Monoclonal antibody against bovine mycoplasma PDHB protein, its preparation method, and its application in identifying B cell epitopes.

Abstract

This invention discloses a monoclonal antibody against bovine mycoplasma PDHB protein, its preparation method, and its application in identifying B-cell epitopes. The bovine mycoplasma PDHB protein monoclonal antibody is either monoclonal antibody B15-41 or monoclonal antibody E20-5. Monoclonal antibody B15-41 contains a heavy chain variable region as shown in SEQ ID No. 1 and a light chain variable region as shown in SEQ ID No. 2; monoclonal antibody E20-5 contains a heavy chain variable region as shown in SEQ ID No. 3 and a light chain variable region as shown in SEQ ID No. 4. The bovine mycoplasma PDHB protein monoclonal antibody provided by this invention has a high titer, ranging from 1:1,024,000 to 1:2,048,000. Furthermore, this invention also used the bovine mycoplasma PDHB protein monoclonal antibody to screen and identify two B-cell epitopes of PDHB protein, with amino acid sequences aa... 10 GALNHA 15 and aa 52 DQRVWD 57 Furthermore, both of the above epitopes are highly conserved and specific, and can be used for the development of bovine mycoplasma vaccines and the establishment of specific serological methods.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a monoclonal antibody against bovine mycoplasma PDHB protein, its preparation method, and its application in identifying B cell epitopes. Background Technology

[0002] Mycoplasma bovis (M. bovis) is a major pathogen causing mastitis, pneumonia, and arthritis in dairy and beef cattle worldwide, severely impacting bovine health and productivity, and causing significant economic losses to the cattle industry. Due to the lack of a cell wall, Mycoplasma bovis exhibits natural resistance to β-lactam antibiotics (such as penicillin), limiting the effectiveness of antibiotic treatment. Furthermore, its high variability and antigenic diversity pose significant challenges to vaccine development; currently, there are no highly effective commercial vaccines. Given these challenges, developing highly sensitive and specific diagnostic tools and effective vaccines is crucial for controlling and preventing Mycoplasma bovis infection.

[0003] Similar to other mycoplasma genera, *Mycoplasma bovis* lacks the tricarboxylic acid cycle (TCA) and primarily relies on glycolysis to generate ATP. In this metabolic process, pyruvate dehydrogenase (E1), a key component of the pyruvate dehydrogenase complex (PDC), promotes the oxidative decarboxylation of pyruvate to acetyl-CoA, thus 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 metabolism. Studies have found that the pyruvate dehydrogenase β subunit (PDHB) protein in *Mycoplasma bovis* possesses strong immunogenicity and high conservation. Further research indicates that PDHB is not only located in mitochondria, participating in biosynthesis and energy metabolism, but also exists on the cell membrane, suggesting potential additional functions in bacterial virulence and host interactions. These findings highlight the significant value 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] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a monoclonal antibody against bovine mycoplasma PDHB protein, which is either monoclonal antibody B15-41 or monoclonal antibody E20-5.

[0008] The monoclonal antibody B15-41 contains 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 contains 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 bovine mycoplasma PDHB gene sequence was cloned into the pET-32a vector, and two restriction enzyme 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. The PDHB protein obtained by induction was expressed in Escherichia coli BL21 cells and then purified.

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

[0015] S5. The collected mouse spleen cells were fused with myeloma cells SP2 / 0, and the resulting fused cells were cultured to screen hybridoma cells that stably secrete monoclonal antibodies against PDHB protein.

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

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

[0018] A third aspect of the present invention provides the application of the above-mentioned bovine mycoplasma PDHB protein monoclonal antibody in identifying B-cell epitopes of 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] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0026] Figure 2The results of identifying positive clones transformed into BL21 by the pET-32a-PDHB recombinant plasmid are shown. Lanes 1-8 are positive clones, and lane 9 is the empty pET-32a vector.

[0027] Figure 3 Results of expression, purification, and identification of PDHB recombinant protein: Among them, 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 represents the Western blot 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 represents the Western blot analysis of the reaction between recombinant PDHB protein and bovine mycoplasma-positive serum; lane 7: purified recombinant PDHB protein.

[0028] Figure 4 The results of the preparation and characterization of monoclonal antibodies are as follows: Figure 4 A represents the results of mouse serum titer assay; Figure 4 B represents the titer results of monoclonal antibodies B15-41 and E20-5 determined using the indirect ELISA method; Figure 4 C represents the subtype identification results of monoclonal antibodies B15-41 and E20-5; Figure 4 D represents the Western blotting analysis of the reactivity of monoclonal antibodies B15-41 and E20-5 with purified recombinant PDHB protein, where M represents the protein molecular weight standard; lane 1 represents the purified recombinant PDHB 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 Results for identifying the minimal B-cell epitopes recognized by monoclonal antibody B15-41: Among them, Figure 5A is a schematic diagram of the minimal epitope localization 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 localization recognized by E20-5; the complete PDHB protein (328 amino acids) is marked in red; the results of the first round of screening are marked in yellow, the second round in green, the third round in light blue, 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 predictive model for molecular docking has ipTM = 0.89 and pTM = 0.92. Figure 9 B represents monoclonal antibody E20-5 and epitope aa. 52 DQRVWD 57 The prediction model for molecular docking has ipTM = 0.94 and pTM = 0.95. Detailed Implementation

[0034] In the following description, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of the invention. However, those skilled in the art will understand that the invention may 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 bovine mycoplasma PDHB gene (GenBank accession number NC_014760.1), codons were optimized to improve its expression efficiency in *E. coli*. The optimized bovine mycoplasma PDHB gene sequence was cloned into the pET-32a vector, and BamHI and SacI restriction enzyme sites, along with protective bases, were introduced at the 5' and 3' ends, respectively, to construct a recombinant plasmid named pET-32a-PDHB recombinant plasmid. 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 culture was identified using the specific universal primers for the pET-32a vector (T7-F: 5'-TAATACGACTCACTATAGGG-3'; T7-R: 5'-TGCTAGTTATTG CTCAGCGG-3'), confirming successful transformation of the pET-32a-PDHB recombinant plasmid into BL21 competent cells. The correct band size was 1693 bp, consistent with expectations.

[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 concentration reached approximately 0.6. Isopropyl β-d-1-thiogalactoside (IPTG) was then added to a final concentration of 100 mM, and the mixture was cultured at 16 °C and 120 rpm for 12 h to induce recombinant protein expression. After induction, soluble recombinant protein was collected and purified using HisGrip Elite affinity chromatography. Finally, the expression of the recombinant protein was analyzed using SDS-PAGE and Western blot. The results are shown in the table below. 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 showed 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 indicated that the PDHB protein was successfully purified at a concentration of approximately 3 mg / ml (lane 4). Figure 3 As shown in B and 3C, the Western blot (WB) assay demonstrated that the purified recombinant PDHB protein not only binds to the anti-His-tagged antibody but also reacts strongly with serum from bovine mycoplasma-positive animals. These results fully confirm the immunogenicity of this recombinant protein and its potential application 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 subtypes were identified using a mouse monoclonal antibody isotype ELISA kit. The specificity and reactivity of the prepared monoclonal antibodies were detected using 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 Results showed that the serum antibody titers of the four immunized mice ranged from 1:24,800 to 1:2,048,000, 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 The 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 Results showed that the heavy chain subclasses of B15-41 and E20-5 were IgG2b and IgG1, respectively, and the light chain type was kappa for both.

[0054] The specificity and reactivity of the prepared monoclonal antibodies were detected using Western blot (WB) and indirect immunofluorescence assay (IFA).

[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 and denatured at 100°C for 10 minutes, followed by SDS-PAGE. After electrophoresis, the protein gel was transferred to an NC membrane using a transfer apparatus. The NC membrane was then blocked with 5% skim milk on a shaker at room temperature for 2 hours to reduce non-specific binding. After blocking, the NC membrane was washed three times with PBST, and incubated overnight at 4°C with a 1:3000 dilution of anti-His tag antibody or monoclonal antibody. After incubation, the membrane was washed three times with PBST and incubated for 1 hour at room temperature with a 1:3000 dilution of goat anti-rabbit IgG or goat anti-mouse IgG enzyme-labeled solution. Finally, after three washes with PBST, the enhanced ECL chemiluminescence detection kit was used for color development. The luminescent solution was prepared according to the instructions, added to the NC membrane in the dark, and observed using a chemiluminescence analyzer after standing for 1 minute. Results are shown below. Figure 4 D and Figure 4 E.

[0057] Figure 4 Results showed that the two monoclonal antibodies, B15-41 and E20-5, could specifically bind to the purified recombinant PDHB protein, indicating that they have high affinity and specificity for the target antigen.

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

[0059] (b) The reactivity and specificity of monoclonal antibodies B15-41 and E20-5 were evaluated using the IFA method.

[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 *Mycoplasma bovis* at a concentration of CFU / mL, and bacterial adhesion was promoted by brief centrifugation at 1000×g for 5 minutes, followed by incubation for 4 hours. After incubation, the medium was replaced with fresh growth medium, and the cells were cultured at 37°C for another 72 hours. Subsequently, the cells were washed three times with PBST and fixed with 4% paraformaldehyde (300 μL / well) at 4°C for 10 minutes. The cells were then washed three times with PBST again, and treated with 0.5% Triton X-100 (300 μL / well) at room temperature for 5 minutes to achieve cell infiltration. After infiltration, the cells were washed three times with PBST again, and 5% BSA (300 μL / well) was added to each well for blocking at 37°C for 2 hours. After blocking, the cells were washed three times with PBST, and monoclonal antibody diluted 1:1000 with PBST (150 μL / well) was added to each well for incubation at 37°C for 1 hour. After incubation with the primary antibody, the cells were washed three times with PBST, and then 150 μL / well of goat anti-mouse IgG (H+L)-Alexa Fluor 488 fluorescent secondary antibody diluted 1:500 with PBST was added. The cells were incubated at 37°C in the dark for 40 minutes. After washing three times with PBST, 150 μL / well of DAPI 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. Results are shown below. 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 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]. Figures 5-6 .

[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) was successfully expressed in Escherichia coli BL21(DE3) cells.

[0071] Subsequently, using the monoclonal antibodies B15-41 and E20-5 prepared in this invention as primary antibodies, the expressed truncated overlapping peptides were detected and analyzed by Western blotting (WB) assay:

[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) indicate that the epitopes recognized by the two monoclonal antibodies 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), it is suggested that the smallest epitope identified by B15-41 should be between aa 8 and 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 demonstrate that the smallest epitope amino acid sequence that monoclonal antibody B15-41 can recognize is aa. 10 GALNHA 15 .

[0078] Similarly, taking monoclonal antibody E20-5 as an example, the results of the Western blot experiment are shown below. Figure 6 C, Figure 6 The 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 demonstrates that the smallest epitope recognized by the monoclonal antibody E20-5 is aa. 52 DQRVWD 57 .

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

[0083] To assess the epitope conservation of different isolates, 24 reference strains of Mycoplasma bovis 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. 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 homology analysis was performed on the identified epitopes to assess their homology. Results are shown below. Figure 7 .

[0088] Figure 7 The results showed that these two epitopes exhibited significant conservation in 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, the characteristics of the B-cell epitopes of bovine mycoplasma PDHB protein were analyzed using DNASTAR Protean software. The results are shown below. Figure 8 A.

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

[0091] To further analyze the spatial distribution characteristics of the two epitopes in the PDHB protein structure, this invention constructed a three-dimensional model of the PDHB protein using the I-TASSER online server (https: / / seq2fun.dcmb.med.umich.edu / I-TASSER / ) via homology modeling, and visualized the three-dimensional structural model and its antigenic epitopes using PyMOL software. The results are shown below. Figure 8 B and Figure 8 C.

[0092] Figure 8 B and Figure 8 The 3D model structure constructed in C++ shows that epitope aa 10 GALNHA 15 It exhibits a typical α-helix conformation, while 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 antigenic epitope to analyze antibody-antigen interactions. The ipTM index was used to assess the accuracy of predicting the relative positions of subunits in the complex. Specifically, a score higher than 0.8 indicates robust and high-quality predictions, while a score lower than 0.6 generally signifies prediction failure. Furthermore, an pTM score greater than 0.5 indicates that the predicted complex fold is generally similar to the actual structure. Results are shown in [link to results]. Figure 9 .

[0094] Figure 9 Results A showed multiple amino acid interactions between residues 10G, 12L, 13N, 14H, and 15A and the complementarity-determining region (CDR) of mAb B15-41 light and heavy chains.

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

[0096] This invention is not limited to the specific embodiments described above. Any modifications made by those skilled in the art based on the above concept without creative effort are within the scope of protection of this invention.

Claims

1. A monoclonal antibody against bovine mycoplasma PDHB protein, characterized in that, It is either monoclonal antibody B15-41 or monoclonal antibody E20-5; The monoclonal antibody B15-41 contains 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 contains 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. The application of the monoclonal antibody against bovine mycoplasma PDHB protein as described in claim 1 in identifying B-cell epitopes of PDHB protein, characterized in that, When the monoclonal antibody against bovine mycoplasma PDHB protein is monoclonal antibody B15-41, the amino acid sequence of the B cell epitope of the PDHB protein identified by it is GALNHA; when the monoclonal antibody against bovine mycoplasma PDHB protein is monoclonal antibody E20-5, the amino acid sequence of the B cell epitope of the PDHB protein identified by it is DQRVWD.

3. The application of the monoclonal antibody against bovine mycoplasma PDHB protein as described in claim 1 in the preparation of drugs for diagnosing bovine mycoplasma infection.

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