Monoclonal antibody of african swine fever virus b602l protein, antigen epitope and application

Abstract

This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody, antigenic epitope, and applications of African swine fever virus (ASFV) B602L protein. The monoclonal antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:4, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:6. This invention provides a monoclonal antibody for ASFV B602L protein, which specifically recognizes the target antigen B602L protein. The antigenic epitope specifically recognized by this monoclonal antibody has been successfully identified, providing a clear target for vaccine and diagnostic reagent development. The monoclonal antibody and antigenic epitope peptide of this invention can not only be used for vaccine development but also for preparing highly sensitive detection kits, enabling rapid and accurate diagnosis of ASFV infection. Furthermore, the product can also be used for the prevention and treatment of ASFV infection, showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody, antigen table, and application of African swine fever virus B602L protein. Background Technology

[0002] African swine fever virus (ASFV) is a widespread hemorrhagic and highly contagious disease of pigs, with a mortality rate as high as 100% in the most acute and acute forms. Since its first report in 1921, ASF has primarily been prevalent in parts of sub-Saharan Africa. An outbreak of ASF occurred in Georgia in 2007, and subsequently spread rapidly throughout the Caucasus and Russia. In 2014, ASF was introduced into most Eastern European countries and gradually showed a trend of expansion. my country has listed ASF as a Class A animal infectious disease requiring priority prevention and control, and the World Organisation for Animal Health (OIE) has listed it as a reportable animal disease. Currently, there is no commercially available vaccine for the effective control of ASF. my country experienced its first ASF outbreak in 2018, followed by 69 outbreaks in 18 provinces within three months, resulting in the culling of over 500,000 pigs and direct economic losses exceeding several billion yuan.

[0003] Due to the high infectivity of ASF and the lack of effective vaccines and drugs, current prevention and control mainly rely on rapid diagnosis, making early and accurate testing particularly important. Summary of the Invention

[0004] Given the current lack of effective measures to prevent African swine fever virus, early diagnosis and control remain the most important preventative strategies. The purpose of this invention is to provide a monoclonal antibody against African swine fever virus, its antigen table, and its applications.

[0005] The specific solution of the present invention is as follows: A monoclonal antibody against the African swine fever virus B602L protein, the monoclonal antibody comprising a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 4; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 6.

[0006] The heavy chain type of the monoclonal antibody is IgG1, and the light chain type is Lambda.

[0007] A polynucleotide encoding the aforementioned monoclonal antibody.

[0008] An antigenic epitope peptide specifically recognized by the above-mentioned monoclonal antibody, characterized in that the amino acid sequence of the antigenic epitope peptide is KYKDQIF.

[0009] The above-mentioned monoclonal antibody is used in the preparation of products for detecting African swine fever virus infection.

[0010] Based on the application of the aforementioned antigenic epitope peptides in products for the prevention of African swine fever virus infection.

[0011] Compared to existing technologies, the advantages of this invention are as follows: This invention provides a monoclonal antibody against the African swine fever virus (ASFV) B602L protein. This monoclonal antibody specifically recognizes the target antigen B602L protein, and the antigenic epitope specifically recognized by this monoclonal antibody has been successfully identified. This provides a clear target for the development of vaccines and diagnostic reagents. The monoclonal antibody and antigenic epitope peptide of this invention can not only be used for vaccine development but also for the preparation of highly sensitive detection kits, enabling rapid and accurate diagnosis of ASFV infection. Furthermore, the product can also be used for the prevention and treatment of ASFV infection, showing broad application prospects. Attached Figure Description

[0012] Figure 1 The image shows the expression identification results of recombinant B602L protein. In the image, A is the identification by Coomassie Brilliant Blue staining; B is the identification by Western blot using ASFV positive serum as the primary antibody; C is the identification by Western blot using His-tag as the primary antibody; M is the marker; 1 is the supernatant of uninduced bacterial culture; 2 is the precipitate of uninduced bacterial culture; 3 is the supernatant after disruption; and 4 is the precipitate after disruption.

[0013] Figure 2 The image shows the SDS-PAGE results of the recombinant B602L protein, where M is the protein marker and 1 is the purified recombinant B602L protein.

[0014] Figure 3 The figure shows the serum titer of Balb / C mice after immunizing them with purified B602L protein using indirect ELISA.

[0015] Figure 4 The figure shows the Western blot results for the expression of the recombinant p3×FLAG-CMV-14-B602L eukaryotic plasmid.

[0016] Figure 5 This is a graph showing the Western blot validation results of monoclonal antibody mAb 7A10.

[0017] Figure 6 The image shows the IFA identification results of monoclonal antibody mAb 7A10.

[0018] Figure 7This is a diagram showing the subtype identification results of monoclonal antibody mAb 7A10.

[0019] Figure 8 The image shows the amplification results of the variable region gene of monoclonal antibody mAb 7A10, where M is the marker; 1 is the VH PCR product; and 2 is the VL PCR product.

[0020] Figure 9 The image shows the titer test results for monoclonal antibody mAb 7A10.

[0021] Figure 10 The image shows the purification results of monoclonal antibody mAb 7A10, where M is the marker; 1 is the stock solution; 2 is the flow-through solution; 3 is the equilibration solution; 4 is the washing solution; and 5 is the elution solution.

[0022] Figure 11 The figures show the Western blot validation results of recombinant proteins B602L-B1-GS, B602L-B2-GST, B602L-B3-GST, and B602L-B4-GST. In the figures, A represents GST-Tag as the primary antibody and HRP-goat anti-mouse as the secondary antibody; B represents monoclonal antibody as the primary antibody and HRP-goat anti-mouse as the secondary antibody; M represents the marker; 1 represents GST; 2 represents B602L-B1-GST; 3 represents B602L-B2-GST; 4 represents B602L-B3-GST; and 5 represents B602L-B4-GST.

[0023] Figure 12 The figures show the Western blot validation results of recombinant proteins B602L-C1-GST and B602L-C2-GST. In the figure, A is GST-Tag as the primary antibody and HRP-goat anti-mouse as the secondary antibody; B is monoclonal antibody as the primary antibody and HRP-goat anti-mouse as the secondary antibody; M is marker; 1 is GST; 2 is B602L-C1-GST; and 3 is B602L-C2-GST.

[0024] Figure 13 The image shows the Western blot validation results of recombinant proteins B602L-D1-GST, B602L-D2-GST, and B602L-D3-GST. In the image, A represents GST-Tag as the primary antibody and HRP-goat anti-mouse as the secondary antibody; B represents a monoclonal antibody as the primary antibody and HRP-goat anti-mouse as the secondary antibody; M represents the marker; 1 represents GST; 2 represents B602L-D1-GST; 3 represents B602L-D2-GST; and 4 represents B602L-D3-GST.

[0025] Figure 14The figures show the Western blot validation results of recombinant proteins B602L-E1-GST, B602L-E2-GST, B602L-E3-GST, and B602L-E4-GST. In the figure, A is the primary antibody with GST-Tag and the secondary antibody with HRP-goat anti-mouse antibody; B is the primary antibody with monoclonal antibody and the secondary antibody with HRP-goat anti-mouse antibody; M is the marker; 1 is GST; 2 is B602L-E1-GST; 3 is B602L-E2-GST; 4 is B602L-E3-GST; and 5 is B602L-E4-GST.

[0026] Figure 15 The image shows the Western blot validation results for recombinant proteins B602L-F1-GST, B602L-F2-GST, B602L-F3-GST, and B602L-F4-GST. In the image, A represents GST-Tag as the primary antibody and HRP-goat anti-mouse as the secondary antibody; B represents a monoclonal antibody as the primary antibody and HRP-goat anti-mouse as the secondary antibody; M represents the marker; 1 represents GST; 2 represents B602L-F1-GST; 3 represents B602L-F2-GST; 4 represents B602L-F3-GST; and 5 represents B602L-F4-GST.

[0027] Figure 16 A schematic diagram of the procedure for identifying the B-cell epitope of ASFV B602L protein.

[0028] Figure 17 The diagram shows the physicochemical properties of ASFV B602L protein.

[0029] Figure 18 A molecular docking model for ASFV B602L protein and monoclonal antibody mAb7A10. Detailed Implementation

[0030] These are examples, not all examples. All other examples obtained by those skilled in the art based on the examples in this invention without inventive effort are within the scope of protection of this invention. It should be noted that any processes not specifically described below are implementations or understandings that can be made or understood by those skilled in the art by referring to existing technology. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0031] I. Prokaryotic Expression and Purification of ASFV B602L Protein The ASFV isolate *African swine fever virus isolate China / 2018 / AnhuiXCGQ* was downloaded from GenBank of the National Center for Biotechnology Information (NCBI), and the complete genome (GenBank: MK128995.1) was obtained, with a full-length genome of 189,393 bp. The B602L gene sequence (GenBank: AYW34051.1) is 1593 bp long and contains 530 amino acids.

[0032] To improve protein translation efficiency and expression levels, the base codons of the ASFV B602L gene were optimized using GenSmart™ Codon Optimization, an online codon optimization tool from GenScript Biotech. Based on the codon preference of E. coli, synonymous codon substitutions were performed on rare codons and a small number of tandem rare codons without changing the amino acids, resulting in the codon-optimized base sequence shown in SEQ ID NO: 1.

[0033] SEQ ID NO: 1: Based on the codon-optimized full-length B602L base sequence, N-termini and C-termini were introduced respectively. deI (CATATG) and X hoI The two restriction enzyme sites (CTCGAG) were synthesized by Nanjing Genscript Biotech Co., Ltd. and cloned into the pET-28a(+) vector to obtain the recombinant pET28a-B602L plasmid. The recombinant pET28a-B602L plasmid was then transformed into BL21(DE3) competent cells. The transformation steps are as follows: (1) Remove BL21(DE3) competent cells from -80℃ and place them in ice for 5 min; (2) Add 100 ng pET28a-B602L plasmid and let it stand on ice for 25 min. (3) Heat shock the EP (Eppendorf) tube in a 42°C water bath for 45 seconds, then quickly insert it into ice and let it stand for 5 minutes; (4) Add 700 μL of LB medium to the EP tube and incubate at 37℃ and 200 rpm for 60 min with shaking. (5) Centrifuge at 5000 rpm for 1 min to collect the bacterial cells, discard 700 μL of supernatant, resuspend the bacterial cells and spread them on LB-Kana solid medium; (6) Invert the plate and incubate it in a 37°C incubator; (7) Pick single colonies and preserve the correctly transformed pET28a-B602L bacterial culture.

[0034] The following steps were taken to induce expression and collect samples from the correctly transformed pET28a-B602L bacterial culture: (1) Activation: Add the preserved pET28a-B602L bacterial culture at a ratio of 1:50 to LB-Kana medium and culture at 37℃ and 220 rpm with shaking. (2) Scale-up: Add the activated pET28a-B602L bacterial culture at a ratio of 1:100 to LB-Kana medium and culture at 37°C and 220 rpm with shaking. (3) Induction: OD of the bacterial culture 600 nm Once the concentration reaches 0.6-0.8, add IPTG to the bacterial culture to a final concentration of 0.1 mmol / L, and continue to incubate at 16℃ and 160 rpm for 12 h with shaking. (4) Collection: Collect the bacterial cells by centrifuging the induced bacterial culture at low speed at 4°C and washing with PBS 3 times; (5) Disruption: The bacterial suspension was disrupted by ultrasonic disruption using an ultrasonic disruptor; (6) Preservation: Centrifuge the broken suspension at 4°C to separate the supernatant and precipitate. Resuspend the precipitate with an equal volume of PBS. Store the supernatant and precipitate suspension at -20°C.

[0035] The expression of recombinant B602L protein was identified by Coomassie brilliant blue staining and Western blot analysis, respectively. The results are as follows: Figure 1 As shown, a distinct band appeared in the supernatant and precipitate of the fragmented sample between 60 and 75 kDa, consistent with the predicted size of 64 kDa, and was mainly expressed in the supernatant under the induction condition of 16℃.

[0036] To obtain high-purity recombinant B602L protein, this invention utilizes His-Tag, which can interact with Ni... 2+ Based on the principle of selective binding through coordination, soluble recombinant B602L protein was purified using a Ni affinity chromatography column (Ni Sepharose™ excel). The purification results were identified by SDS-PAGE, as shown below. Figure 2 As shown, the protein has a molecular weight of approximately 64 kDa, which is consistent with the expected size, indicating that the expressed and purified protein can be used as an antigen for immunizing mice.

[0037] II. Screening of hybridoma cells The purified B602L protein was dialyzed into physiological saline, concentrated to 0.5 μg / μL, and then mixed 1:1 with a rapid immunization adjuvant as an antigen. This mixture was used to immunize 6-8 week old female Balb / C mice via intramuscular injection in the hind leg (100 μL / mouse). A booster immunization was administered on day 14 using the same method. On day 21, a small amount of tail blood was collected for indirect ELISA to detect the mouse serum titer. The results are as follows: Figure 3 As shown, the serum antibody titers of four mice were detected by indirect ELISA, with negative mouse serum as a control. The serum antibody titers of three mice all reached 1:512000. In fact, the serum titer of mouse 1 was the highest at 1:1024000. Mouse 1 was selected for subsequent experiments.

[0038] Mice were euthanized 3 days after booster immunization, and their blood was excised through the eyeballs to collect positive serum for later use. The spleen of the mice was removed and splenic lymphocytes were isolated. Mouse myeloma cells (SP2 / 0) were mixed with splenic lymphocytes, and 1 mL of PEG-1500 was added under 37°C water bath conditions to fuse the two cell types. The fused cells were cultured in hybridoma cell culture medium containing HAT. The fused cells were evenly seeded into 96-well cell culture plates and placed in a 37°C, 5% CO2 incubator. On the 7th day after cell fusion, the old culture medium was discarded and replaced with new HT-containing culture medium for continued culture.

[0039] On day 14 after cell fusion, positive hybridoma cells were screened using an indirect ELISA method. The antigen was 100 ng B602L protein, the primary antibody was 50 μL of hybridoma cell supernatant, and the secondary antibody was HRP-goat anti-mouse. The hybridoma cells from the initially screened positive wells were expanded and cultured in serum-free hybridoma medium containing HT at 37°C in a 5% CO2 incubator.

[0040] 100 μL of the initially screened positive hybridoma cells were serially diluted in a 96-well cell culture plate containing HT medium. The cells were incubated for 30 min and counted under a microscope. When the total cell count was approximately 100, all cells from each well were extracted and added to 20 mL of fresh HT medium. The mixed cells were then added to 200 μL / well of a 96-well cell culture plate and cultured at 37°C with 5% CO2 for 7–14 days. Cell growth was observed, and subcloning was repeated until hybridoma cells capable of stably secreting a single antibody were selected and named 7A10. The monoclonal antibody secreted by hybridoma cells 7A10 was named mAb 7A10.

[0041] III. Identification of the Reactivity Characteristics of Monoclonal Antibodies Using homologous recombination technology, a recombinant B602L eukaryotic plasmid was constructed using the p3×FLAG-CMV-14 plasmid vector, named recombinant p3×FLAG-CMV-14-B602L eukaryotic plasmid. The successfully constructed recombinant p3×FLAG-CMV-14-B602L eukaryotic plasmid was transfected into HEK 293T cells. After 24 h of transfection, the cells were removed from the culture plate, the cell supernatant was discarded, 2× protein loading buffer was added, and the mixture was thoroughly stirred and then heated at 99℃ for 10 min to obtain the eukaryotic B602L protein. Western blot identification was performed using β-Actin, ASFV positive serum, and DYKDDDDK-tag as primary antibodies, and HRP-goat anti-rabbit, HRP-goat anti-pig, and HRP-goat anti-mouse as secondary antibodies, respectively. The results are as follows: Figure 4 As shown, the β-Actin detection results showed a distinct band between 35 and 50 kDa, consistent with the expected size of 42 kDa. The ASFV positive serum and DYKDDDDK-tag detection results showed a distinct band between 60 and 75 kDa, consistent with the expected size of 64 kDa. This indicates that the eukaryotic protein was successfully expressed and could specifically bind to ASFV positive serum and DYKDDDDK-Tag.

[0042] The eukaryotic B602L protein was validated by Western blot. The primary antibody was the supernatant of a hybridoma cell line 7A10 selected from subcloning, and the secondary antibody was HRP-goat anti-mouse. The results are as follows: Figure 5 As shown, the monoclonal antibody exhibits a specific reaction with the B602L protein in the range of 60–75 kDa, consistent with the expected size of 64 kDa, indicating that the monoclonal antibody can specifically react with the B602L protein.

[0043] The successfully constructed recombinant p3×FLAG-CMV-14-B602L eukaryotic plasmid was transfected into HEK 293T cells. After 24 h, transfected cells were harvested, the cell supernatant was discarded, and the cells were washed three times with PBS before IFA identification. Hybridoma cell supernatant and DYKDDDDK-Tag were used as primary antibodies, and FITC-goat anti-mouse was used as a secondary antibody. Results were observed under a fluorescence microscope, with DYKDDDDK-Tag as a positive control and HEK S93T cell supernatant as a negative control. The results are as follows: Figure 6 As shown, the results of monoclonal antibody mAb7A10 and DYKDDDDK-Tag are consistent, both of which can produce specific reactions.

[0044] IV. Sequencing and Subtype Identification of Monoclonal Antibodies The heavy chain and light chain isotypes of monoclonal antibodies in hybridoma cells 7A10 were identified using a mouse monoclonal antibody isotype identification kit (Wuhan Sanying Biotechnology Co., Ltd.). OD values ​​were measured using a microplate reader. 450 nm The result is as follows Figure 4-14 As shown, the heavy chain type of the monoclonal antibody is IgG1, and the light chain type is Lambda. The results are as follows: Figure 7 As shown.

[0045] RNA was extracted from hybridoma cells 7A10, reverse transcribed into cDNA, and the target sequence was amplified using primers VH-F mix (VL-F) and VH-R mix (VL-R) (Takara). The results are as follows. Figure 8 As shown, VH and VL each have a band between 250 and 500 bp.

[0046] The amplified target sequences VH and VL were ligated into the pMD™19-T Vector and the resulting plasmid was transformed into DH5α competent cells. Single colonies were selected for sequencing.

[0047] The monoclonal antibody includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 2; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 3; the monoclonal antibody includes a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO: 4; and a light chain variable region nucleotide sequence as shown in SEQ ID NO: 5.

[0048] SEQ ID NO: 2 QVKLEQSGAELARPGASVKLSCKASGYTFTRYWMQWVKQRPGQGLEWIGAVYPGDGDTLYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARRAQVGRGWYFDVWGAGTTVTVSSAKTTPPSVYNL SEQ ID NO: 3 CAGGTGAAGCTGGAGCAGTCTGGGGCTGAGCTGGCAAGACCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGATACTGGATGCAGTGGGTTAAACAGAGGCCTGGACAGGGTTCTGGAATGGATTGGGGCTGTTTATCCTGGAGATGGTGATACTCTTTACACTCAGAAGTTCAAGGGC AAGGCCACATTGACTGCAGATAAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCTTGGCATCTGAGGACTCTGCGGTCTATTACTGTGCCAGACGGGCCCAAGTGGGACGCGGCTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATAATCTC SEQ ID NO: 4 DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK SEQ ID NO: 5 GACATTTGTGATGACACAGTCTCCAGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTT GTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAA V. Preparation and Detection of Monoclonal Antibodies Female Balb / C mice aged 8-12 weeks were injected intraperitoneally with liquid paraffin (500 μL / mouse). Seven days later, the selected positive hybridoma cells were centrifuged, the cell supernatant was discarded, and the cells were resuspended in PBS for viable cell counting. Mice in good condition were then injected intraperitoneally with 1 × 10⁻⁶ ppm of liquid paraffin. 6 ~5×10 6 A number of positive hybridoma cells were collected. The mice were observed for 7-14 days. Ascites fluid was extracted from the peritoneal cavity of the mice, the supernatant was collected by centrifugation, dialyzed into PBS, and stored at -20°C.

[0049] The titer of the prepared monoclonal antibody was determined using an indirect ELISA method. The coating antigen was 100 ng of B602L protein, the primary antibody was a serially diluted monoclonal antibody, and the secondary antibody was HRP-goat anti-mouse antibody. The OD values ​​were read using a microplate reader. 450 nm Numerical values ​​were obtained, with negative mouse serum serving as a negative control. The results were as follows: Figure 9 As shown, the monoclonal antibody titer reached 1:2048000.

[0050] The monoclonal antibody was dialyzed into a equilibration buffer and then purified using an antibody purification column. The purification steps for the monoclonal antibody are as follows: (1) Cleaning the column: Clean 5 column volumes with ddH2O; (2) Equilibrium column: Equilibrium the volumes of 5 columns with equilibrium fluid; (3) Antibody loading: Add the prepared monoclonal antibody sample to the gravity chamber and cycle the loading 5 times; (4) Antibody washing: Add 10-15 column volumes of washing buffer; (5) Antibody elution: Add 5-10 column volumes of elution buffer; (6) Antibody neutralization: The elution buffer needs to be adjusted to neutral immediately, and neutralization is performed using a neutralization solution with a volume of 1 / 10 of the elution buffer. (7) Column cleaning: Clean 5 column volumes with equilibration solution and then clean 5 column volumes with ddH2O; (8) Column preservation: Seal the column with 20% ethanol and store at 4°C; (9) Sample preservation: Dialyze the purified monoclonal antibody into PBS and store at -20°C. The purified results are as follows: Figure 10 As shown, the eluent is the purified monoclonal antibody, with distinct bands at 45-60 kDa and around 25 kDa, consistent with the antibody heavy chain size of 55 kDa and the light chain size of 25 kDa, indicating successful purification of the monoclonal antibody.

[0051] VI. Identification of B-cell epitopes of ASFV B602L protein (I) Preliminary localization: The full-length B602L sequence was truncated, and two suitable restriction enzyme sites were selected in the multiple cloning site (MCS) region of the pGEX-5X-2 plasmid. Based on the truncated B602L sequence and the two restriction enzyme sites B... amHI (GGATCC) and E coRI (GAATTC) designed primers with homologous arms (Table 1).

[0052] Table 1 PCR Primers

[0053] First localization: The target sequence was amplified using PCR; restriction endonuclease B was used. amHI and E coRI The vector plasmid was digested with two enzymes; the sequences B602L-B1, B602L-B2, B602L-B3, and B602L-B4 with homologous arms were amplified by PCR. The target fragments and the vector were ligated using homologous recombination ligase to obtain recombinant plasmids pGEX-5X-2-B602L-B1, pGEX-5X-2-B602L-B2, pGEX-5X-2-B602L-B3, and pGEX-5X-2-B602L-B4. These recombinant plasmids were transformed into BL21(DE3) competent cells for induced expression. After collection, Western blot analysis was performed using GST-Tag and the purified monoclonal antibody as primary antibodies, and HRP-goat anti-mouse antibody as secondary antibody. The results are as follows: Figure 11As shown, the GST-Tag detection results showed a clear band between 45 and 60 kDa, consistent with the expected size of 48 kDa, indicating successful expression of recombinant proteins B602L-B1-GS, B602L-B2-GST, B602L-B3-GST, and B602L-B4-GST. The B602L protein monoclonal antibody mAb 7A10 detection results showed that B602L protein monoclonal antibody mAb 7A10 only reacted with B602L-B3-GST, indicating that the B cell epitope is located at aa236-412.

[0054] Second localization: The truncated recombinant plasmids pGEX-5X-2-B602L-C1 and pGEX-5X-2-B602L-C2 were constructed and transformed into BL21(DE3) competent cells for induced expression. After sample collection, Western blot analysis was performed using GST-Tag and the purified monoclonal antibody as primary antibodies, and HRP-goat anti-mouse antibody as secondary antibody. The results are as follows: Figure 12 As shown, the GST-Tag detection results showed a clear band between 25 and 35 kDa, consistent with the expected size of 32 kDa, indicating successful expression of recombinant proteins B602L-C1-GST and B602L-C2-GST. The B602L protein monoclonal antibody detection results showed that the B602L protein monoclonal antibody only reacted with B602L-C2-GST, indicating that the B cell epitope is located at aa315-353.

[0055] Precise localization: The recombinant protein identified by the monoclonal antibody mAb7A10 was further truncated and analyzed. Using point mutation technology, the target sequence was mutated in the MCS of the pGEX-5X-2 plasmid using PCR. Primer design is shown in Table 2.

[0056] Table 2 PCR Primers

[0057] Third localization: To improve the purity of the recombinant plasmids, after site-directed mutagenesis, it is necessary to distinguish between the unmutated original plasmid and the mutated target DNA. The original plasmid containing methylated template was removed using DpnI enzyme, while the unmethylated mutant plasmid was retained, yielding recombinant plasmids pGEX-5X-2-B602L-D1, pGEX-5X-2-B602L-D2, and pGEX-5X-2-B602L-D3. These recombinant plasmids were then transformed into BL21(DE3) competent cells for induced expression. After sample collection, Western blot analysis was performed using GST-Tag and the purified monoclonal antibody as primary antibodies, and HRP-goat anti-mouse antibody as secondary antibodies. The results are as follows: Figure 13As shown, the GST-Tag detection results showed a clear band between 25 and 35 kDa, consistent with the expected size of 29 kDa, indicating successful expression of recombinant proteins B602L-D1-GST, B602L-D2-GST, and B602L-D3-GST. The B602L protein monoclonal antibody detection results showed that the B602L protein monoclonal antibody could react with B602L-D2-GST and B602L-D3-GST, indicating that the B cell epitope is located at aa339-348.

[0058] Fourth localization: The original plasmid containing the methylated template was removed using DpnI enzyme, and the unmethylated mutant plasmid was retained to obtain recombinant plasmids pGEX-5X-2-B602L-E1, pGEX-5X-2-B602L-E2, pGEX-5X-2-B602L-E3, and pGEX-5X-2-B602L-E4. These recombinant plasmids were transformed into BL21(DE3) competent cells for induced expression. After sample collection, Western blot analysis was performed using GST-Tag and the purified monoclonal antibody as primary antibodies, and HRP-goat anti-mouse antibody as secondary antibody. The results are as follows: Figure 14 As shown, the GST-Tag detection results revealed a distinct band between 25 and 35 kDa, consistent with the expected size of 28 kDa, indicating successful expression of recombinant proteins B602L-E1-GST, B602L-E2-GST, B602L-E3-GST, and B602L-E4-GST. The B602L protein monoclonal antibody detection results showed that the B602L protein monoclonal antibody could react with B602L-E3-GST and B602L-E4-GST, indicating that the B cell epitope is located at aa341-348.

[0059] Fifth localization: The original plasmid containing the methylated template was removed using DpnI enzyme, and the unmethylated mutant plasmid was retained to obtain recombinant plasmids pGEX-5X-2-B602L-F1, pGEX-5X-2-B602L-F2, pGEX-5X-2-B602L-F3, and pGEX-5X-2-B602L-F4. These recombinant plasmids were transformed into BL21(DE3) competent cells for induced expression. After sample collection, Western blot analysis was performed using GST-Tag and the purified monoclonal antibody as primary antibodies, and HRP-goat anti-mouse antibody as secondary antibody. The results are as follows: Figure 15As shown, the GST-Tag detection results revealed a distinct band between 25 and 35 kDa, consistent with the expected size of 28 kDa, indicating successful expression of recombinant proteins B602L-F1-GST, B602L-F2-GST, B602L-F3-GST, and B602L-F4-GST. The B602L protein monoclonal antibody detection results showed that the B602L protein monoclonal antibody reacted only with B602L-F1-GST, indicating that the epitope is located at aa341-347, meaning the final B-cell epitope of the B602L protein is located at aa341-347. 341 KYKDQIF 347 The amino acid sequence KYKDQIF is the antigenic epitope peptide specifically recognized by monoclonal antibody mAb 7A10. The identification procedure is as follows: Figure 16 As shown.

[0060] SEQ ID NO: 40 KYKDQIF The secondary structure (α-helix, β-sheet, β-turn, Ω-loop, and random coil), surface residue hydrophilicity / hydrophobicity, and antigenic index of the identified ASFV B602L protein monoclonal antibody 7A10 were analyzed using the Protein software of DNA Star. The results are as follows: Figure 17 (The dashed box indicates the antigenic epitope) shows the B-cell epitope recognized by monoclonal antibody 7A10. 341 KYKDQIF 347 It has high hydrophilicity and high antigenic index, and is most likely located on the protein surface.

[0061] The monoclonal antibodies VH and VL of the ASFV B602L protein were modeled using AlphaFold Serve (https: / / deepmind.google / technologies / alphafold / alphafold-server / ). The interaction between the B602L protein and the monoclonal antibodies VH and VL was analyzed using PyMOL. The results are as follows: Figure 18 As shown, the green portion represents the ASFVB602L protein, the red portion represents B-cell epitopes, and the blue portion represents the monoclonal antibody VH and VL complex. The epitope amino acids and the VH and VL complexes achieve precise regulation of structure and function through the synergy of hydrogen bonds, electrostatic interactions, van der Waals forces, and hydrophobic interactions, resulting in highly efficient adaptation between biomolecules. The spatial distribution and intensity differences of these interactions can provide a theoretical basis for drug design (such as targeting hydrogen bond networks with small molecule inhibitors) or protein engineering (optimizing salt bridges to enhance thermal stability).

[0062] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the technical solutions and core ideas of this application. Those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A monoclonal antibody against the African swine fever virus B602L protein, characterized in that, The monoclonal antibody includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 4; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

6.

2. The monoclonal antibody against African swine fever virus B602L protein according to claim 1, characterized in that, The heavy chain type of the monoclonal antibody is IgG1, and the light chain type is Lambda.

3. A polynucleotide encoding the monoclonal antibody of claim 1 or claim 2.

4. An antigenic epitope peptide specifically recognized by a monoclonal antibody according to claim 1 or claim 2, characterized in that, The amino acid sequence of the antigenic epitope peptide is KYKDQIF.

5. The use of the monoclonal antibody according to claim 1 or claim 2 in the preparation of products for detecting African swine fever virus infection.

6. The use of the antigenic epitope peptide according to claim 4 in products for the prevention of African swine fever virus infection.

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