Monoclonal antibody BY017 combined with African swine fever virus P30 protein and application thereof

Abstract

The invention discloses a monoclonal antibody BY017 combined with African swine fever virus P30 protein and application of the monoclonal antibody BY017. The invention provides an IgG antibody which is composed of a light chain and a heavy chain. CDR1, CDR2 and CDR3 in the heavy chain variable region are sequentially shown as the 47 to 54 , the 72 to 79 and the 118 to 126 in SEQ ID NO: 1; cDR1, CDR2 and CDR3 in a light chain variable region are sequentially as shown in the 49 to 60 , the 78 to 80 and the 117 to 124 in SEQ ID NO: 3. The invention also protects the application of any one of the IgG antibodies in preparation of products. The product has the functions that the product is used for being combined with an African swine fever virus P30 protein fragment; the antibody is used for binding African swine fever virus P30 protein; the antibody is used for binding African swine fever virus. The IgG antibody provided by the invention can be used for multiple purposes of western blot, immunohistochemistry, flow cytometry, ELISA (Enzyme-Linked Immunosorbent Assay) and the like, and can be used as an important tool for ASFV detection and structural analysis of P30 protein of ASFV detection.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a monoclonal antibody BY017 binding to the P30 protein of African swine fever virus and an application thereof. Background Art

[0002] African swine fever (ASF) is an acute, severe, and highly contagious disease of swine caused by the African swine fever virus (ASFV). Clinically characterized by high fever, loss of appetite, cyanosis of the skin, and internal bleeding, it is characterized by a short course, high morbidity, and a mortality rate of up to 100%. As a notifiable animal disease designated by the World Organization for Animal Health (WOAH) and a Category I animal disease in my country, ASF has garnered significant attention worldwide. First confirmed in Kenya, Africa in 1921, ASF has spread and become endemic in numerous countries worldwide since 2007, reaching my country in 2018, causing significant direct economic losses and profound industrial impacts. Despite nearly a century of discovery, ASF remains globally lacking effective vaccines and treatments. Prevention and control efforts primarily rely on early diagnosis, culling of infected pigs, strict quarantine, and biosafety management.

[0003] ASFV is the only member of the genus Asfivirus in the family Asfarviridae and the only known DNA arbovirus with a double envelope. The virion has icosahedral symmetry and is approximately 260 nm in diameter. Its genome consists of 170-194 kb of double-stranded linear DNA, containing 151-167 open reading frames encoding 150-200 proteins. The virus has a unique replication cycle, primarily infecting mononuclear leukocytes and macrophages via endocytosis and macropinocytosis. Genome replication, viral assembly, and release all occur in the host cytoplasm.

[0004] The P30 protein, a core structural protein of ASFV, is located in the inner membrane of the viral particle. Encoded by the CP204L gene, it has a molecular mass of approximately 23.6 kDa and is a phosphorylated protein. This protein plays a key role in viral invasion of host cells, mediating viral internalization and participating in the initiation of infection. P30 protein is detected 2-4 hours after ASFV infection. It is an early expressed protein that persists throughout the infection cycle and can induce the production of neutralizing antibodies in the host. Due to its immunological properties, P30 protein has been widely used as a candidate antigen for ASF serological diagnosis. However, as a major viral structural protein and an important protective antigen, its three-dimensional structure remains elusive. Therefore, the preparation of P30 protein-specific monoclonal antibodies and epitope screening are of great significance not only for elucidating the biological function of this protein but also for the development of ASFV detection technologies and structural biology research. Summary of the Invention

[0005] The purpose of the present invention is to provide a monoclonal antibody BY017 that binds to the African swine fever virus P30 protein and its application.

[0006] The present invention provides an IgG antibody consisting of a light chain and a heavy chain; CDR1, CDR2 and CDR3 in the heavy chain variable region of the heavy chain are shown, respectively, at positions 47-54 (GYTFTNSY), positions 72-79 (INPRNGDT) and positions 118-126 (TRGGSQFDY) in SEQ ID NO: 1; CDR1, CDR2 and CDR3 in the light chain variable region of the light chain are shown, respectively, at positions 49-60 (QSLLNSRTRKNY), positions 78-80 (WAS) and positions 117-124 (KQSYNLQT) in SEQ ID NO: 3.

[0007] The IgG antibody is an IgG antibody that binds to the P30 protein of the African swine fever virus, or the IgG antibody is an IgG antibody that binds to the P30 protein fragment of the African swine fever virus, or the IgG antibody is an IgG antibody that binds to the African swine fever virus.

[0008] Specifically, the heavy chain variable region is shown in positions 22-137 of SEQ ID NO: 1.

[0009] Specifically, the light chain variable region is shown in positions 23-134 of SEQ ID NO: 3.

[0010] Specifically, the heavy chain is as follows (a1) or (a2): (a1) the protein shown at positions 22-467 in SEQ ID NO: 1; (a2) the protein shown in SEQ ID NO: 1.

[0011] Specifically, the light chain is (b1) or (b2) as follows: (b1) the protein shown at positions 23-241 in SEQ ID NO: 3; (b2) the protein shown in SEQ ID NO: 3.

[0012] The present invention also protects the gene encoding the IgG antibody.

[0013] Specifically, the gene encoding the heavy chain is as follows (c1) or (c2) or (c3) or (c4): (c1) a DNA molecule having a coding region as shown in positions 64 to 1401 of SEQ ID NO: 2; (c2) a DNA molecule having a coding region as shown in positions 64-1404 of SEQ ID NO: 2; (c3) a DNA molecule having a coding region as shown in positions 1 to 1401 of SEQ ID NO: 2; (c4) A DNA molecule whose coding region is shown in SEQ ID NO: 2.

[0014] Specifically, the gene encoding the light chain is as follows (d1) or (d2) or (d3) or (d4): (d1) a DNA molecule having a coding region as shown in positions 67-723 of SEQ ID NO: 4; (d2) a DNA molecule having a coding region as shown in positions 67-726 of SEQ ID NO: 4; (d3) A DNA molecule having a coding region as shown in positions 1 to 723 of SEQ ID NO: 4.

[0015] (d4) A DNA molecule whose coding region is shown in SEQ ID NO: 4.

[0016] The present invention also protects the use of any of the above IgG antibodies in preparing a product; the function of the product is as follows (e1) or (e2) or (e3): (e1) for binding to the P30 protein fragment of African swine fever virus; (e2) for binding to African swine fever virus P30 protein; (e3) is used to bind African swine fever virus.

[0017] The present invention also protects the use of any of the above-mentioned IgG antibodies in the preparation of products for identifying African swine fever virus P30 protein.

[0018] The present invention also protects the use of any of the above-mentioned IgG antibodies in the preparation of products for identifying African swine fever virus.

[0019] The present invention also protects the use of any of the above-mentioned IgG antibodies in the preparation of drugs for inhibiting African swine fever virus.

[0020] The present invention also protects the use of any of the above-mentioned IgG antibodies in the preparation of a drug for neutralizing African swine fever virus.

[0021] The present invention also protects the use of any of the above-mentioned IgG antibodies in the preparation of drugs for preventing and / or treating African swine fever virus infectious diseases.

[0022] Specifically, the African swine fever virus P30 protein is shown in positions 1-194 of SEQ ID NO: 5.

[0023] Specifically, the African swine fever virus P30 protein is shown in SEQ ID NO: 5.

[0024] Specifically, the African swine fever virus P30 protein fragment is shown in positions 110-131 in SEQ ID NO: 5.

[0025] The naming system of the CDRs is IMGT.

[0026] The IgG antibody provided by the present invention can be used for various purposes such as western blot, immunohistochemistry, flow cytometry staining, ELISA, etc., and can be used as an important tool for ASFV detection and structural analysis of its P30 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the SDS-PAGE electrophoresis diagram in Example 1.

[0028] Figure 2 This is a diagram showing the results of monoclonal antibody subtype identification in Example 3.

[0029] Figure 3 This is a diagram showing the results of identifying the antigen epitope recognized by the monoclonal antibody in Example 3.

[0030] Figure 4 This is a diagram showing the results of verifying the effect of the monoclonal antibody in Example 3.

[0031] Figure 5 This is the SDS-PAGE electrophoresis diagram in Example 5.

[0032] Figure 6 This is a diagram showing the results of specific identification of the genetically engineered antibody in Example 6.

[0033] Figure 7 This is a graph showing the results of titer identification of the genetically engineered antibody in Example 7. DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0035] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. 1640 complete medium: 1640 medium containing 10% FBS. 1640 medium, also known as RPMI 1640 medium. Enzyme-labeled secondary antibody (horseradish enzyme-labeled goat anti-mouse IgG): Beijing Zhongshan Jinqiao Company, product catalog number ZB-2305; dilute 1:10,000 for use to obtain the enzyme-labeled secondary antibody working solution. Hi-exp medium: Aupuma Company, product catalog number AC601501. ACK red blood cell lysis buffer: Contains 150mM NH₄Cl, 10mM KHCO₃, and 0.1mM Na₂-EDTA, with the balance being water; sterilize by filtration through a 0.22μm filter before use. PBST solution: PBS buffer containing 0.05% (volume percentage) Tween-20. Blocking solution: PBST solution containing 0.2g / 100mL BSA. Unless otherwise specified, cell cultures were performed in a cell culture incubator (37°C, 5% CO2). Unless otherwise specified, the PBS buffer used in the examples was pH 8.0. Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0036] Example 1. Expression and purification of P30 protein 1. Construction of recombinant plasmids and acquisition of recombinant bacteria In SEQ ID NO:6, nucleotides 1-582 represent the CP204L gene (encoding the P30 protein) from the genome of the ASFV epidemic strain Pig / HLJ / 2018 in China. The double-stranded DNA molecule represented by SEQ ID NO:6 encodes the protein represented by SEQ ID NO:5. In SEQ ID NO:5, amino acid residues 1-194 constitute the P30 protein, and amino acid residues 205-233 constitute the Twin-Strep tag.

[0037] The double-stranded DNA molecule shown in SEQ ID NO: 6 was used to replace the pET-28a (+) vector. Nco I and XhoI restriction enzyme sites, while keeping the rest of the sequence unchanged, to obtain the recombinant plasmid P30-Strep. The recombinant plasmid has been sequenced and verified.

[0038] The recombinant plasmid P30-Strep was introduced into Escherichia coli BL-21 (DE3) to obtain recombinant bacteria.

[0039] 2. Protein Preparation and Purification 1. Inoculate the recombinant bacteria obtained in step 1 into 1L of liquid LB medium and culture at 37°C and 200 rpm until the OD 600nm The value was 0.8-1.0, and then the cells were shaken and cultured at 20°C and 200 rpm for 1 hour.

[0040] 2. After completing step 1, add IPTG to the culture system and make its concentration in the system 0.5mM, then culture at 20°C and 200rpm with shaking for 14-16 hours, then centrifuge at 4000rpm for 10 minutes to collect the bacterial precipitate.

[0041] 3. Resuspend the bacterial pellet obtained in step 2 with 25 mL of Buffer I, then disrupt the cells using a high-pressure homogenizer. Centrifuge at 18,000 rpm for 30 min at 4°C, and collect the supernatant.

[0042] Buffer I: Contains 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl, with the balance being water.

[0043] 4. Use an affinity chromatography column (5 mL; STarm Streptactin Beads 4FF, Changzhou Tiandi Renhe Biotechnology Co., Ltd., catalog number SA092100). First, equilibrate with buffer I for 10 column volumes, then load the supernatant from step 3 (load volume 25 mL). Wash with buffer I for 10 column volumes, and then elute with buffer II for 5 column volumes. Collect the post-column solution to obtain the target protein solution.

[0044] Buffer II: contains 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 2.5 mM desthiobiotin, with the balance being water.

[0045] The target protein is the protein shown in SEQ ID NO:5.

[0046] The SDS-PAGE electrophoresis of the target protein solution is shown in Figure 1 .

[0047] Example 2: Obtaining target hybridoma cells by myeloma cell fusion and subcloning screening 1. Animal Immunization SPF 6-8 week old Balb / c mice were immunized according to the following procedure: Day 1: First immunization, multiple subcutaneous injections of the immunizing agent (consisting of the immunogen and Freund's complete adjuvant); Day 29: Second immunization, multiple subcutaneous injections of the immunizing agent (consisting of the immunogen and Freund's incomplete adjuvant); Day 57: The third immunization, multiple subcutaneous injections of the immunizing agent (consisting of the immunogen and Freund's incomplete adjuvant); Day 64: Orbital venous blood was collected to separate serum and used as test antibody for titer detection (for the method of detecting antibody titer, see Example 7, the only difference is that the test antibody is used instead of the antibody diluent). Mice with high titer were selected for the fourth immunization and the immunogen was injected intraperitoneally.

[0048] The immunogen was the target protein prepared in Example 1, and the single immunization dose for a single mouse was 10 μg (calculated as protein amount).

[0049] 2. Preparation and Screening of Hybridoma Cells 1. Preparation of feeder cells One day before fusion, well-developed Balb / c mice were killed by cervical dislocation and placed in a 75% ethanol aqueous solution for disinfection. 8-10 ml of pre-cooled 0.34 M sucrose aqueous solution was drawn up with a pre-cooled syringe and injected into the peritoneum at the lower right corner of the mouse. The needle did not come out of the peritoneum, and the abdominal cavity was massaged with fingers for about 1 minute. The fluid in the mouse peritoneal cavity was then aspirated and added to a pre-cooled 50 ml centrifuge tube. Pre-cooled 1640 complete medium was then added, and the cells were centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in HAT medium to a cell concentration of 1×10 5 The cell suspension was then added to a 96-well cell culture plate (100 μl / well) and cultured.

[0050] 2. Preparation of splenocytes Four days after the fourth immunization in step 1, the mice were killed by removing their eyeballs, and the blood and spleen were collected separately. Serum was separated from the blood and used as a positive control. Take a culture dish with a diameter of 10 cm, add 10 ml of 1640 medium and DNase, then add the mouse spleen, grind and pipette to a single cell suspension, then filter with a 70 μm filter and collect the filtrate into a 50 ml centrifuge tube, then centrifuge at 1500 rpm for 5 minutes, discard the supernatant, resuspend the cell pellet with ACK red blood cell lysis buffer, incubate at room temperature for 2 minutes, then wash the cells with 1640 medium, then add 20 ml of 1640 medium to resuspend the cells, pipette to mix, and count.

[0051] 3. Cell fusion Collect Sp2 / 0 mouse myeloma cells in good condition and in the logarithmic growth phase and wash them with 1640 medium. Mix Sp2 / 0 mouse myeloma cells and spleen cells in a ratio of 1:1-1:3, then centrifuge at 1500 rpm for 10 minutes. Discard the supernatant and beat the cells to form a paste. Then, incubate in a 37°C water bath and add 1 ml of preheated 50% PEG solution dropwise. Then, slowly add 40 ml of preheated 1640 medium. Centrifuge at 1500 rpm for 10 minutes, discard the supernatant, add 10 ml of HAT medium and pipette up and down several times. Then add HAT medium to approximately 90 ml. Mix thoroughly and add 2 drops per well to the feeder cell culture plate for incubation.

[0052] 4. Screening of hybridoma cell positive wells Four days after cell fusion, half of the medium was replaced with HAT medium. After about 7-10 days, the hybridoma cells grew to a certain size. One day before the test, about 200 μl of medium was aspirated and 200 μl of fresh HT medium was added.

[0053] On the day of testing, culture medium of the hybridoma cell mass is aspirated as the test antibody, the positive control is used as the positive control, and the serum of unimmunized mice is used as the negative control. The titer is tested (the method for testing the antibody titer is described in Example 7, the only difference is that the test antibody or control is used instead of the antibody diluent), and positive clones are screened.

[0054] 5. Subcloning of positive hybridoma cells Use HT medium for the first subcloning. For the first subcloning, select positive clone wells and observe the cell status and cell cluster size under a microscope. Under sterile conditions, gently pipette and mix the cells to be subcloned to avoid blowing out bubbles. Pipette 10μl of the counted cells and add 10μl of 0.04% trypan blue solution. After mixing, count the cells in 8 large grids and calculate the cell concentration. Based on the cell count results, pipette 100-150 cells by limiting dilution and add them to 9.5ml of culture medium. After mixing, add dropwise to the feeder cell culture plate that has been cultured for 1 day.

[0055] After about 5 days of subcloning, count the hybridomas in each well under an inverted microscope. When the hybridoma cells grow to an appropriate size, test again and screen the positive monoclones for a second subcloning. Replace the culture medium with 1640 complete culture medium. Continue subcloning 2-3 times until the obtained monoclonal hybridoma cell line can stably secrete the desired antibody.

[0056] 6. Expansion culture and cryopreservation of hybridoma cells ① The positive hybridoma cells obtained after identification were expanded and transferred to 24-well cell culture plates and cultured until the confluence reached about 80%.

[0057] ②After completing step ①, transfer the cells to a T25 cell culture flask and culture until the cell confluence reaches about 80%.

[0058] ③After completing step ②, repeatedly blow the inner wall of the culture flask with culture medium, then transfer to a sterile centrifuge tube, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, resuspend the cells in 3 ml of cell freezing solution, mix thoroughly, and transfer the cell suspension to a cryopreservation tube. Transfer it to a programmed cooling box, let it stand at -80℃ for 24 hours, and then transfer it to liquid nitrogen for long-term storage.

[0059] Based on the above steps, multiple hybridoma cells secreting the target monoclonal antibody (i.e., a monoclonal antibody that binds to the P30 protein of African swine fever virus) were obtained, one of which was named BY017 hybridoma cell.

[0060] Example 3. Preparation and identification of monoclonal antibodies 1. Obtaining Monoclonal Antibody Supernatant Hybridoma cells BY017 were cultured in 1640 complete medium (culture time was 2-3 days), and then the supernatant was collected, which was the solution containing the monoclonal antibody BY017, referred to as BY017 solution.

[0061] 2. Identification of Monoclonal Antibody Subtypes Take the BY017 solution and use a monoclonal antibody isotyping kit according to the manufacturer's instructions. Monoclonal antibody isotyping kit (Isotyping Kit for Mouse Monoclonal Antibody): Beijing Sino Biological Science & Technology Co., Ltd., catalog number SEK003.

[0062] See the results Figure 2 Monoclonal antibody BY017 is of IgG2b subtype.

[0063] 3. Identification of Antigen Epitopes Recognized by Monoclonal Antibodies 1. Design and preparation of peptides Based on the amino acid sequence of the ASFV P30 protein, peptides were designed, as shown in Table 1. The peptide names correspond to the positional range of the peptide corresponding to SEQ ID NO: 5 (e.g., peptide 110-131 refers to the peptide at positions 110-131 in SEQ ID NO: 5). Each peptide in Table 1 was prepared separately.

[0064] Table 1

[0065] 2. Preparation of coating The polypeptides prepared in step 1 were coupled to ovalbumin (OVA) to obtain coatings.

[0066] 3. ELISA test Test coating source: each coating source prepared in step 2 or the target protein prepared in Example 1. Take the test coating source and dilute it with PBS buffer to a protein concentration of 1 μg / ml, which is the coating solution.

[0067] ① Take a 96-well plate, add coating solution (100 μl / well), incubate at 4°C for 12 hours, discard the supernatant, wash three times with PBST solution, and pat dry.

[0068] ② Take the 96-well plate prepared in step ①, add blocking solution (200 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash three times with PBST solution, and pat dry.

[0069] ③ Take the 96-well plate prepared in step ②, add BY017 solution (100 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash three times with PBST solution, and pat dry.

[0070] ④ Take the 96-well plate prepared in step ③, add enzyme-labeled secondary antibody working solution (100 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash 5 times with PBST solution, and pat dry.

[0071] ⑤ Take the 96-well plate prepared in step ④, add TMB colorimetric solution (100 μl / well), and react for 5-10 minutes in the dark.

[0072] ⑥ Take the 96-well plate prepared in step ⑤, add 2M sulfuric acid solution (50 μl / well), and then measure the absorbance at 450 nm (OD 450 ).

[0073] See the results Figure 3 Monoclonal antibody BY017 reacts with peptide 110-131.

[0074] 4. Verification of the effect of monoclonal antibodies The test cells were: PAM cells or PAM cells infected with ASFV virus HLJ / 18 strain.

[0075] Lyse the test cells and extract total protein, then perform Western blotting. Primary antibody working solution: Dilute BY017 solution with PBS buffer to an antibody concentration of 1 μg / ml. Secondary antibody working solution: Enzyme-labeled secondary antibody working solution.

[0076] See the results Figure 4 (PAM represents the total protein of PAM cells, and ASFV+PAM represents the total protein of PAM cells infected with the HLJ / 18 strain of ASFV.) Western blot results showed that BY017 solution could recognize the P30 protein in PAM cells infected with ASFV.

[0077] Example 4. Obtaining the variable region sequence of an antibody BY017 hybridoma cells were lysed, and total RNA was extracted and reverse transcribed to obtain cDNA. Using the cDNA as a template, PCR amplification was performed using primers targeting the antibody variable region. The PCR amplification product was then recovered and sequenced to obtain the gene sequence of the antibody's heavy chain variable region and the gene sequence of the antibody's light chain variable region. Furthermore, the amino acid sequence of the antibody's heavy chain variable region and the amino acid sequence of the antibody's light chain variable region were obtained.

[0078] The amino acid sequence of the antibody's heavy chain variable region is shown at positions 22-137 of SEQ ID NO: 1 (the three CDRs are: GYTFTNSY, INPRNGDT, TRGGSQFDY, in order). The amino acid sequence of the antibody's light chain variable region is shown at positions 23-134 of SEQ ID NO: 3 (the three CDRs are: QSLLNSRTRKNY, WAS, KQSYNLQT, in order).

[0079] The coding sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 2, positions 64 to 411. The coding sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 4, positions 67 to 402.

[0080] Example 5: Preparation of genetically engineered antibodies 1. Preparation of recombinant expression vector The double-stranded DNA molecule shown in SEQ ID NO: 2 was substituted for the PTT3 vector EcoR I and Not I enzyme cuts the small fragment between the recognition sites, keeping other sequences unchanged, to obtain the heavy chain expression plasmid. The double-stranded DNA molecule shown in SEQ ID NO: 4 replaces the PTT3 vector. EcoR I and Not I enzyme digested the small fragment between the recognition sites, leaving the rest of the sequence unchanged, to generate the light chain expression plasmid. The recombinant plasmid was sequenced and verified.

[0081] The PTT3 vector is a circular plasmid, and its complete sequence is shown in SEQ ID NO: 7.

[0082] The double-stranded DNA molecule represented by SEQ ID NO: 2 encodes the protein represented by SEQ ID NO: 1. In SEQ ID NO: 1, amino acid residues 1-21 constitute a signal peptide (which is cleaved during extracellular secretion), amino acid residues 22-137 constitute the heavy chain variable region, and amino acid residues 138-467 constitute the heavy chain constant region.

[0083] The double-stranded DNA molecule represented by SEQ ID NO: 4 encodes the protein represented by SEQ ID NO: 3. In SEQ ID NO: 3, amino acid residues 1 to 22 constitute a signal peptide (which is cleaved during extracellular secretion), amino acid residues 23 to 134 constitute the light chain variable region, and amino acid residues 135 to 241 constitute the light chain constant region.

[0084] 2. Preparation of transfection complexes Preparation of PEI transfection reagent solution: Add 100 mg of linear PEI transfection reagent (Mw 40,000; LABLEAD, catalog number P4000) to 90 mL of Milli-Q ultrapure water and stir until completely dissolved. Adjust the pH to 6.9-7.1, dilute to 100 mL with Milli-Q ultrapure water, and filter through a 0.22 μm filter to collect the filtrate.

[0085] Preparation of transfection complex (for 1L cell suspension): Add 500 μg of heavy chain expression plasmid and 500 μg of light chain expression plasmid to 5 mL of Hi-exp medium and mix thoroughly by pipetting to prepare liquid A. Add 3 mL of PEI transfection reagent solution to 5 mL of Hi-exp medium and mix thoroughly by pipetting to prepare liquid B. Add liquid B to liquid A and mix thoroughly by pipetting, then incubate at room temperature for 5 minutes.

[0086] 3. Preparation of cell suspension Take the 293F cells in culture, count the cells, centrifuge at 800 rpm for 5 minutes, discard the supernatant, and resuspend in Hi-exp medium to a cell content of 1×10 6 cells / mL.

[0087] 4. Add the transfection complex dropwise to 1 L of cell suspension, shake and culture at 130 rpm for 5 days (environmental conditions: 37°C, 8% CO2), then centrifuge at 4000 rpm for 20 min, collect the supernatant, and then filter through a 0.45 μm pore size filter membrane to collect the filtrate.

[0088] 5. Using an affinity chromatography column (column volume, 10 mL; packing material: Protein At Beads LX, Changzhou Tiandi Renhe Biotechnology Co., Ltd., catalog number SA08501L), first equilibrate the column with PBS buffer, then load the filtrate obtained in step 4 (load volume, 950 mL), and then wash with PBS buffer for 10-15 column volumes. Then, elute with glycine buffer (pH 2.5-3.0, 0.1 M) and collect the post-column solution. Mix 10 parts by volume of the post-column solution with 1 part by volume of neutralization buffer (i.e., Tris-HCl buffer, pH 9.0, 1 M) to obtain a mixed solution.

[0089] 6. Take the mixed solution obtained in step 5 and use a 30K ultrafiltration tube (Millipore, UFC903096) to concentrate and replace the buffer system with PBS buffer to obtain a genetically engineered antibody solution.

[0090] The SDS-PAGE electrophoresis of the genetically engineered antibody solution is shown in Figure 5 .

[0091] Example 6: Specificity Identification of Genetically Engineered Antibodies Test coating agent: Each coating agent prepared in step 2 of Example 3 or the target protein prepared in Example 1. Dilute the test coating agent with PBS buffer to a protein concentration of 1 μg / ml to prepare the coating solution. Antibody diluent: Dilute the genetically engineered antibody solution prepared in Example 5 with PBS buffer to a concentration of 2 μg / ml.

[0092] ① Take a 96-well plate, add coating solution (100 μl / well), incubate at 4°C for 12 hours, discard the supernatant, wash three times with PBST solution, and pat dry.

[0093] ② Take the 96-well plate prepared in step ①, add blocking solution (200 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash three times with PBST solution, and pat dry.

[0094] ③ Take the 96-well plate prepared in step ②, add antibody diluent (100 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash three times with PBST solution, and pat dry.

[0095] ④ Take the 96-well plate prepared in step ③, add enzyme-labeled secondary antibody working solution (100 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash 5 times with PBST solution, and pat dry.

[0096] ⑤ Take the 96-well plate prepared in step ④, add TMB colorimetric solution (100 μl / well), and react for 5-10 minutes in the dark.

[0097] ⑥ Take the 96-well plate prepared in step ⑤, add 2M sulfuric acid solution (50 μl / well), and then measure the absorbance at 450 nm (OD 450 ).

[0098] See the results Figure 6 The genetically engineered antibody prepared in Example 5 reacted with polypeptide 110-131.

[0099] Example 7: Identification of the potency of genetically engineered antibodies The target protein solution prepared in Example 1 was diluted with PBS buffer to a protein concentration of 1 μg / ml, which was used as the coating solution. Preparation of the antibody diluent: The genetically engineered antibody solution prepared in Example 5 was first diluted with PBS buffer to an antibody concentration of 2 μg / ml, and then diluted 2-fold with PBS buffer.

[0100] 1. Take a 96-well plate, add coating solution (100 μl / well), incubate at 4°C for 24 hours, discard the supernatant, wash three times with PBST solution, and pat dry.

[0101] 2. Take the 96-well plate prepared in step 1, add blocking solution (200 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash three times with PBST solution, and pat dry.

[0102] 3. Take the 96-well plate prepared in step 2, add antibody diluent (100 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash three times with PBST solution, and pat dry.

[0103] 4. Take the 96-well plate prepared in step 3, add enzyme-labeled secondary antibody working solution (100 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash 5 times with PBST solution, and pat dry.

[0104] 5. Take the 96-well plate prepared in step 4, add TMB colorimetric solution (100 μl / well), and react for 5-10 minutes in the dark.

[0105] 6. Take the 96-well plate prepared in step 5, add 2M sulfuric acid solution (50 μl / well), and measure the absorbance at 450 nm (OD 450 ).

[0106] See the results Figure 7 .

[0107] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. An IgG antibody, comprising a light chain and a heavy chain; wherein the CDR1, CDR2, and CDR3 in the heavy chain variable region of the heavy chain are as shown, in sequence, at positions 47-54, 72-79, and 118-126 of SEQ ID NO: 1; and the CDR1, CDR2, and CDR3 in the light chain variable region of the light chain are as shown, in sequence, at positions 49-60, 78-80, and 117-124 of SEQ ID NO:

3.

2. The IgG antibody according to claim 1, wherein: The heavy chain variable region is shown in positions 22-137 of SEQ ID NO: 1; The light chain variable region is shown in positions 23-134 of SEQ ID NO:

3.

3. The IgG antibody according to claim 2, wherein: The heavy chain is as follows (a1) or (a2): (a1) the protein shown at positions 22-467 of SEQ ID NO: 1; (a2) the protein shown at positions 22-467 of SEQ ID NO: 1; The light chain is (b1) or (b2) as follows: (b1) the protein shown at positions 23-241 in SEQ ID NO: 3; (b2) the protein shown in SEQ ID NO:

3.

4. A gene encoding the IgG antibody according to any one of claims 1 to 3.

5. Use of the IgG antibody according to claim 1, 2 or 3 in preparing a product; the function of the product is as follows (e1) or (e2) or (e3): (e1) used to bind to the P30 protein fragment of African swine fever virus; (e2) for binding to African swine fever virus P30 protein; (e3) is used to bind African swine fever virus.

6. Use of the IgG antibody according to claim 1, 2 or 3 in the preparation of a product for identifying the P30 protein of African swine fever virus.

7. Use of the IgG antibody according to claim 1, 2 or 3 in the preparation of a product for identifying African swine fever virus.

8. Use of the IgG antibody according to claim 1, 2 or 3 in the preparation of a medicament for inhibiting African swine fever virus.

9. Use of the IgG antibody according to claim 1, 2 or 3 in the preparation of a medicament for neutralizing African swine fever virus.

10. Use of the IgG antibody according to claim 1, 2 or 3 in the preparation of a medicament for preventing and / or treating African swine fever virus infectious diseases.

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