BY017, a monoclonal antibody against the African swine fever virus P30 protein, and its application.

Abstract

This invention discloses a monoclonal antibody BY017 that binds to the African swine fever virus (ASFV) P30 protein and its applications. This invention provides an IgG antibody composed of a light chain and a heavy chain; CDR1, CDR2, and CDR3 in the variable region of the heavy chain are as shown in positions 47-54, 72-79, and 118-126 of SEQ ID NO: 1, respectively; CDR1, CDR2, and CDR3 in the variable region of the light chain are as shown in positions 49-60, 78-80, and 117-124 of SEQ ID NO: 3, respectively. This invention also protects the application of any of the above-described IgG antibodies in the preparation of products; the functions of the products are: binding to the ASFV P30 protein fragment; binding to the ASFV P30 protein; and binding to ASFV. The IgG antibody provided by this invention can be used for various applications such as Western blot, immunohistochemistry, flow cytometry, and ELISA, and can serve as an important tool for ASFV detection and structural analysis of the P30 protein.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a monoclonal antibody BY017 that binds to the P30 protein of African swine fever virus and its applications. Background Technology

[0002] African swine fever (ASF) is an acute, highly contagious, and deadly infectious disease of pigs caused by the African swine fever virus (ASFV). Clinically, it is characterized by high fever, loss of appetite, cyanosis of the skin, and internal organ hemorrhage. The disease has a short course, high morbidity, and a mortality rate that can reach 100%. As a notifiable animal disease of the World Organization for Animal Health (WOAH) and a Class A animal disease in my country, ASF has received high attention from countries around the world. The disease was first confirmed in Kenya, Africa in 1921. Since 2007, African swine fever has occurred, spread, and become prevalent in many countries globally, and entered my country in 2018, causing huge direct economic losses and far-reaching industrial impacts. Although ASF has been around for nearly a century, there is still a lack of effective vaccines and treatments globally. Prevention and control measures mainly rely on early diagnosis, culling of infected pigs, strict quarantine, and biosecurity management.

[0003] ASFV is the sole member of the genus *Asfivirus* within the family Asfarviridae, and the only known DNA arbovirus with a double-layered envelope structure. The virus particle is icosahedral in shape, approximately 260 nm in diameter, and its genome is a 170-194 kb double-stranded linear DNA containing 151-167 open reading frames encoding 150-200 proteins. This virus has a unique replication cycle, primarily infecting monocytes and macrophages through endocytosis and macropinocytosis. Its genome replication, viral assembly, and release all occur within the host cytoplasm.

[0004] The P30 protein, a core structural protein of ASFV, is located on the inner membrane of the viral particle. Encoded by the CP204L gene, it has a molecular weight of approximately 23.6 kDa and is a phosphorylated protein. This protein plays a crucial role in viral invasion of host cells, participating in the initiation phase of infection by mediating viral internalization. The P30 protein can be detected 2-4 hours after ASFV infection, making it 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, the P30 protein has been widely used as a candidate antigen for the serological diagnosis of ASF. However, as a major structural protein and important protective antigen of the virus, its three-dimensional structure remains unresolved. Therefore, the preparation of P30 protein-specific monoclonal antibodies and the screening of antigenic epitopes are not only significant for elucidating the biological function of this protein but also provide a key tool for the development of ASFV detection technology and its structural biology research. Summary of the Invention

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

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

[0007] The IgG antibody is an IgG antibody that binds to the African swine fever virus P30 protein, or an IgG antibody that binds to the African swine fever virus P30 protein fragment, or 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 variable region of the light chain is shown in positions 23-134 of SEQ ID NO: 3.

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

[0011] Specifically, the light chain is as follows (b1) or (b2): (b1) the protein shown at positions 23-241 of 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 (c1) or (c2) or (c3) or (c4):

[0014] (c1) A DNA molecule whose coding region is shown as positions 64-1401 in SEQ ID NO: 2;

[0015] (c2) DNA molecules with coding regions as shown in positions 64-1404 of SEQ ID NO: 2;

[0016] (c3) DNA molecules with coding regions as shown in positions 1-1401 of SEQ ID NO: 2;

[0017] (c4) DNA molecules with coding regions as shown in SEQ ID NO: 2.

[0018] Specifically, the gene encoding the light chain is as follows (d1) or (d2) or (d3) or (d4):

[0019] (d1) A DNA molecule whose coding region is shown as positions 67-723 in SEQ ID NO: 4;

[0020] (d2) DNA molecules with coding regions as shown in positions 67-726 of SEQ ID NO: 4;

[0021] (d3) The coding region is shown as positions 1-723 in SEQ ID NO: 4 of the DNA molecule.

[0022] (d4) The coding region is shown in the DNA molecule as shown in SEQ ID NO:4.

[0023] This invention also protects the use of any of the above-described IgG antibodies in the preparation of products; the functions of said products are as follows (e1) or (e2) or (e3):

[0024] (e1) is used to bind to the African swine fever virus P30 protein fragment;

[0025] (e2) is used to bind to the African swine fever virus P30 protein;

[0026] (e3) is used to bind to African swine fever virus.

[0027] This invention also protects the use of any of the above-described IgG antibodies in the preparation of products for identifying the African swine fever virus P30 protein.

[0028] This invention also protects the use of any of the above-described IgG antibodies in the preparation of products for identifying African swine fever virus.

[0029] The present invention also protects the use of any of the above-described IgG antibodies in the preparation of medicaments for inhibiting African swine fever virus.

[0030] The present invention also protects the use of any of the above-described IgG antibodies in the preparation of medicaments for neutralizing African swine fever virus.

[0031] The present invention also protects the use of any of the above-described IgG antibodies in the preparation of medicaments for the prevention and / or treatment of African swine fever virus infection.

[0032] Specifically, the African swine fever virus P30 protein is shown in positions 1-194 of SEQ ID NO: 5.

[0033] Specifically, the African swine fever virus P30 protein is shown in SEQ ID NO: 5.

[0034] Specifically, the African swine fever virus P30 protein fragment is shown in positions 110-131 of SEQ ID NO: 5.

[0035] The naming system for the CDR is IMGT.

[0036] The IgG antibody provided by this invention can be used for various purposes such as western blot, immunohistochemistry, flow cytometry, and ELISA, and can serve as an important tool for ASFV detection and structural analysis of its P30 protein. Attached Figure Description

[0037] Figure 1 This is an SDS-PAGE electrophoresis image from Example 1.

[0038] Figure 2 This is a diagram showing the results of monoclonal antibody subtype identification in Example 3.

[0039] Figure 3 This is a diagram showing the results of the identification of antigen epitopes by monoclonal antibodies in Example 3.

[0040] Figure 4 This is a graph showing the results of the monoclonal antibody efficacy verification in Example 3.

[0041] Figure 5 This is an SDS-PAGE electrophoresis image from Example 5.

[0042] Figure 6This is a diagram showing the results of the specificity identification of the genetically engineered antibody in Example 6.

[0043] Figure 7 This is a graph showing the results of titer identification of the genetically engineered antibody in Example 7. Detailed Implementation

[0044] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0045] Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this 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 is 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; diluted 1:10000 before use to obtain the enzyme-labeled secondary antibody working solution. Hi-exp medium: Optimum Biotech, product catalog number AC601501. ACK erythrocyte lysis buffer: containing 150mM NH4Cl, 10mM KHCO3, and 0.1mM Na2-EDTA, with the remainder being water; filtered through a 0.22μm filter for sterilization before use. PBST solution: PBS buffer containing 0.05% (v / v) Tween-20. Blocking solution: PBST solution containing 0.2 g / 100 mL 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, all quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0046] Example 1: Expression and purification of P30 protein

[0047] I. Constructing recombinant plasmids and obtaining recombinant bacteria

[0048] In SEQ ID NO: 6, nucleotides 1-582 represent the CP204L gene (encoding the P30 protein) from the genome of the Chinese prevalent ASFV strain Pig / HLJ / 2018. The double-stranded DNA molecule shown in SEQ ID NO: 6 encodes the protein shown in 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.

[0049] The pET-28a(+) vector was replaced with the double-stranded DNA molecule shown in SEQ ID NO: 6. Nco I and Xho A small fragment between the I restriction enzyme sites was extracted, keeping other sequences unchanged, to obtain the recombinant plasmid P30-Strep. The recombinant plasmid has been sequenced and verified.

[0050] The recombinant plasmid P30-Strep was introduced into Escherichia coli BL-21(DE3) to obtain the recombinant bacteria.

[0051] II. Preparation and purification of proteins

[0052] 1. Inoculate the recombinant bacteria obtained in step one into 1L of liquid LB medium and incubate at 37ºC with shaking at 200rpm until OD. 600nm The value was 0.8-1.0, and then the mixture was incubated at 20℃ and 200rpm for 1 hour with shaking.

[0053] 2. After completing step 1, add IPTG to the culture system and make its concentration in the system 0.5mM. Then, culture at 20℃ and 200rpm for 14-16 hours with shaking. Then, centrifuge at 4000rpm for 10min and collect the bacterial pellet.

[0054] 3. Resuspend the bacterial precipitate obtained in step 2 with 25 mL of Buffer I, then homogenize the bacterial cells using a high-pressure homogenizer, and then centrifuge at 4°C and 18000 rpm for 30 min, and collect the supernatant.

[0055] Buffer I: Contains 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl, with the remainder being water.

[0056] 4. Use an affinity chromatography column (column volume 5 mL; packing material: STarm Streptactin Beads 4FF, Changzhou Tiandi Renhe Biotechnology Co., Ltd., product catalog number SA092100). First, equilibrate with 10 column volumes using buffer I, then load the supernatant obtained in step 3 (loading volume 25 mL), then wash with 10 column volumes using buffer I, and finally elute with 5 column volumes using buffer II. Collect the post-column solution, which is the target protein solution.

[0057] Buffer II: Contains 25 mM Tris-HCl (pH 7.5), 150 mM NaCl and 2.5 mM desulfurized biotin, with the balance being water.

[0058] The target protein is the protein shown in SEQ ID NO: 5.

[0059] SDS-PAGE electrophoresis image of the target protein solution is shown below. Figure 1 .

[0060] Example 2: Obtaining target hybridoma cells using myeloma cell fusion and subclonal screening

[0061] I. Animal Immunization

[0062] SPF-grade 6-8 week old Balb / c mice were immunized according to the following procedure:

[0063] Day 1: First immunization, multiple subcutaneous injections of immunizing agent (composed of immunogen and Freund's complete adjuvant).

[0064] Day 29: Second immunization, multiple subcutaneous injections of immunizing agents (composed of immunogen and Freund's incomplete adjuvant);

[0065] Day 57: Third immunization, multiple subcutaneous injections of immunizing agents (composed of immunogen and Freund's incomplete adjuvant);

[0066] Day 64: Orbital venous blood was collected to separate serum, which was then used to test the titer of the antibody (see Example 7 for the method of antibody titer testing, the only difference being that the test antibody was used instead of the antibody diluent). Mice with high titers were selected for the fourth immunization, and the immunogen was injected intraperitoneally.

[0067] The immunogen was the target protein prepared in Example 1, and the single immunization dose per mouse was 10 μg (based on protein content).

[0068] II. Preparation and Screening of Hybridoma Cells

[0069] 1. Preparation of feeder cells

[0070] The day before fusion, well-developed Balb / c mice were euthanized by cervical dislocation and sterilized in 75% ethanol aqueous solution. Using a pre-chilled syringe, 8-10 ml of pre-chilled 0.34M sucrose aqueous solution was injected through the lower right peritoneum of the mouse, ensuring the needle did not exit the peritoneum. The peritoneal cavity was massaged with a finger for approximately 1 minute. The fluid from the peritoneal cavity was then aspirated and added to a pre-chilled 50 ml centrifuge tube, followed by pre-chilled 1640 complete culture medium. The tube was 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 1×10⁻⁶. 5 Cells / ml were then added to a 96-well cell culture plate (100 μl / well) and cultured.

[0071] 2. Preparation of spleen cells

[0072] Four days after the fourth immunization in step one, mice were euthanized by enucleation, and blood and spleen were collected separately. Serum was isolated from the blood as a positive control. A 10cm diameter culture dish was prepared, with 10ml of 1640 medium and DNase added, followed by the addition of mouse spleen. The mixture was ground and pipetted to form a single-cell suspension. The suspension was then filtered through a 70μm filter and collected into a 50ml centrifuge tube. The tube was centrifuged at 1500rpm for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in ACK erythrocyte lysis buffer. The cells were incubated at room temperature for 2 minutes, washed with 1640 medium, and then resuspended in 20ml of 1640 medium. After mixing, the cells were counted.

[0073] 3. Cell fusion

[0074] Collect healthy Sp2 / 0 mouse myeloma cells in the logarithmic growth phase and wash them with 1640 medium. Mix Sp2 / 0 mouse myeloma cells and spleen cells at a ratio of 1:1 to 1:3, centrifuge at 1500 rpm for 10 min, discard the supernatant, and agitate the cells to form a paste. Incubate at 37°C, add 1 ml of preheated 50% PEG solution dropwise, then slowly add 40 ml of preheated 1640 medium. Centrifuge at 1500 rpm for 10 min, discard the supernatant, add 10 ml of HAT medium and agitate several times, then add HAT medium to approximately 90 ml. Mix well and drop the mixture into feeder cell culture plates, 2 drops per well, and incubate.

[0075] 4. Screening of hybridoma cell positive wells

[0076] Four days after cell fusion, half of the medium was replaced with HAT medium. After about 7-10 days, the hybridoma cell clusters grew to a certain size. About 200 μl of medium was aspirated the day before the test and 200 μl of fresh HT medium was added back.

[0077] On the day of testing, the culture medium of the hybridoma cell cluster was used as the test antibody, the positive control was used as the positive control, and the serum of unimmunized mice was used as the negative control. The titer was tested (the method for testing antibody titer is described in Example 7, the only difference being that the test antibody or control was used instead of the antibody diluent), and positive clones were screened.

[0078] 5. Subcloning of positive hybridoma cells

[0079] The first subcloning was performed using HT medium. Positive cloning wells were selected for the first subcloning, and cell state and cell cluster size were observed under a microscope. Under aseptic conditions, the cells to be subcloned were gently pipetted to mix, avoiding air bubbles. 10 μl of the cell count was aspirated, and 10 μl of 0.04% trypan blue solution was added. After mixing, the cells were counted in 8 large squares, and the cell concentration was calculated. Based on the cell count results, 100-150 cells were added to 9.5 ml of medium using the limiting dilution method. After mixing, the mixture was dropped into feeder cell culture plates cultured for 1 day.

[0080] After about 5 days of subcloning, the hybridomas in each well are counted under an inverted microscope. Once the hybridoma cells have grown to a suitable size, they are tested again and positive monoclonal cells are selected for a second subcloning. The culture medium is then replaced with 1640 complete medium. Subcloning is repeated 2-3 times until the obtained monoclonal hybridoma cell line can stably secrete the required antibody.

[0081] 6. Expansion culture and cryopreservation of hybridoma cells

[0082] 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%.

[0083] ② After completing step ①, transfer the cells to a T25 cell culture flask and culture until the cell confluence reaches approximately 80%.

[0084] ③ After completing step ②, repeatedly blow the culture flask with culture medium, then transfer it to a sterile centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend the cells with 3 ml of cell cryopreservation solution, mix thoroughly, transfer the cell suspension to a cryopreservation tube, transfer it to a programmed cooling box, incubate at -80℃ for 24 h, and then transfer it to liquid nitrogen for long-term storage.

[0085] Based on the above steps, multiple hybridoma cells secreting target monoclonal antibodies (i.e., monoclonal antibodies that bind to the African swine fever virus P30 protein) were obtained, one of which was named BY017 hybridoma cell.

[0086] Example 3: Preparation and Identification of Monoclonal Antibodies

[0087] I. Obtaining Monoclonal Antibody Supernatant

[0088] Hybridoma cells BY017 were cultured in 1640 complete medium (culture time was 2-3 days), and then the supernatant was collected, which is the solution containing monoclonal antibody BY017, referred to as BY017 solution.

[0089] II. Identification of Monoclonal Antibody Subtypes

[0090] Take the BY017 solution and use the monoclonal antibody subtype identification kit according to the instructions. Monoclonal antibody subtype identification kit (Isotyping Kit for Mouse Monoclonal Antibody): Beijing Yiqiao Shenzhou Technology Co., Ltd., product catalog number SEK003.

[0091] See results Figure 2 The monoclonal antibody BY017 is the IgG2b subtype.

[0092] III. Identification of Antigenic Epitopes Recognized by Monoclonal Antibodies

[0093] 1. Design and prepare peptides

[0094] 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 position range in SEQ ID NO: 5 (e.g., peptide 110-131 is the peptide shown at positions 110-131 in SEQ ID NO: 5). Each peptide in Table 1 was prepared.

[0095] Table 1

[0096]

[0097] 2. Preparation of coated antigens

[0098] Each polypeptide prepared in step 1 was conjugated with ovalbumin (OVA) to obtain each coated antigen.

[0099] 3. ELISA testing

[0100] Test coating agents: These are either the coating agents prepared in step 2 or the target protein prepared in Example 1. Take the test coating agents and dilute them with PBS buffer to a protein concentration of 1 μg / ml to obtain the coating solution.

[0101] ① Take a 96-well plate, add coating solution (100 μl / well), incubate at 4℃ for 12 hours, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0102] ② Take the 96-well plate from step ①, add blocking solution (200 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0103] ③ Take the 96-well plate from step ②, add BY017 solution (100 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0104] ④ Take the 96-well plate from 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.

[0105] ⑤ Take the 96-well plate from step ④, add TMB colorimetric solution (100 μl / well), and react in the dark for 5-10 minutes.

[0106] ⑥ Take the 96-well plate from step ⑤, add 2M sulfuric acid solution (50 μl / well), and then measure the absorbance (OD) at 450 nm. 450 ).

[0107] See results Figure 3 Monoclonal antibody BY017 reacted with peptide 110-131.

[0108] IV. Validation of the efficacy of monoclonal antibodies

[0109] The test cells were either PAM cells or PAM cells infected with the ASFV virus strain HLJ / 18.

[0110] Total protein was extracted from lysed cells and then subjected to Western blot. Primary antibody working solution: BY017 solution was diluted with PBS buffer to an antibody concentration of 1 μg / ml. Secondary antibody working solution: Enzyme-labeled secondary antibody working solution.

[0111] See results Figure 4 (PAM represents the total protein in PAM cells, and ASFV+PAM represents the total protein in PAM cells infected with the ASFV HLJ / 18 strain). Western blot results showed that BY017 solution could recognize the P30 protein in PAM cells infected with ASFV.

[0112] Example 4: Obtaining the variable region sequence of the antibody

[0113] BY017 hybridoma cells were lysed, and total RNA was extracted and reverse transcribed to obtain cDNA. Using cDNA as a template, PCR amplification was performed using primers targeting the antibody variable region. The PCR amplification products were then recovered and sequenced to obtain the gene sequences of the antibody heavy chain variable region and the antibody light chain variable region. The amino acid sequences of the antibody heavy chain variable region and the antibody light chain variable region were further obtained.

[0114] The amino acid sequence of the heavy chain variable region of the antibody is shown in positions 22-137 of SEQ ID NO: 1 (the three CDRs are: GYTFTNSY, INPRNGDT, TRGGSQFDY). The amino acid sequence of the light chain variable region of the antibody is shown in positions 23-134 of SEQ ID NO: 3 (the three CDRs are: QSLLNSRTRKNY, WAS, KQSYNLQT).

[0115] The coding sequence of the variable region of the heavy chain of the antibody is shown in positions 64-411 of SEQ ID NO: 2. The coding sequence of the variable region of the light chain of the antibody is shown in positions 67-402 of SEQ ID NO: 4.

[0116] Example 5: Preparation of genetically engineered antibodies

[0117] 1. Preparation of recombinant expression vectors

[0118] Replace the PTT3 vector with the double-stranded DNA molecule shown in SEQ ID NO: 2 EcoR I and Not The small fragment between the recognition sites was digested with enzyme I, leaving other sequences unchanged, to obtain the heavy chain expression plasmid. The double-stranded DNA molecule shown in SEQ ID NO: 4 was then used to replace the PTT3 vector. EcoR I and Not The small fragment between the recognition sites was digested with enzyme I, while keeping other sequences unchanged, to obtain a light chain expression plasmid. The recombinant plasmid has been sequenced and verified.

[0119] The PTT3 vector is a circular plasmid, and its full sequence is shown in SEQ ID NO: 7.

[0120] The double-stranded DNA molecule shown in SEQ ID NO: 2 encodes the protein shown in SEQ ID NO: 1. In SEQ ID NO: 1, amino acid residues 1-21 form the signal peptide (which is cleaved when secreted extracellularly), amino acid residues 22-137 form the variable region of the heavy chain, and amino acid residues 138-467 form the constant region of the heavy chain.

[0121] The double-stranded DNA molecule shown in SEQ ID NO:4 encodes the protein shown in SEQ ID NO:3. In SEQ ID NO:3, amino acid residues 1 to 22 form the signal peptide (which is cleaved when secreted extracellularly), amino acid residues 23 to 134 form the variable region of the light chain, and amino acid residues 135 to 241 form the constant region of the light chain.

[0122] 2. Preparation of transfection complex

[0123] Preparation method of PEI transfection reagent solution: Add 100 mg of linear PEI transfection reagent (Mw 40000; LABLEAD, catalog number P4000) to 90 mL of Milli-Q ultrapure water, stir until completely dissolved, adjust the pH to 6.9-7.1, and make up to 100 mL with Milli-Q ultrapure water. Then filter through a 0.22 μm filter membrane and collect the filtrate.

[0124] Preparation of transfection complex (1L cell suspension): Add 500μg heavy chain expression plasmid and 500μg light chain expression plasmid to 5mL Hi-exp medium and mix well by pipetting to obtain phase A; add 3mL PEI transfection reagent solution to 5mL Hi-exp medium and mix well by pipetting to obtain phase B; add phase B to phase A, mix well by pipetting, and then incubate at room temperature for 5min.

[0125] 3. Preparation of cell suspension

[0126] Take 293F cells from the culture, count the cells, centrifuge at 800 rpm for 5 minutes, discard the supernatant, and resuspend in Hi-exp medium to make the cell content 1×10⁻⁶. 6 Cells / mL.

[0127] 4. Add the transfection complex dropwise to 1L of cell suspension and incubate at 130rpm for 5 days (environmental conditions: 37℃, 8% CO2). Then centrifuge at 4000rpm for 20min, collect the supernatant, and filter it through a 0.45μm filter membrane and collect the filtrate.

[0128] 5. Using an affinity chromatography column (column volume 10 mL; packing material: protein At Beads LX, Changzhou Tiandi Renhe Biotechnology Co., Ltd., product catalog number SA08501L), first equilibrate the chromatography column with PBS buffer, then load the filtrate obtained in step 4 (loading volume 950 mL), 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 volumes of the post-column solution with 1 volume of neutralization buffer (i.e., pH 9.0, 1 M Tris-HCl buffer) to obtain a mixture.

[0129] 6. Take the mixture obtained in step 5 and concentrate it using a 30K ultrafiltration tube (Millipore, UFC903096). Replace the buffer system with PBS buffer to obtain the genetically engineered antibody solution.

[0130] SDS-PAGE electrophoresis image of the genetically engineered antibody solution is shown below. Figure 5 .

[0131] Example 6: Specificity Identification of Genetically Engineered Antibodies

[0132] Test coating agents: These are either the coating agents prepared in step 3.2 of Example 3 or the target protein prepared in Example 1. Take the test coating agents and dilute them with PBS buffer to a protein concentration of 1 μg / ml; this is the coating solution. Antibody dilution solution: Take the genetically engineered antibody solution prepared in Example 5 and dilute it with PBS buffer to an antibody concentration of 2 μg / ml.

[0133] ① Take a 96-well plate, add coating solution (100 μl / well), incubate at 4℃ for 12 hours, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0134] ② Take the 96-well plate from step ①, add blocking solution (200 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0135] ③ Take the 96-well plate from step ②, add antibody dilution buffer (100 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0136] ④ Take the 96-well plate from 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.

[0137] ⑤ Take the 96-well plate from step ④, add TMB colorimetric solution (100 μl / well), and react in the dark for 5-10 minutes.

[0138] ⑥ Take the 96-well plate from step ⑤, add 2M sulfuric acid solution (50 μl / well), and then measure the absorbance (OD) at 450 nm. 450 ).

[0139] See results Figure 6 The genetically engineered antibody prepared in Example 5 reacted with polypeptide 110-131.

[0140] Example 7: Identification of the titer of genetically engineered antibodies

[0141] Take the target protein solution prepared in Example 1 and dilute it with PBS buffer to a protein concentration of 1 μg / ml to obtain the coating solution. Preparation method of antibody dilution buffer: Take the genetically engineered antibody solution prepared in Example 5, first dilute it with PBS buffer to an antibody concentration of 2 μg / ml, and then perform a 2-fold serial dilution with PBS buffer.

[0142] 1. Take a 96-well plate, add coating buffer (100 μl / well), incubate at 4°C for 24 hours, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0143] 2. Take the 96-well plate from step 1, add blocking buffer (200 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0144] 3. Take the 96-well plate from step 2, add antibody dilution buffer (100 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0145] 4. Take the 96-well plate from 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.

[0146] 5. Take the 96-well plate after completing step 4, add TMB colorimetric solution (100 μl / well), and react in the dark for 5-10 minutes.

[0147] 6. Take the 96-well plate from step 5, add 2M sulfuric acid solution (50 μl / well), and then measure the absorbance (OD) at 450 nm. 450 ).

[0148] See results Figure 7 .

[0149] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. An IgG antibody that specifically binds to the P30 protein of African swine fever virus, comprising a light chain and a heavy chain; wherein the CDR1, CDR2, and CDR3 in the variable region of the heavy chain are as shown in positions 47-54, 72-79, and 118-126 of SEQ ID NO: 1, respectively; and the CDR1, CDR2, and CDR3 in the variable region of the light chain are as shown in positions 49-60, 78-80, and 117-124 of SEQ ID NO: 3, respectively.

2. The IgG antibody as described in claim 1, characterized in that: The heavy chain variable region is shown in bits 22-137 of SEQ ID NO: 1; The variable region of the light chain is shown in positions 23-134 of SEQ ID NO:

3.

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

3.

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

5. The use of the IgG antibody according to claim 1, 2, or 3 in the preparation of the product; wherein the product has the following function (e1), (e2), or (e3): (e1) is used to bind to the African swine fever virus P30 protein fragment; (e2) is used to bind to the African swine fever virus P30 protein; (e3) is used to bind to African swine fever virus.

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

7. The use of the IgG antibody according to claim 1, 2 or 3 in the preparation of products for identifying African swine fever virus.

8. The 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. The use of the IgG antibody of claim 1, 2 or 3 in the preparation of a medicament for neutralizing African swine fever virus.

10. The use of the IgG antibody of claim 1, 2 or 3 in the preparation of a medicament for the prevention and / or treatment of African swine fever virus infection.

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