 5) showed typical pulmonary consolidation and other lesions characteristic of bovine mycoplasma, while the FtsZ group showed no significant lesions (0%, 0 / 5), providing good immunoprotection. Serum antibody levels were detected by diluting the rFtsZ recombinant protein to 300 ng / well using carbonate coating buffer and coating 96-well plates at 4°C. After overnight coating, the liquid was discarded, and the plate was washed three times with PBST on a microplate shaker, 3 min each time. The plate was dried, and 200 μL of 2% casein was added to each well. The plate was blocked at 37°C for 2 h. After washing away the blocking solution, 2% casein was used as a diluent to dilute the serum from days 0, 7, 14, 21, and 28 post-immunization to 1:400, and the plate was incubated at 37°C for 1 h. Goat anti-rabbit HRP-IgG at a dilution of 1:5000 was used as a secondary antibody and the plate was incubated at 37°C for 1 h. After washing away the secondary antibody diluent, 100 μL of TMB was added to each well, and the plate was developed in the dark at room temperature. After the development was complete, 50 μL of ELISA stop solution was added to each well to stop the development, and the OD value was measured at 450 nm using a microplate reader. The results showed that the PBS group remained essentially unchanged throughout the experiment. Compared with the PBS control group, the FtsZ protein level was significantly higher than that of the PBS control group 14 days after immunization (p<0.0001), and the serum specific antibody level remained high. This indicates that FtsZ protein stimulation produced high levels of antibodies, providing good immunoprotection.

[0040] Table 1. ELISA results of bovine mycoplasma-specific antibody levels in different experimental groups (OD450nm value).

[0041]

[0042] In summary, the bovine mycoplasma FtsZ protein can bind to EBL cell membrane proteins and extracellular matrix components (fibronectin, fibronectin, laminin, and type IV collagen); the FtsZ protein has a direct adhesion effect on host cells; the FtsZ protein is highly conserved, has good immunogenicity and reactivity, good antigenicity, and stimulates the production of high levels of antibodies, providing good immunoprotection against bovine mycoplasma infection.

[0043] The above description is merely a preferred embodiment of the present invention and is 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. Bovine mycoplasma adhesion protein FtsZ, characterized in that, The nucleic acid sequence is shown in SEQ ID NO.

1.

2. The recombinant plasmid pET-30a-ftsZ, characterized in that... By cloning the bovine mycoplasma FtsZ protein-coding gene into the prokaryotic expression vector pET30a(+) restriction enzyme site BamH I and Xho I obtained it.

3. A plasmid containing the recombinant plasmid pET-30a- as described in claim 2. ftsZ Escherichia coli.

4. The use of the bovine mycoplasma adhesion protein FtsZ as described in claim 1 in the preparation of drugs for the prevention and treatment of bovine mycoplasma.

5. The use of the bovine mycoplasma adhesion protein FtsZ as described in claim 1 in the preparation of a vaccine for the prevention and treatment of bovine mycoplasma.

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