Mycoplasma bovis PDHB protein monoclonal antibody, preparation method and application thereof in identification of B cell epitope

Abstract

The invention discloses a mycoplasma bovis PDHB protein monoclonal antibody, a preparation method of the mycoplasma bovis PDHB protein monoclonal antibody and application of the mycoplasma bovis PDHB protein monoclonal antibody in identification of B cell epitopes. The mycoplasma bovis PDHB protein monoclonal antibody is a monoclonal antibody B15-41 or a monoclonal antibody E20-5; wherein the monoclonal antibody B15-41 comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID No. 1 and a light chain variable region with an amino acid sequence as shown in SEQ ID No. 2; the monoclonal antibody E20-5 comprises a heavy chain variable region of which the amino acid sequence is shown as SEQ ID No.3 and a light chain variable region of which the amino acid sequence is shown as SEQ ID No.4. The mycoplasma bovis PDHB protein monoclonal antibody provided by the invention is relatively high in titer, and the titer ranges from 1: 1,024,000 to 1: 2,048,000; in addition, two B cell epitopes of the PDHB protein are further screened and identified by using the mycoplasma bovis PDHB protein monoclonal antibody, the amino acid sequences are aa10GALNHA15 and aa52DQRVWD57 respectively, and the two epitopes both have high conservative property and specificity and can be used for development of mycoplasma bovis vaccines, establishment of specific serological methods and the like.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a monoclonal antibody against a Mycoplasma bovis PDHB protein, a preparation method thereof, and an application thereof in identifying B cell epitopes. Background Art

[0002] Mycoplasma bovis (M. bovis) is a major pathogen globally causing diseases such as mastitis, pneumonia, and arthritis in dairy and beef cattle, severely impacting cattle health and productivity, and causing significant economic losses to the cattle industry. Due to its lack of a cell wall, M. bovis is naturally resistant to β-lactam antibiotics (such as penicillin), making antibiotic treatment very limited. Furthermore, its high variability and diverse antigenic diversity pose significant challenges to vaccine development, and currently no highly effective commercial vaccine exists. Given these challenges, the development of highly sensitive and specific diagnostic tools and effective vaccines is crucial for the control and prevention of M. bovis infection.

[0003] Like other mycoplasmas, M. bovis lacks a tricarboxylic acid (TCA) cycle and relies primarily on glycolysis to generate ATP. Within this metabolic process, pyruvate dehydrogenase (E1), a key component of the pyruvate dehydrogenase complex (PDC), promotes the oxidative decarboxylation of pyruvate to acetyl-CoA, thereby tightly linking glycolysis with subsequent energy production pathways. Pyruvate dehydrogenase E1 typically exists as a heterotetramer (α2-β2) and plays a central role in regulating metabolic processes. Studies have found that the pyruvate dehydrogenase β subunit (PDHB) protein of M. bovis is highly immunogenic and highly conserved. Further studies have shown that PDHB is not only localized within mitochondria, participating in biosynthesis and energy metabolism, but is also present on the cell membrane, suggesting that it may have additional functions in bacterial virulence and host interactions. These findings highlight the importance of PDHB as a potential candidate for vaccine development and targeted immunotherapy.

[0004] However, our understanding of the structure and function of bovine mycoplasma PDHB protein is currently limited, and no research reports have been published on its epitope distribution. This knowledge gap not only hinders the further application of PDHB as a vaccine target but also restricts in-depth research on its function. Therefore, a comprehensive analysis of the structure, function, and epitope distribution of PDHB protein will provide important theoretical basis for the diagnosis and development of bovine mycoplasma vaccines. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the purpose of this invention is to provide a monoclonal antibody against bovine mycoplasma PDHB protein, its preparation method, and its application in identifying B cell epitopes.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a monoclonal antibody against the PDHB protein of Mycoplasma bovis, which is monoclonal antibody B15-41 or monoclonal antibody E20-5;

[0008] The monoclonal antibody B15-41 comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID No. 1 and a light chain variable region with an amino acid sequence as shown in SEQ ID No. 2;

[0009] The monoclonal antibody E20-5 comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID No. 3 and a light chain variable region with an amino acid sequence as shown in SEQ ID No. 4.

[0010] A second aspect of the present invention provides a method for preparing the above-mentioned monoclonal antibody against bovine mycoplasma PDHB protein, comprising the following steps:

[0011] S1. Construction of pET-32a-PDHB recombinant plasmid: The optimized Mycoplasma bovis PDHB gene sequence was cloned into the pET-32a vector, and two restriction sites, BamHI and SacI, and protective bases were introduced at the 5' and 3' ends, respectively, to construct the pET-32a-PDHB recombinant plasmid;

[0012] S2. Expression and purification of PDHB protein: pET-32a-PDHB recombinant plasmid was transformed into BL21 competent cells to obtain PDHB protein;

[0013] S3, inducing the expression of the obtained PDHB protein in E. coli BL21 cells, and then purifying;

[0014] S4. Immunize mice with purified PDHB protein and then collect spleen cells from the mice;

[0015] S5. Fusing the collected mouse spleen cells with myeloma SP2 / 0 cells, culturing the resulting fused cells, and screening hybridoma cells that stably secrete PDHB protein monoclonal antibodies;

[0016] S6. The selected hybridoma cells were injected into mice to obtain a monoclonal antibody against bovine mycoplasma PDHB protein.

[0017] Preferably, triple-immunized mice are used with purified PDHB protein.

[0018] The third aspect of the present invention provides the use of the above-mentioned monoclonal antibody against the PDHB protein of Mycoplasma bovis in identifying B cell epitopes of the PDHB protein.

[0019] Preferably, when the bovine mycoplasma PDHB protein monoclonal antibody is monoclonal antibody B15-41, the minimum B-cell epitope amino acid sequence of the PDHB protein it identifies is aa. 10 GALNHA 15 When the monoclonal antibody against bovine mycoplasma PDHB protein is monoclonal antibody E20-5, the amino acid sequence of the smallest B-cell epitope of the PDHB protein it identifies is aa. 52 DQRVWD 57 .

[0020] The fourth aspect of the present invention provides the application of the above-mentioned bovine mycoplasma PDHB protein monoclonal antibody in the preparation of drugs for diagnosing bovine mycoplasma virus.

[0021] The present invention has the following beneficial effects:

[0022] (1) This invention successfully obtained two highly specific monoclonal antibodies, namely monoclonal antibody E20-5 and monoclonal antibody B15-41. Experimental results show that the titers of the two monoclonal antibodies against bovine mycoplasma PDHB protein are both high, ranging from 1:1,024,000 to 1:2,048,000. They can be used for virus diagnosis, such as WB and IFA, and have the characteristics of precision and efficiency. They can be applied to reagent kit detection and drug preparation, laying the foundation for the study of the mechanism of bovine mycoplasma virus, disease diagnosis and prevention.

[0023] (2) In this invention, two B-cell epitopes of PDHB protein were screened and identified using monoclonal antibodies against bovine mycoplasma PDHB protein (monoclonal antibody E20-5 and monoclonal antibody B15-41, respectively), with amino acid sequences aa 10 GALNHA 15 and aa 52 DQRVWD 57 Furthermore, it exhibits high conservation and specificity for both of the aforementioned epitopes, making it suitable for applications such as the development of bovine mycoplasma vaccines and the establishment of specific serological methods. Attached Figure Description

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is a schematic diagram of the structure of the constructed pET-32a-PDHB recombinant plasmid;

[0026] Figure 2The results of positive clone identification of pET-32a-PDHB recombinant plasmid transformed into BL21, where lanes 1-8 are positive clones and lane 9 is pET-32a empty vector;

[0027] Figure 3 The results of expression, purification and identification of PDHB recombinant protein are as follows: Figure 3 A is an SDS-PAGE image of purified PDHB protein. Lane 1: Unpurified soluble expression product; Lane 2: Inclusion body expression product; Lane 3: Product eluted with 30mM imidazole; Lane 4: Product eluted with 250mM imidazole; Lane 5: Product eluted with 500mM imidazole. Figure 3 B is the WB analysis of the reaction between PDHB recombinant protein and anti-His tag monoclonal antibody, lane 6: purified PDHB recombinant protein with His tag; Figure 3 C is the WB analysis of the reaction between PDHB recombinant protein and Mycoplasma bovis positive serum, lane 7: purified PDHB recombinant protein;

[0028] Figure 4 The preparation and characterization results of monoclonal antibodies: Figure 4 A represents the results of mouse serum titer assay; Figure 4 B is the titer results of monoclonal antibodies B15-41 and E20-5 determined by indirect ELISA method; Figure 4 C represents the subtype identification results of monoclonal antibodies B15-41 and E20-5; Figure 4 D is the reactivity of monoclonal antibodies B15-41 and E20-5 with purified recombinant PDHB protein by WB analysis, where M is the protein molecular weight standard; lane 1 is the purified PDHB recombinant protein; Figure 4 E represents the specificity results of the monoclonal antibody analysis by Western blotting; lane 1: Mycoplasma bovis; lane 2: Pasteurella multocida; lane 3: Mansonia hemolyticus; lane 4: Staphylococcus aureus; lane 5: Streptococcus agalactiae; lane 6: Klebsiella pneumoniae; lane 7: Mycoplasma synoviae; lane 8: Mycoplasma ovis; channel 9: BVDV-1; Figure 4 F represents the specificity of the monoclonal antibody as assessed by IFA (scale bar, 100 μm); where green signals represent the reaction of monoclonal antibodies B15-41 and E20-5 with MDBK cells infected with Mycobacterium bovis, and blue signals represent the nuclei of MDBK cells after DAPI staining.

[0029] Figure 5 To identify the minimal B cell epitope recognized by monoclonal antibody B15-41: Figure 5A is a schematic diagram of the minimal epitope recognized by monoclonal antibody B15-41; the complete PDHB protein (328 amino acids) is marked in red, the first round of screening results are marked in yellow, the second round in green, the third round in light blue, and the fourth and fifth rounds in purple; the truncated fragments N1 and N1-1 specifically recognized by monoclonal antibody B15-41 are clearly marked in blue and orange, respectively; Figure 5 BD represents the results of identifying the antigenic epitopes recognized by monoclonal antibody B15-41 using a Western blot assay with an anti-his tag antibody as a positive control; lane 1 represents the reaction of full-length PDHB protein with monoclonal antibody B15-41.

[0030] Figure 6 Results for identifying the minimal B-cell epitopes recognized by monoclonal antibody E20-5: Among them, Figure 6 A is a schematic diagram of the minimal epitope recognized by E20-5; the complete PDHB protein (328 amino acids) is marked in red; the first round of screening results are marked in yellow, the second round in green, the third round in light cyan, and the fourth and fifth rounds in brown; the truncated fragments N1 and N1-2 specifically recognized by monoclonal antibody B15-41 are highlighted in blue and pink, respectively; Figure 6 BE represents the results of identifying the antigenic epitopes recognized by E20-5 using a Western blot assay with an anti-his tag antibody as a positive control; lane 1 represents the reaction of full-length PDHB protein with monoclonal antibody E20-5.

[0031] Figure 7 The results of the conservation analysis of the identified minimal epitopes are as follows: The identified epitopes showed high conservation among *Mycoplasma bovis* reference strains from different countries and regions. Epitope aa 10 GALNHA 15 Marked with a red box, and the epitope aa 52 DQRVWD 57 Then mark it with a blue box;

[0032] Figure 8 The predicted results of antigenicity and spatial structure of the identified minimal epitope are as follows: Figure 8 A represents the predicted secondary structure of the PDHB protein. Epitope 10GALNHA15 is marked with a red box, and epitope aa... 52 DQRVWD 57 Marked with a blue box; Figure 8 B is a linear structure diagram of the protein; Figure 8 C is the three-dimensional structure diagram of the protein, with epitope aa. 10 GALNHA 15 (Red area) and epitope aa 52 DQRVWD 57 (Blue areas) are all exposed on the surface of the protein structure;

[0033] Figure 9 The analysis results for antigen-antibody interactions are as follows: Figure 9 A represents monoclonal antibody B15-41 and epitope aa. 10 GALNHA 15 The prediction model of molecular docking is ipTM=0.89pTM=0.92; Figure 9 B represents monoclonal antibody E20-5 and epitope aa. 52 DQRVWD 57 The prediction model of molecular docking was ipTM=0.94 and pTM=0.95. DETAILED DESCRIPTION

[0034] In the following description, specific details such as specific system structures and technologies are provided for the purpose of illustration rather than limitation to facilitate a thorough understanding of the embodiments of the present invention. However, it should be clear to those skilled in the art that the present invention may also be implemented in other embodiments without these specific details.

[0035] Example 1

[0036] A monoclonal antibody against bovine mycoplasma PDHB protein includes the following steps:

[0037] S1. Constructing the pET-32a-PDHB recombinant plasmid:

[0038] Based on the reference sequence of the Mycoplasma bovis PDHB gene (GenBank accession number NC_014760.1), codons were optimized to improve its expression efficiency in Escherichia coli. The optimized Mycoplasma bovis PDHB gene sequence was cloned into the pET-32a vector, and two restriction sites, BamHI and SacI, and protective bases were introduced at the 5' and 3' ends, respectively, to construct a recombinant plasmid named pET-32a-PDHB. Figure 1 As shown;

[0039] Expression and purification of S2 and PDHB recombinant proteins:

[0040] The pET-32a-PDHB recombinant plasmid was transformed into E. coli BL21(DE3) competent cells and cultured on LB solid agar plates containing amp+ (100 μg / mL) for 8–12 h. The bacterial suspension was identified using universal primers specific for the pET-32a vector (T7-F: 5'-TAATACGACTCACTATAGGG-3'; T7-R: 5'-TGCTAGTTATTG CTCAGCGG-3'). The pET-32a-PDHB recombinant plasmid was successfully transformed into BL21 competent cells, and the correct band size was 1693 bp, as expected.

[0041] Positive clones were selected and inoculated into LB liquid medium containing amp+ (100 μg / mL), and cultured with shaking at 37°C and 220 rpm until OD was reached. 600 The expression of recombinant protein was induced by isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 100 mM. The cells were shaken at 16°C and 120 rpm for 12 h to induce recombinant protein expression. After induction, the soluble recombinant protein was collected and purified using HisGrip Elite affinity chromatography. Finally, the expression of the recombinant protein was analyzed by SDS-PAGE and Western blot. The results are shown in Figure 2-3 .

[0042] Depend on Figure 2 The results show that the pET-32a-PDHB recombinant plasmid has been successfully transformed into BL21 competent cells, and the correct band size is 1693 bp, which is in line with expectations.

[0043] Depend on Figure 3 (A) The results show that the pET-32a-PDHB recombinant plasmid was successfully expressed in E. coli BL21 cells. Subsequently, the soluble recombinant protein was purified using a HisGrip Elite affinity chromatography column. SDS-PAGE analysis results showed that the PDHB protein was successfully purified with a concentration of approximately 3 mg / ml (lane 4). Figure 3 As shown in Figures B and 3C, western blot analysis demonstrated that the purified recombinant PDHB protein not only bound to anti-His tag antibodies but also reacted strongly with serum from animals positive for Mycoplasma bovis. These results fully demonstrate the immunogenicity of this recombinant protein and its potential as a diagnostic and vaccine target.

[0044] S3. Preparation and characterization of monoclonal antibodies:

[0045] 100 μg of purified and concentrated PDHB protein was emulsified with an equal volume of complete Freund's adjuvant (CFA) and subcutaneously injected multiple times into 6-week-old female BALB / c mice. Two weeks later, a second immunization was performed using half the antigen dose of the first immunization emulsified with an equal volume of incomplete Freund's adjuvant. A third immunization was performed two weeks after the second immunization, using the same dose and procedure. One week after the third immunization, serum samples were collected, and serum antibody titers were determined by indirect ELISA. Mice with the highest antibody titers were selected for booster immunization via intraperitoneal injection of 100 μg of antigen. Three days after the booster immunization, the mice were euthanized, and spleen cells were collected. These cells were then fused with SP2 / 0 myeloma cells using polyethylene glycol (PEG). The fused cells were then incubated with a solution containing 20% ​​fetal bovine serum (FBS) and 2%... Hybridoma cells were cultured in HAT RPMI-1640 medium. After 7 days of culture, the medium was changed. When the cells grew to about one-third of each well, fusion cells that secreted positive antibodies were screened by indirect ELISA. At least three rounds of subcloning were performed using limiting dilution to ensure stable secretion of monoclonal antibodies. Subsequently, the selected hybridoma cells were injected intraperitoneally into 8-week-old BALB / c mice at a concentration of 1×10^6 cells per mouse. About two weeks later, ascites fluid was collected and purified by Protein G HP affinity chromatography.

[0046] Monoclonal antibody isotypes were identified using a mouse monoclonal antibody isotype ELISA kit, and the specificity and reactivity of the prepared monoclonal antibodies were tested by Western blot (WB) and indirect immunofluorescence assay (IFA). Finally, total RNA was extracted from hybridoma cells, and the light and heavy chain variable regions of the monoclonal antibodies were amplified and subjected to high-throughput sequencing.

[0047] The indirect ELISA method was used to assess serum antibody titers in immunized mice, identify and screen positive hybridoma cell subclones, and determine monoclonal antibody titers. The assay steps were as follows:

[0048] The purified PDHB recombinant protein was diluted to 2 μg / mL in carbonate buffer (50 mM, pH 9.6) and coated onto microplates at a rate of 100 μL / well, then incubated overnight at 4°C. Subsequently, the plates were washed three times with PBST (1×PBS, containing 0.05% Tween 20, pH 7.4), followed by the addition of 200 μL of 5% skim milk and blocking at 37°C for 2 hours to reduce nonspecific binding. After washing three more times with PBST, 100 μL of diluted sample (including mouse serum, hybridoma cell supernatant, or monoclonal antibody, with SP2 / 0 cell supernatant used as a negative control) was added to each well, and the plates were incubated at 37°C for 1 hour. After three washes, 100 μL of goat anti-mouse IgG enzyme-labeled secondary antibody (dilution ratio 1:3000) was added, and the plates were incubated at 37°C for 1 hour. Finally, after three washes, 200 μL of TMB substrate chromogenic solution was added, and the mixture was incubated at room temperature in the dark for 15 minutes. The enzymatic reaction was then terminated by adding 50 μL of 1M sulfuric acid, and the OD was measured. 450nm The P / N ratio is defined as ≥2.1, where a P / N value ≥2.1 is considered positive. The P / N value is the serum concentration of the experimental group mice at OD2000. 450 nm The absorbance of the serum under the negative control group at OD 450 nm The ratio of absorbance was calculated, and the results are shown in [the table]. Figure 4 A.

[0049] Figure 4 A results showed that the serum antibody titers of the four immunized mice ranged from 1:24800 to 1:2048000, indicating a strong humoral immune response.

[0050] Subsequently, spleen cells from mice with the highest antibody titers (experimental group 1) were fused with SP2 / 0 myeloma cells. Through screening for fusion cells secreting positive antibodies and performing at least three rounds of subcloning using limiting dilution, two hybridoma cell lines secreting anti-PDHB monoclonal antibodies were successfully established, named B15-41 and E20-5. The reactivity of monoclonal antibodies B15-41 and E20-5 with purified recombinant PDHB protein was evaluated using a 2-fold serial dilution method (starting dilution 1:500). The results are shown in [Figure 1]. Figure 4 B.

[0051] Figure 4 BThe results showed that both monoclonal antibodies B15-41 and E20-5 had high antibody titers, ranging from 1:1,024,000 to 1:2,048,000.

[0052] The monoclonal antibodies were identified using a mouse monoclonal antibody subtype identification ELISA kit, and the results are shown below. Figure 4 C.

[0053] Figure 4 The results of C showed that the heavy chain subclasses of B15-41 and E20-5 were IgG2b and IgG1, respectively, and the light chain types of both were kappa.

[0054] Among them, Western blot (WB) and indirect immunofluorescence assay (IFA) were used to detect the specificity and reactivity of the prepared monoclonal antibodies:

[0055] (a) Western blot (WB) analysis of the immunogenicity of recombinant PDHB protein, assessment of the reactivity and specificity of monoclonal antibodies B15-41 and E20-5, and precise identification of the minimal linear epitopes recognized by the monoclonal antibodies.

[0056] Specifically, the protein sample was mixed with 5× loading buffer, heated at 100°C for 10 minutes for denaturation, and then subjected to SDS-PAGE experiments. After electrophoresis, the membrane was transferred using a transfer apparatus to transfer the protein gel to the NC membrane. The NC membrane was then blocked with 5% skim milk on a shaker at room temperature for 2 hours to reduce nonspecific binding. After blocking, the NC membrane was washed three times with PBST, and a 1:3000 diluted anti-His tag antibody or monoclonal antibody was added and incubated overnight at 4°C. After the incubation was completed, the membrane was washed three times with PBST and incubated with a 1:3000 diluted goat anti-rabbit IgG or goat anti-mouse IgG enzyme-labeled secondary label for 1 hour at room temperature. Finally, after washing three times with PBST, an enhanced ECL chemiluminescence detection kit was used for color development. After preparing the luminescent solution according to the instructions, it was added to the NC membrane in the dark and allowed to stand for 1 minute before the results were observed using a chemiluminescence instrument. See the results. Figure 4 D and Figure 4 E.

[0057] Figure 4 D The results showed that two monoclonal antibodies, namely monoclonal antibodies B15-41 and E20-5, were able to specifically bind to the purified recombinant PDHB protein, indicating that they had high affinity and specificity for the target antigen.

[0058] Figure 4 The results showed that two monoclonal antibodies, namely monoclonal antibodies B15-41 and E20-5, reacted only with Mycoplasma bovis, but had no cross-reaction with other pathogens causing similar clinical symptoms (such as Pasteurella multocida, Mannheimia haemolytica and Staphylococcus aureus) or other mycoplasma species of the same genus (such as Mycoplasma synoviae and Mycoplasma ovipneumoniae).

[0059] (b) IFA was used to evaluate the reactivity and specificity of mAb B15-41 and mAb E20-5.

[0060] MDBK(NBL-1) cells were seeded in 24-well cell culture plates and cultured to approximately 80% cell density. Subsequently, at 10... ^6 mdbk cells were infected with M. bovis at a concentration of CFU / mL and briefly centrifuged at 1000 × g for 5 minutes to promote bacterial adhesion, followed by incubation for 4 hours. After incubation, the cells were replaced with fresh growth medium and cultured at 37°C for an additional 72 hours. Subsequently, the cells were washed three times with PBST and fixed with 4% paraformaldehyde (300 μL / well) for 10 minutes at 4°C. The cells were washed again three times with PBST and treated with 0.5% Triton X-100 (300 μL / well) for 5 minutes at room temperature to achieve cell permeabilization. After permeabilization, the cells were washed again three times with PBST, and 5% BSA (300 μL / well) was added to each well and blocked at 37°C for 2 hours. After blocking, the cells were washed three times with PBST, and monoclonal antibodies diluted to 1:1000 in PBST were added to each well (150 μL / well) and incubated at 37°C for 1 hour. After incubation with the primary antibody, the cells were washed three times with PBST and a goat anti-mouse IgG (H+L)-AlexaFluor 488 fluorescent secondary antibody (150 μL / well) diluted to 1:500 with PBST was added and incubated at 37°C in the dark for 40 minutes. After washing three times with PBST, DAPI (150 μL / well) was added to each well and incubated at 4°C for 5 minutes. Finally, the fluorescence signal was observed and recorded using an inverted fluorescence microscope. Figure 4 F.

[0061] Figure 4 The results showed that monoclonal antibodies B15-41 and E20-5 could specifically bind to MDBK cells infected with Mycoplasma bovis, while no fluorescent signal was observed in uninfected MDBK cells.

[0062] In summary, the results indicate that both monoclonal antibodies B15-41 and E20-5 possess good immunogenicity and specificity, and have potential value for serological detection or vaccine development.

[0063] II. Identification of the smallest antigenic epitope

[0064] In order to accurately identify the smallest B-cell linear epitope of the PDHB protein recognized by the monoclonal antibody generated in this invention, a series of overlapping peptides covering the full-length PDHB sequence were designed (see Table 1 below).

[0065]

[0066]

[0067]

[0068] Table 1. Primers for identifying minimal antigenic epitopes

[0069] The truncated overlapping peptides were cloned into the pET-32a prokaryotic expression vector containing BamHI and XhoI restriction sites, respectively. The constructed recombinant plasmids were then transformed into *E. coli* BL21 competent cells for induced expression. The results are shown in [Figure 1]. Figure 5-Figure 6 .

[0070] Figure 5 A and Figure 6 Results A showed that, based on the amino acid sequence (328aa) of the PDHB protein, a series of overlapping truncated fragments covering the full length were designed ( Figure 5 A, 6A) were successfully expressed in Escherichia coli BL21 (DE3) cells.

[0071] Subsequently, the monoclonal antibody B15-41 and the monoclonal antibody E20-5 prepared by the present invention were used as primary antibodies, and the expressed truncated overlapping peptides were detected and analyzed by WB test:

[0072] First, preliminary screening was performed, truncating the PDHB protein into three overlapping polypeptides: N1 (1-134 aa), N2 (121-246 aa), and N3 (233-328 aa). Western blot results showed that mAbs B15-41 and E20-5 specifically recognized N1 only, without reacting with N2 or N3. Figure 5 B and 5C), indicating that the epitopes recognized by the two mAbs are located within aa 1-134;

[0073] In the second round of localization, the N1 protein was divided into N1-1 (1-48 aa), N1-2 (33-91 aa), and N1-3 (73-134 aa). Western blot results showed that mAb B15-41 specifically recognizes N1-1, while E20-5 specifically recognizes N1-2 (1-48 aa). Figure 5 B and Figure 6 B) indicates that the epitope recognized by monoclonal antibody B15-41 is located at aa 1-48, and the epitope recognized by monoclonal antibody E20-5 is located at aa 33-91;

[0074] In the third round of localization, taking monoclonal antibody B15-41 as an example, N1-1 (1-48 aa) was further divided into N1-1-1 (1-23 aa), N1-1-2 (8-33 aa), and N1-1-3 (18-48 aa). The results showed that monoclonal antibody B15-41 could recognize the two short peptides N1-1-1 and N1-1-2, but not N1-1-3 (18-48 aa). Figure 5 B) indicates that the epitope should exist in the overlapping segment (8-23aa) of N1-1-1 and N1-1-2;

[0075] In the fourth round of localization, to accurately identify the shortest epitope, the N-terminus of the aforementioned overlapping short peptides (8-23 aa) was fixed, and two amino acids were sequentially truncated starting from the C-terminus, resulting in D1 (8-21 aa), D2 (8-19 aa), D3 (8-17 aa), and D4 (8-15 aa). The results showed that monoclonal antibody B15-41 could recognize all four truncated fragments, E1-4. Figure 5 C), suggesting that the minimum epitope recognized by B15-41 should be between aa 8-15.

[0076] Finally, in the fifth round of localization, the N-terminus of E4 was still fixed, and one amino acid was truncated sequentially from the C-terminus, resulting in D5 (8-14 aa), D6 (8-13 aa), and D7 (8-12 aa). The results showed that B15-41 did not react with E5, E6, or E7, indicating that the smallest epitope that B15-41 could recognize was the 15th amino acid at the C-terminus. Therefore, the C-terminus of the short peptide E4 was fixed again, and one amino acid was truncated sequentially from the N-terminus, resulting in D8 (9-15 aa), D9 (10-15 aa), and D10 (11-15 aa). The results showed that the monoclonal antibody B15-41 could recognize E8 and E9, but did not react with E10. Figure 5 D).

[0077] The above results show that the minimum epitope amino acid sequence that can be recognized by monoclonal antibody B15-41 is aa 10 GALNHA 15 .

[0078] Similarly, taking the monoclonal antibody E20-5 as an example, the western blot test results are shown in Figure 6 C, Figure 6 C Results showed that the third round of monoclonal antibody E20-5 could recognize two short peptides, N1-2-1 (33-63aa) and N1-2-2 (48-83aa), but did not react with N1-2-3 (68-91aa).

[0079] Figure 6 Results D: In the fourth round, the monoclonal antibody E20-5 could recognize E1 (48-61aa), E2 (48-59aa), and E3 (48-57aa), but could not recognize E4 (48-55aa) and E5 (48-53aa).

[0080] Figure 6 The results showed that the fifth round of monoclonal antibody E20-5 could recognize E7 (49-57aa), E8 (50-57aa), E9 (51-57aa) and E10 (52-57aa), but could not recognize E6 (48-56aa) and E11 (53-57aa).

[0081] The above proves that the minimum epitope that monoclonal antibody E20-5 can recognize is aa 52 DQRVWD 57 .

[0082] (2) Bioinformatics analysis of minimal B-cell epitopes

[0083] To evaluate the epitope conservation of different isolates, 24 Mycoplasma bovis reference strains from different countries and regions around the world were collected from the NCBI database (https: / / www.ncbi.nlm.nih.gov) (see Table 2 below).

[0084] Table 2 Information on reference strains of Mycoplasma bovis

[0085]

[0086]

[0087] First, the amino acid sequences of 24 Mycoplasma bovis reference strains from different geographical regions were compared using MEGA12 software, and the identified epitopes were analyzed to assess their homology. Figure 7 .

[0088] Figure 7 The results showed that these two epitopes were significantly conserved among the selected strains. Specifically, in the Chinese strain 16M (GenBank accession number CA29804.1), epitope aa 10 GALNHA 15 There is only one amino acid substitution (A→T) at position 15. In contrast, epitope aa 52 DQRVWD 57 The strains were completely conserved across all selected strains, further highlighting their significant potential as diagnostic biomarkers and vaccine development candidates.

[0089] Then, DNASTAR Protean software was used to analyze the characteristics of B cell epitopes of Mycoplasma bovis PDHB protein. Figure 8 A.

[0090] Figure 8 The result indicates that epitope aa 10 GALNHA 15 Located in the flexible α-helical region and has high hydrophilicity; in contrast, epitope aa 52 DQRVWD 57 Despite its low hydrophilicity, its antigenic index is high, suggesting that it may still have strong immunogenicity.

[0091] In order to further analyze the spatial distribution characteristics of the above two epitopes in the PDHB protein structure, the present invention constructed a three-dimensional model of the PDHB protein through the I-TASSER online server (https: / / seq2fun.dcmb.med.umich.edu / I-TASSER / ) by homology modeling method, and visualized the three-dimensional structure model and its antigenic epitopes with the help of PyMOL software. Figure 8 B and Figure 8 C.

[0092] Figure 8 B and Figure 8 The three-dimensional model structure constructed in C shows that epitope aa 10 GALNHA 15 It presents a typical α-helical conformation, and the epitope aa 52 DQRVWD 57 This results in a mixed secondary structure consisting of β-sheets and random coils. Notably, both epitopes are exposed on the surface of the PDHB protein, suggesting that it is more easily recognized by the host immune system.

[0093] Finally, Alphafold 3 (https: / / alphafoldserver.com) was used to predict the complex structure of the monoclonal antibody Fab fragment and the antigen epitope to analyze the antibody-antigen interaction. Among them, the ipTM indicator is used to evaluate the accuracy of the predicted relative positions of the subunits in the complex. Specifically, a score higher than 0.8 indicates that the prediction result is robust and of high quality, and a score less than 0.6 usually means that the prediction failed. In addition, a pTM score greater than 0.5 indicates that the predicted complex fold is generally similar to the true structure. The results are shown in Figure 9 .

[0094] Figure 9 A The results show that residues 10G, 12L, 13N, 14H, and 15A interact with multiple amino acids in the complementarity determining regions (CDRs) of the light and heavy chains of mAb B15-41.

[0095] Figure 9 The results in B show that residues 55Q, 56R, 57V, 58W and 59D interact with multiple amino acids in the CDR regions of the light and heavy chains of mAb E20-5.

[0096] The present invention is not limited to the above-mentioned specific implementation methods. Various changes made by ordinary technicians in this field based on the above-mentioned concept without creative work are all within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against the PDHB protein of Mycoplasma bovis, characterized in that: is monoclonal antibody B15-41 or monoclonal antibody E20-5; The monoclonal antibody B15-41 comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID No. 1 and a light chain variable region with an amino acid sequence as shown in SEQ ID No. 2; The monoclonal antibody E20-5 comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID No. 3 and a light chain variable region with an amino acid sequence as shown in SEQ ID No.

4.

2. A method for preparing a monoclonal antibody against the PDHB protein of Mycoplasma bovis according to claim 1, characterized in that: The following steps are involved: S1. Construction of pET-32a-PDHB recombinant plasmid: The optimized Mycoplasma bovis PDHB gene sequence was cloned into the pET-32a vector, and two restriction sites, BamHI and SacI, and protective bases were introduced at the 5' and 3' ends, respectively, to construct the pET-32a-PDHB recombinant plasmid; S2. Expression and purification of PDHB protein: The pET-32a-PDHB recombinant plasmid was transformed into BL21 competent cells to obtain PDHB protein; S3, inducing the expression of the obtained PDHB protein in E. coli BL21 cells, and then purifying; S4. Immunize mice with purified PDHB protein and then collect spleen cells from the mice; S5. Fusing the collected mouse spleen cells with myeloma SP2 / 0 cells, culturing the resulting fused cells, and screening hybridoma cells that secrete PDHB protein monoclonal antibodies; S6. Inject the screened hybridoma cells into mice to obtain monoclonal antibodies against the PDHB protein of Mycoplasma bovis.

3. The method for preparing a monoclonal antibody against the PDHB protein of Mycoplasma bovis according to claim 2, wherein: Mice were triple-immunized with purified PDHB protein.

4. Use of the monoclonal antibody against the PDHB protein of Mycoplasma bovis according to claim 1 in identifying B cell epitopes of the PDHB protein.

5. The use according to claim 4, characterized in that When the monoclonal antibody against the bovine Mycoplasma PDHB protein is monoclonal antibody B15-41, the minimum B cell epitope amino acid sequence of the PDHB protein identified by it is aa 10 GALNHA 15 When the monoclonal antibody against the bovine Mycoplasma PDHB protein is monoclonal antibody E20-5, the amino acid sequence of the minimal B cell epitope of the PDHB protein identified by it is aa 52 DQRVWD 57 .

6. Use of the monoclonal antibody against the PDHB protein of Mycoplasma bovis according to claim 1 in the preparation of a drug for diagnosing Mycoplasma bovis virus.

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