ASFV p49 protein linear epitope peptide, its specific antibody, hybridoma cells and their applications

Abstract

This application discloses an ASFV p49 protein linear epitope peptide, its specific antibody, hybridoma cells, and their applications, aiming to solve the technical problems of imperfect and incomplete ASFV serotype detection, diagnosis, prevention, and treatment reagents or drugs. This invention identifies an ASFV p49 linear B-cell epitope: 333 YQTHYMENIVTLVPR 347 and / or 383 NNYIPKYTGGIGDSK 397 The study induced the production of monoclonal antibodies that specifically recognize this epitope. This linear B-cell epitope enriches the immunological function of the ASFV p49 protein, providing a reference for subsequent research on the structural and antigenic properties of the p49 protein, and can be applied to the development of antiviral drugs and vaccines.

Description

Technical Field

[0001] This invention application relates to the field of molecular immunology, specifically to the linear epitope peptide of ASFV p49 protein, its specific antibody, hybridoma cells, and their applications. Background Technology

[0002] African swine fever (African swine fever) African swine fever ASF is caused by the African swine fever virus (ASF). African swine fever virus African swine fever (ASF) is a highly contagious, hemorrhagic, and fatal infectious disease in pigs. Morbidity and mortality rates can reach 100%. The World Organisation for Animal Health (OIE) lists it as a notifiable animal disease. As there are currently no effective treatments for African swine fever, its spread and prevention remain a global concern.

[0003] ASFV is a double-stranded DNA virus belonging to the African swine fever virus family (ASFV). Asfarviridae African swine fever virus (ASV) genus ( Asfarvirus The only member of the ASFV family. The genome size of different ASFV strains varies, ranging from 170 to 190 kbp, containing more than 150 open reading frames encoding multiple proteins. Currently, there are 24 known ASFV genotypes and 8 serogroups globally. African swine fever virus particles contain approximately 70 different polypeptide segments, including various structural proteins and various enzymes and factors involved in viral transcription.

[0004] The p49 protein is a structural protein encoded by the B438L gene, located within the EcoRI B genome segment of the ASFV BA71V strain. It is approximately 49.3 kDa in size, expressed late post-infection, and contains no transmembrane domains with post-translational modifications. As a capsid structural protein, p49 participates in the late-stage viral assembly process and is located at the viral particle assembly site. In the absence of p49, viral particles form tubular structures and lose their icosahedral symmetry, indicating that p49 plays a crucial role in capsid assembly. Therefore, it holds promise as a target protein for vaccine development. The preparation of monoclonal antibodies against the ASFV p49 protein and the study of its B-cell epitopes can provide important technical support for the research and application of ASF-related diagnostic reagents, preventive and therapeutic drugs, and lay the foundation for protein structure analysis and epitope vaccine preparation.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] Based on the shortcomings of existing research and practical needs, this application aims to provide a linear B-cell epitope of ASFV p49 protein and induce the production of specific neutralizing antibodies, so as to establish and improve the identification method of ASFV serotypes and provide technical support for the preparation and research of ASF-related diagnostic reagents, preventive and therapeutic drugs.

[0007] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0008] Based on long-term and extensive experimental research, a linear B-cell epitope of ASFV p49 was identified: 333 YQTHYMENIVTLVPR 347 and / or 383 NNYIPKYTGGIGDSK 397 .

[0009] In this application, mice were immunized with ASFV p49 protein expressed in a prokaryotic system. Using cell fusion technology and ASFV p49 protein as a detection source, monoclonal cell lines resistant to ASFV p49 protein were screened by indirect ELISA and named 3B12, 6F1 and 7C5, respectively.

[0010] The monoclonal antibodies produced by the screened monoclonal cell lines are able to specifically recognize and bind to the ASFVp49 protein. 333 YQTHYMENIVTLVPR 347 and 383 NNYIPKYTGGIGDSK 397 Sequence region.

[0011] This application uses the expressed ASFV p49 protein as an immunogen to immunize mice, and the serum titer after immunization, as determined by indirect ELISA, can reach 1:102400. The prepared monoclonal antibodies 3B12, 6F1, and 7C5 have titers as high as 1:128000, 1:128000, and 1:1024000, respectively, and exhibit good specificity.

[0012] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0013] 1. This application comprehensively utilizes molecular biology, virology, and immunology technologies to screen and identify a linear B-cell epitope of the ASFV p49 protein, providing technical support for the application of the ASFV p49 protein in vaccine development; and further utilizes three monoclonal antibodies prepared by induction to recognize the polypeptide, p49 protein, and inactivated ASF virus containing the linear B-cell epitope of the ASFV p49 protein, indicating that the linear B-cell epitope of the ASFV p49 protein can be specifically recognized.

[0014] 2. The linear B-cell epitopes of the ASFV p49 protein identified in this application enrich the immunological function of the ASFV p49 protein, providing a reference for subsequent research on the structural and antigenic characteristics of the p49 protein, and can be applied to the development of antiviral drugs.

[0015] 3. The monoclonal antibodies produced by the selected monoclonal cell lines can specifically recognize and bind to the ASFV p49 protein. 333 YQTHYMENIVTLVPR 347 and 383 NNYIPKYTGGIGDSK 397 Sequence region.

[0016] 4. The monoclonal antibodies induced and prepared can specifically recognize ASF inactivated virus, providing a new technical means for the differential diagnosis of ASFV. Attached Figure Description

[0017] Figure 1 SDS-PAGE identification is performed in one embodiment of this application; where M is a marker; 1-3 are whole bacteria before induction, sonicated supernatant after induction, and sonicated precipitate after induction, respectively.

[0018] Figure 2 This is a Western Blot identification in one embodiment of this application; where M is a marker; 1 is an empty vector protein; and 2 is the whole bacteria after induction.

[0019] Figure 3 This is an example of SDS-PAGE identification in one embodiment of this application; where M is a marker; 1-2 are the target protein before purification and after refolding, respectively.

[0020] Figure 4 This is a Western Blot identification in one embodiment of this application; where M is a marker; and 1 is the reaction of the target protein with positive porcine serum.

[0021] Figure 5 The serum titer of mice immunized with p49 recombinant protein before fusion in one embodiment of this application.

[0022] Figure 6 In one embodiment of this application, an indirect ELISA screening method was used to select positive hybridoma cell lines after fusion.

[0023] Figure 7 In one embodiment of this application, an IFA experiment was conducted to verify the specific binding of ASFV p49 protein to monoclonal antibodies 3B12, 6F1, and 7C5.

[0024] Figure 8The ascites titers of the 3B12, 6F1 and 7C5 monoclonal antibodies prepared in one embodiment of this application.

[0025] Figure 9 This is a schematic diagram of the truncated full-length ASFV p49 protein in one embodiment of this application.

[0026] Figure 10 The results of Peptide-ELISA and Dot-ELISA experiments of monoclonal antibodies 3B12, 6F1 and 7C5 with designed and synthesized polypeptide fragments (P1-P10) in one embodiment of this application are shown.

[0027] Figure 11 The results of Peptide-ELISA and Dot-ELISA experiments on epitope peptides P1 and P6 screened in one embodiment of this application with positive porcine serum are shown. Detailed Implementation

[0028] The specific embodiments of the present invention will be described below with reference to the accompanying drawings and examples. However, the following examples are only used to illustrate the present invention in detail and do not limit the scope of the present invention in any way.

[0029] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; and the test methods involved are all conventional methods unless otherwise specified.

[0030] Example 1: Immunogen Preparation

[0031] The ASFV p49 protein is located in the "virus factory" and can generate an immune response in animals. Therefore, the full-length sequence of the ASFV p49 protein was expressed using a prokaryotic expression system.

[0032] 1.1 Primer Design

[0033] Based on the gene sequence information of ASFV p49 protein on NCBI (http: / / www.ncbi.nlm.nih.gov) (GenBank accession No. QIE07029.1), primers were designed using SnapGene software, and selected... Bsa I and Xho I represents the restriction enzyme site (underlined part), and protective bases are added (italic part) to amplify the ASFV p49 gene. Primer sequences are shown in Table 1.

[0034] Table 1 Primer sequence listing

[0035] .

[0036] 1.2 PCR amplification of the p49 gene

[0037] The synthesized ASFV p49 gene sequence was used as a template for PCR amplification. The PCR amplification system is shown in Table 2.

[0038] Table 2 PCR amplification system

[0039] .

[0040] 1.3 Purification and recovery of PCR products

[0041] After the target gene amplification was completed, the PCR amplification products were verified by 1% agarose gel electrophoresis. The amplification products at the target band position were excised and recovered using a DNA purification and recovery kit. The concentration of the recovered DNA solution was determined using Nano Drop, and the solution was labeled and stored at -20℃ for later use.

[0042] 1.4 Double enzyme digestion of the target gene and vector

[0043] (1) The p49 gene / pE-SUMO vector was double-digested with enzymes. The enzyme digestion reaction system is shown in Table 3.

[0044] Table 3. Double enzyme digestion reaction system

[0045] .

[0046] (2) After the components are mixed evenly, they are placed in a metal bath at 37°C for double enzyme digestion for 3 h.

[0047] 1.5 Recovery of Enzyme Digestion Products

[0048] The double-digested products were recovered using a DNA purification and recovery kit. 1 µL of the recovered product was taken to determine the concentration of the recovered DNA, labeled, and stored at -20℃ for later use.

[0049] 1.6 Construction of recombinant plasmids

[0050] (1) Ligation of p49 gene with pE-SUMO vector

[0051] Add the following components to a 200 µL EP tube:

[0052] Table 4 Connection System

[0053] .

[0054] After mixing the above solution in the centrifuge tube, place it in a 16°C connection instrument for overnight connection.

[0055] (2) Transformation: In a clean bench, 10 μL of the ligation product was added to 100 μL of DH5α competent cells, gently mixed, and incubated on ice for 40 min. After incubation, the centrifuge tube was heat-shocked in a 42℃ metal bath for 90 s, then incubated in an ice-water mixture for 4 min. 900 µL of LB liquid medium was added to the centrifuge tube, and the mixture was incubated at 37℃, 220 r / min on a shaker for 40 min, followed by centrifugation at 8000 rpm for 2 min. After centrifugation, the supernatant was discarded, and the mixture was resuspended in 100 µL of LB liquid medium. 50 µL of the resuspended mixture was then spread onto LB solid medium (ampicillin-resistant) and incubated overnight in a 37℃ incubator.

[0056] (3) Screening positive clones by bacterial culture PCR: Select a smooth single colony and inoculate it into LB liquid medium (ampicillin resistant). Incubate it on a shaker at 37°C and 220 r / min until it becomes turbid. Use the turbid bacterial culture as a template for bacterial culture PCR for verification.

[0057] Add the components listed in Table 5 to a 200 µL EP tube.

[0058] Table 5. Bacterial PCR Reaction System

[0059] .

[0060] After mixing all components thoroughly, the mixture was placed in a PCR instrument for PCR amplification. After the bacterial culture PCR was completed, the products were identified using a 1% agarose gel, and single-clone strains whose band positions matched the target gene positions were selected and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0061] (4) Extraction of recombinant plasmid: Selected positive strains that were successfully sequenced were inoculated into 20 mL LB liquid medium (ampicillin resistant) and cultured overnight at 37°C and 220 rpm. The pE-SUMO-p49 recombinant plasmid was extracted using the Tiangen plasmid mini-prep kit according to the kit instructions.

[0062] 1.7 Expression, purification, and identification of the target protein

[0063] (1) Expression of the target protein

[0064] a. Transform the pE-SUMO-p49 recombinant plasmid into Escherichia coli Rosetta competent cells to construct the pE-SUMO-p49-Rosetta expression bacterium.

[0065] b. The constructed pE-SUMO-p49-Rosetta expression bacteria were inoculated at a ratio of 1:100 into 80 mL of LB liquid medium (ampicillin resistant) for secondary activity.

[0066] c. After inoculation, incubate at 37°C and 220 rpm for approximately 2 hours on a shaker. When the OD... 600 When the pH value is between 0.6 and 0.8, add the inducing agent IPTG (0.2 mmol / L) and continue culturing at 220 rpm in a shaker at 16°C until the next day.

[0067] d. The next day, the bacteria were collected and resuspended in cell disruption buffer (20 mmol / L Tris, 100 mmol / L NaCl, pH 8.0) for ultrasonic disruption.

[0068] e. After ultrasonic disruption, centrifuge at 12000 rpm for 10 min at 4℃. Take 40 μL of the ultrasonic supernatant and precipitate respectively, add 10 μL of 5×SDS loading buffer, boil for 10 min, and then perform SDS-PAGE and Western Blot analysis. Figure 1 , Figure 2 As shown, the p49 recombinant protein is expressed in the form of inclusion bodies.

[0069] (2) Purification and identification of the target protein

[0070] After sonication, the sonicated precipitate was collected and washed three times with washing buffer (20 mmol / L Tris, 100 mmol / L NaCl, 2 mol / L Urea, pH 8.0), stirring for 30 min at room temperature each time, followed by centrifugation at 12000 rpm for 10 min at 4°C. Finally, dissolution buffer (20 mmol / L Tris, 100 mmol / L NaCl, 8 mol / L Urea, pH 8.0) was added to the precipitate, and the mixture was stirred at room temperature for 30 min before being stored at 4°C. The next day, the solution was removed, centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was separated. Purification was performed using a nickel affinity chromatography column. Detailed purification steps are as follows:

[0071] a. Equilibrium Ni packing: Rinse the affinity chromatography column with inclusion body denaturing solution; the rinse flow-through buffer can be recycled.

[0072] b. binding of p49 recombinant protein with Ni material: After column equilibration, the denatured supernatant was filtered through a 0.45µm filter and passed through the column at a rate of 1 mL / min. The column was repeated twice, and the column was washed with inclusion body denaturation buffer.

[0073] c. Protein elution: The chromatography column was washed stepwise with inclusion body denaturation buffer containing imidazole at concentrations of 20, 50, 100, 200, and 500 mmol / L, respectively. Each gradient of elution was indicated with a protein indicator. When the protein indicator no longer changed color, the next gradient was replaced.

[0074] d. The purified and collected samples were analyzed by SDS-PAGE.

[0075] e. The purified target protein was subjected to a low-temperature gradient dialyze in inclusion body refolding buffer (20 mmol / L Tris, 100 mmol / L NaCl, pH 8.0) containing different concentrations of urea (6, 3, 1.5, 0 mol / L), followed by low-temperature dialyze in a buffer containing 20 mmol / L Tris and 150 mmol / L NaCl to ensure adequate refolding of the target protein. After refolding, the protein was identified by SDS-PAGE and Western Blot. Figure 3 , Figure 4 As shown, the concentration of p49 recombinant protein after purification reached over 90% and reacted with positive porcine serum.

[0076] Example 2: Analysis of mouse immunization and its effects using p49 recombinant protein

[0077] 2.1 Preliminary evaluation of mouse immunization and its effects

[0078] Four 6-8 week old Balb / c mice were immunized with p49 recombinant protein as an immunogen, 50 μg per mouse, for a total of three immunizations, with each immunization occurring every two weeks. For the first immunization, Freund's complete adjuvant and p49 recombinant protein were mixed and emulsified; for the second and third immunizations, Freund's incomplete adjuvant and p49 recombinant protein were mixed and emulsified. Tail veins were collected from mice at 14, 28, and 42 days after the first immunization, and serum titers were determined by indirect ELISA.

[0079] 2.2 Indirect ELISA determination of serum titer of p49 recombinant protein-immunized mice

[0080] p49 recombinant protein was diluted to 2 μg / mL with CBS and coated onto a 96-well plate (100 μL per well). Coating was carried out at 37°C for 2 h. The coating solution was discarded, and the plate was washed three times with PBST. The plate was then blocked with 5% skim milk at 37°C for 2 h. The 5% skim milk was discarded, and the plate was washed three times with PBST. A 1:100 dilution of immunized mouse serum was added to the first well, with unimmunized mouse serum used as a negative control. The plate was serially diluted from left to right and incubated at 37°C for 1 h. After washing three times with PBST, a 1:5000 dilution of HRP-labeled goat anti-mouse protein was added, and the plate was incubated at 37°C for 1 h. After washing three times with PBST, 100 μL of TMB chromogenic solution was added to each well. After developing the color in the dark for 5 min, 100 μL of 2 mol / L H2SO4 was added to each well to stop the color development. OD was then measured. 450nm Values ​​are used to analyze the immune response. For example... Figure 5 As shown, it produces a good immune effect against the p49 recombinant protein.

[0081] Example 3: Preparation and identification of p49 recombinant protein monoclonal antibody

[0082] 3.1 Cell Fusion

[0083] Three days before cell fusion, mice with the highest titer were selected and injected intraperitoneally with 100 μg of p49 recombinant protein to induce hyperimmunity. Cell fusion was performed three days after hyperimmunity. First, a healthy Kunming mouse was euthanized by cervical insemination and immersed in 75% alcohol. The mouse was fixed on a dissecting board, and its skin was cut open with sterile scissors and forceps in a laminar flow hood. 20 mL of RPMI-1640 medium containing HAT and 10% fetal bovine serum was injected into the peritoneal cavity of the mouse using a sterile syringe. The medium was then aspirated, mixed with 80 mL of medium, and spread into 96-well cell culture plates, 100 μL per well. The next day, ocular blood was collected from the hyperimmune mice. After incubating the blood at 37°C for 2 hours, it was centrifuged at 4000 rpm for 10 minutes. The supernatant was collected as a positive control, aliquoted, and stored at -20°C for later use. Next, the superimmune mice were euthanized by cervical indwelling and immersed in 75% alcohol for 5 minutes. Afterward, they were fixed on a dissecting board, and their fur was cut open using sterile scissors and forceps in a laminar flow hood. A second set of sterile scissors and forceps was used to cut open the mouse's peritoneum. The spleen was then removed and placed on a 200-mesh sterile nylon mesh, and ground with scissors while continuously adding GNK to rinse the cells, allowing them to filter individually into a sterile beaker below. The spleen cells were transferred to a 50 mL sterile centrifuge tube and centrifuged together with cultured SP2 / 0 cells at 1000 rpm for 10 minutes. After centrifugation, the supernatant was discarded, and the cells were gently ejected. Each cell was resuspended in 20 mL of GNK and counted. The spleen cells and SP2 / 0 cells were then mixed together at an 8:1 ratio and centrifuged at 1000 rpm for 10 minutes. After centrifugation, discard the supernatant, gently disperse the cells at the bottom, and place the centrifuge tube in a 37°C water bath. Add 1 mL of 50% PEG1500 to the bottom of the centrifuge tube over 90 seconds, and let it stand for 90 seconds. Then, slowly add 15 mL of GNK to stop the process, and let it stand for another 5 minutes. Next, add 25 mL of GNK and centrifuge at 1000 rpm for 10 minutes. After centrifugation, discard the supernatant, gently disperse the cells at the bottom, add 100 mL of culture medium and mix gently. Spread 100 μL of the mixture into each well of a 96-well cell culture plate containing feeder cells, for a final volume of 200 μL per well. Incubate in a cell culture incubator for 7 days.

[0084] 3.2 Screening of hybridoma cell positive wells

[0085] Seven days after cell fusion, cell cluster size was observed, and the cell supernatant was detected by indirect ELISA. Ocular serum from fusion mice was used as a positive control. Recombinant p49 protein and empty vector bacterial expression protein were used as assays, diluted to 2 μg / mL with CBS, and 50 μL were coated per well at 37°C for 2 h. The coating solution was discarded, the plate was washed three times with PBST, and 200 μL of 5% skim milk was added to each well for blocking at 37°C for 2 h. 50 μL of cell supernatant was collected per well and incubated at 37°C for 1 h. The cell supernatant was discarded, the plate was washed three times with PBST, and 50 μL of 1:5000 diluted HRP-labeled goat anti-mouse antibody was added to each well as a secondary antibody, and incubated at 37°C for 1 h. The secondary antibody was discarded, the plate was washed three times with PBST, and 50 μL of TMB chromogenic solution was added to each well. After developing the color in the dark for 5 min, 50 μL of 2 mol / L H2SO4 was added to each well to stop the reaction. OD was selected. 450nm Cells with high readings were expanded and cultured, and then subcloned in positive hybridoma cell wells using a dilution method. This process was repeated multiple times until a hybridoma cell line stably secreting monoclonal antibodies was obtained. The results are as follows: Figure 6 As shown, three positive hybridoma cell lines were screened and named 3B12, 6F1 and 7C5, respectively.

[0086] 3.3 Indirect Immunofluorescence (IFA) Assay

[0087] 2×10 per hole 4 293T cells were seeded into 96-well cell culture plates, 100 μL per well. When the cell density reached 80%, the 293T cells were transfected with pcDNA3.1-p49 and pcDNA3.1-GFP plasmids and cultured in a cell culture incubator for 48 h. After 48 h, the cell supernatant was discarded, and the cells were fixed with 4% tissue cell fixative, 100 μL per well for 20 min. The fixative was aspirated, the plate was washed 3 times with PBS, and blocked with 5% skim milk for 2 h. The 5% skim milk was aspirated, the plate was washed 3 times with PBS, and 100 μL of cell supernatant containing 0.3% Triton X-100 was added to each well. The plate was incubated at 37°C for 1 h. Ocular serum from fusion mice (1:100) and serum from unimmunized mice (1:100) were used as positive and negative controls, respectively. Aspirate the supernatant, wash the plate three times with PBS, add 100 μL of GoatAnti-Mouse IgG / FITC (1:200) as secondary antibody to each well, and incubate at 37°C in the dark for 1 h. Aspirate the secondary antibody, wash the plate three times with PBS, add 50 μL of DAPI solution to each well, observe the fluorescence using an inverted microscope, and photograph and save the results. Figure 7 As shown, the results indicate that monoclonal antibodies 3B12, 6F1, and 7C5 can also react with eukaryotically expressed ASFV p49 protein.

[0088] 3.4 Large-scale preparation of monoclonal antibodies and purification of ascites fluid

[0089] Subcloning was followed by indirect ELISA screening, and the selected positive hybridoma cell lines 3B12, 6F1, and 7C5 were expanded and cultured. Multiparous Balb / c mice were intraperitoneally injected with 500 μL of incomplete Freund's adjuvant. One week later, the expanded cultured positive hybridoma cells were diluted with 500 μL of RPMI-1640 basal medium and counted. Each mouse received an intraperitoneal injection of 1.0 × 10⁶ cells. 6 One hybridoma cell. Monitor the changes in the mouse's abdomen daily after injection. When the mouse's abdomen continues to enlarge and it has difficulty moving, euthanize the mouse by cervical inclination, cut open the abdominal cavity and collect the ascites.

[0090] 3.5 Monoclonal Antibody Titer Determination

[0091] Ascites fluid was purified according to the instructions of Protein A agarose resin, and the titer of monoclonal antibodies was determined by indirect ELISA. p49 recombinant protein was diluted to 2 μg / mL with CBS, 100 μL per well, and coated at 37°C for 2 h. After coating, 200 μL of 5% skim milk was added to each well for blocking at 37°C for 2 h. The plate was washed three times with PBST. The purified monoclonal antibody was diluted 1:500 as the first well, followed by serial dilutions from left to right. Unimmunized mouse serum was used as a negative control, 100 μL per well, and incubated at 37°C for 1 h. The plate was washed three times with PBST, and 100 μL of HRP-labeled goat anti-mouse antibody diluted 1:5000 was added to each well, and incubated at 37°C for 1 h. The plate was washed three times with PBST, and 100 μL of TMB chromogenic solution was added to each well. The plate was incubated in the dark for 5 min, and the reaction was stopped by adding 100 μL of 2 mol / L H2SO4 to each well. The OD value was then read. 450nm The result is as follows Figure 8 As shown.

[0092] Example 4: Localization of linear B-cell epitopes of ASFV p49 protein

[0093] 4.1 Truncation, purification, and identification of the ASFV p49 protein amino acid sequence

[0094] The ASFV p49 protein was truncated using the overlapping peptide method. The truncation details are shown in [link to details]. Figure 9 As shown, a total of 8 segments were truncated, and they were constructed into the pET-32a vector using the same method described above. BamH I and XhoBetween the I restriction sites, *E. coli* Rosetta competent cells were transformed. Expression was induced using the same method described above, and the expressed protein was purified according to the nickel affinity chromatography column manufacturer's instructions. The purified truncated protein and empty vector expressed protein were diluted to 2 μg / mL with CBS, 50 μL per well, and incubated at 37°C for 2 h. The plates were washed three times with PBST, and blocked with 200 μL of 5% skim milk per well at 37°C for 2 h. After washing three times with PBST, 50 μL of 1:1000 diluted monoclonal antibody was added to each well as the primary antibody, and the plates were incubated at 37°C for 1 h. After washing three times with PBST, 50 μL of 1:5000 diluted HRP-labeled goat anti-mouse antibody was added to each well as the secondary antibody, and the plates were incubated at 37°C for 1 h. After washing three times with PBST, 50 μL of TMB chromogenic buffer was added to each well, and the plates were incubated in the dark for 5 min. The reaction was stopped by adding 50 μL of 2 mol / L H2SO4 to each well, and the OD was read. 450nm The results are shown in Table 6. It was finally determined that the 3B12 and 6F1 monoclonal antibodies recognize the amino acid region from 333 to 390, while the 7C5 monoclonal antibody recognizes the amino acid site from 381 to 438.

[0095] 4.2 Synthesis and Identification of ASFV p49 Protein Peptide

[0096] Based on the above experimental results, the truncated fragments B7 (aa 333-390) and B8 (aa381-438) were further truncated to synthesize polypeptides P1-P10 (Jier Biochemical), as shown in Table 7. The 10 synthesized polypeptide fragments were diluted with PBS to 4 mg / mL, and each polypeptide was coated at a rate of 4 μg per well. The p49 recombinant protein coating served as a positive control, and PBS as a negative control. A 1:1000 dilution of monoclonal antibody was used as the primary antibody, and a 1:5000 dilution of HRP-labeled goat anti-mouse antibody was used as the secondary antibody. The results are as follows: Figure 10 As shown, Peptide-ELISA and Dot-ELISA showed that 3B12 and 6F1 monoclonal antibodies reacted with P1, and 7C5 monoclonal antibody reacted with P6.

[0097] Table 6 Identification of truncated proteins

[0098] .

[0099] Table 7. Amino acid sequences of truncated peptides

[0100] .

[0101] 4.3 Identification of ASFV p49 protein epitope peptide

[0102] The screened epitope peptides were coated using the method described above, with 1:1000 diluted positive porcine serum as the primary antibody and 1:5000 diluted HRP-labeled goat anti-porcine serum as the secondary antibody. The results are as follows: Figure 11 As shown, Peptide-ELISA and Dot-ELISA revealed that both P1 and P6 reacted with positive porcine serum, which is the first reported B-cell epitope.

[0103] The present application has been described in detail above with reference to the accompanying drawings and embodiments. However, those skilled in the art will understand that the implementation of the present application is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the inventive concept of the present application shall be considered equivalent substitutions and shall be included within the protection scope of the present application.

Claims

1. A linear B-cell epitope peptide of ASFV p49 protein, the amino acid sequence of which is YQTHYMENIVTLVPR or NNYIPKYTGGIGDSK.

2. The application of the linear B-cell epitope peptide of ASFV p49 protein according to claim 1 in the preparation of diagnostic reagents for African swine fever (ASF).

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