Monoclonal antibody BY013 combined with African swine fever virus P30 protein and application thereof

Abstract

The invention discloses a monoclonal antibody BY013 combined with African swine fever virus P30 protein and application of the monoclonal antibody BY013. 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 130 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 BY013 that binds to African swine fever virus P30 protein and its application. Background Art

[0002] African swine fever (ASF) is an acute, highly virulent, and highly contagious disease of pigs caused by African swine fever virus (ASFV). Its clinical features include high fever, loss of appetite, skin cyanosis, and bleeding of internal organs. The disease has a short course, a high incidence rate, and a fatality rate that can reach up to 100%. As a legally reportable animal disease of the World Organization for Animal Health (WOAH) and a Class I animal disease in China, ASF has always received high attention from countries around the world. The disease was first confirmed in Kenya, Africa in 1921. Since 2007, ASF has occurred, spread, and prevailed in many countries around the world. It was introduced into China in 2018, bringing huge direct economic losses and far-reaching industrial impacts. Although ASF has been discovered for nearly a century, there is still a lack of effective vaccines and therapeutic drugs globally. Prevention and control measures mainly rely on early diagnosis, culling of diseased pigs, strict quarantine, and biosafety management.

[0003] ASFV is the only member of the genus Asfivirus in the family Asfarviridae and is also the only known DNA arbovirus with a double-layer envelope structure. The virus particles are icosahedral in symmetry, with a diameter of about 260 nm. The genome is a double-stranded linear DNA of 170 - 194 kb, containing 151 - 167 open reading frames and encoding 150 - 200 proteins. The virus has a unique replication cycle and mainly infects monocytes and macrophages through endocytosis and macropinocytosis. The replication of its genome, virus assembly, and release are all completed in the host cytoplasm.

[0004] As a core structural protein of African swine fever virus (ASFV), P30 protein is located in the inner membrane of virus particles, encoded by the CP204L gene, with a molecular mass of approximately 23.6 kDa and is a phosphorylated protein. This protein plays a key role in the process of virus invading host cells and participates in the initial stage of infection by mediating virus internalization. P30 protein can be detected 2 - 4 hours after ASFV infects cells. It is an early - expressed protein, persists throughout the infection cycle, and can induce the host to produce neutralizing antibodies. Given its immunological characteristics, P30 protein has been widely used as a candidate antigen for ASF serological diagnosis. However, as a major structural protein and important protective antigen of the virus, its three - dimensional structure has not been resolved yet. Therefore, preparing specific monoclonal antibodies against P30 protein and conducting epitope screening not only have important significance for elucidating the biological functions of this protein, but also can provide key tools for the research and development of ASFV detection technologies and its structural biology research. Summary of the Invention

[0005] The object of the present invention is to provide a monoclonal antibody BY013 that binds to African swine fever virus P30 protein and its applications.

[0006] The present invention provides an IgG antibody, which consists of a light chain and a heavy chain; CDR1, CDR2, and CDR3 in the heavy - chain variable region of the heavy chain are successively shown as positions 47 - 54 (GYAFPYYL), 72 - 79 (INPGSGGT), and 118 - 130 (AASGYSGKFYFDY) in SEQ ID NO: 1; CDR1, CDR2, and CDR3 in the light - chain variable region of the light chain are successively shown as positions 49 - 60 (QSLLNSGTRKNY), 78 - 80 (WAS), and 117 - 124 (KQSYYLWT) in SEQ ID NO: 3.

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

[0008] Specifically, the heavy - chain variable region is shown as positions 22 - 141 in SEQ ID NO: 1.

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

[0010] Specifically, the heavy chain is as follows (a1) or (a2): (a1) the protein shown as positions 22 - 471 in SEQ ID NO: 1; (a2) the protein shown as SEQ ID NO: 1.

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

[0014] (c1) a DNA molecule whose coding region is shown by positions 64 - 1413 in SEQ ID NO: 2;

[0015] (c2) a DNA molecule whose coding region is shown by positions 64 - 1416 in SEQ ID NO: 2;

[0016] (c3) a DNA molecule whose coding region is shown by positions 1 - 1413 in SEQ ID NO: 2;

[0017] (c4) a DNA molecule whose coding region is shown by SEQ ID NO: 2.

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

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

[0020] (d2) a DNA molecule whose coding region is shown by positions 67 - 726 in SEQ ID NO: 4;

[0021] (d3) a DNA molecule whose coding region is shown by positions 1 - 723 in SEQ ID NO: 4.

[0022] (d4) a DNA molecule whose coding region is shown by SEQ ID NO: 4.

[0023] The present invention also protects the application of any of the above - mentioned IgG antibodies in the preparation of a product; the function of the product is as follows (e1) or (e2) or (e3):

[0024] (e1) for binding to the African swine fever virus P30 protein fragment;

[0025] (e2) for binding to the African swine fever virus P30 protein;

[0026] (e3) for binding to the African swine fever virus.

[0027] The present invention also protects the use of any one of the above-mentioned IgG antibodies in the preparation of a product for identifying African swine fever virus P30 protein.

[0028] The present invention also protects the use of any one of the above-mentioned IgG antibodies in the preparation of a product for identifying African swine fever virus.

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

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

[0031] The present invention also protects the use of any one of the above-mentioned IgG antibodies in the preparation of a drug for preventing and / or treating African swine fever virus infectious diseases.

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

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

[0034] Specifically, the African swine fever virus P30 protein fragment is as shown in positions 74-95 of SEQ ID NO: 5.

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

[0036] [[ID=X]]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 the structural analysis of its P30 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the SDS-PAGE electrophoresis diagram in Example 1.

[0038] Figure 2 It is the result diagram of monoclonal antibody subtype identification in Example 3.

[0039] Figure 3 It is the result diagram of the identification of antigenic epitopes recognized by monoclonal antibodies in Example 3.

[0040] Figure 4 It is the result diagram of the verification of the effect of monoclonal antibodies in Example 3.

[0041] Figure 5 It is the SDS-PAGE electrophoresis diagram in Example 5.

[0042] Figure 6 Note: In the original text, there is an unclear line "[[ID=X]]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 the structural analysis of its P30 protein. " as the ID "X" is not defined in the original. I translated it as best as possible while maintaining the format. If this is an error in the original, it may need to be corrected for a more accurate translation.The figure shows the results of the specificity identification of the genetically engineered antibody in Example 6.

[0043] Figure 7 The figure shows the results of the titer identification of the genetically engineered antibody in Example 7. Detailed implementation manners

[0044] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided examples are only for clarifying the present invention and not for limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements and do not limit the present invention in any way.

[0045] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. 1640 complete medium: 1640 medium containing 10% FBS. 1640 medium, the full name is RPMI 1640 medium. Enzyme-labeled secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG): Zhongshan Golden Bridge Co., Ltd., Beijing, product catalog number ZB-2305; when used, it is diluted 1:10000, which is the working solution of the enzyme-labeled secondary antibody. Hi-exp medium: Optmize Co., Ltd., product catalog number AC601501. ACK red blood cell lysate: containing 150 mM NH4Cl, 10 mM KHCO3 and 0.1 mM Na2-EDTA, the balance is water; filtered and sterilized with a 0.22 μm filter membrane before use. PBST solution: PBS buffer containing 0.05% (volume percentage) Tween-20. Blocking solution: PBST solution containing 0.2 g / 100 mL BSA. Unless otherwise specified, cell culture is carried out in a cell culture incubator (37 °C, 5% CO2). Unless otherwise specified, the PBS buffer used in the examples is PBS buffer with a pH of 8.0. Unless otherwise specified, the quantitative tests in the following examples are all set with three repeated experiments, and the results are averaged.

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

[0047] I. Construction of recombinant plasmid and obtaining of recombinant bacteria

[0048] In SEQ ID NO: 6, nucleotides 1-582 represent the CP204L gene (encoding the P30 protein) in 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.

[0049] The recombinant plasmid P30-Strep was obtained by replacing the small fragment between the NcoI and XhoI restriction sites of the pET-28a(+) vector with the double-stranded DNA molecule shown in SEQ ID NO: 6, while keeping the other sequences unchanged. The recombinant plasmid was sequenced and verified.

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

[0051] 2. Protein Preparation and Purification

[0052] 1. Inoculate the recombinant bacteria obtained in step 1 into 1 L 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.

[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°C and 200rpm with shaking for 14-16 hours, then centrifuge at 4000rpm for 10 minutes to collect the bacterial precipitate.

[0054] 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.

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

[0056] 4. Use an affinity chromatography column (5 mL column volume; 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 obtained in step 3 (load volume 25 mL), then 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.

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

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

[0059] The SDS-PAGE electrophoresis pattern of the target protein solution is shown in Figure 1 .

[0060] Example 2: Obtaining the target hybridoma cells by myeloma cell fusion and subcloning 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: The first immunization, multiple-point subcutaneous injection of the immunizing agent (composed of the immunogen and Freund's complete adjuvant);

[0064] Day 29: The second immunization, multiple-point subcutaneous injection of the immunizing agent (composed of the immunogen and Freund's incomplete adjuvant);

[0065] Day 57: The third immunization, multiple-point subcutaneous injection of the immunizing agent (composed of the immunogen and Freund's incomplete adjuvant);

[0066] Day 64: Collect orbital venous blood to isolate serum and use it as the test antibody to detect the titer (the method for detecting the antibody titer is shown in Example 7, with the only difference being that the test antibody is used instead of the antibody diluent), select the mice with high titers for the fourth immunization, and inject the immunogen intraperitoneally.

[0067] The immunogen is the target protein prepared in Example 1, and the immunization dose for a single mouse per time is 10 μg (calculated based on the protein amount).

[0068] II. Preparation and screening of hybridoma cells

[0069] 1. Preparation of feeder cells

[0070] One day before fusion, decapitate and sacrifice well-developed Balb / c mice, and place them in a 75% ethanol aqueous solution for disinfection. Use a pre-cooled syringe to aspirate 8-10 ml of pre-cooled 0.34 M sucrose aqueous solution, insert the needle into the mouse's right lower abdomen peritoneum, do not let the needle out of the peritoneum, and massage the abdomen with fingers for about 1 min. Then aspirate the liquid in the mouse's abdominal cavity, add it to a pre-cooled 50 ml centrifuge tube, add pre-cooled 1640 complete medium, centrifuge at 1500 rpm for 5 min, discard the supernatant, and resuspend the cell pellet with HAT medium to make the cell concentration 1×10 5cells / ml, and then add the cell suspension to a 96-well cell culture plate (100 μl / well) and culture.

[0071] 2. Preparation of splenocytes

[0072] Four days after the 4th immunization in Step 1, the mice are sacrificed by eye enucleation, and blood and spleens are collected respectively. Serum is isolated from the blood 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 form a single cell suspension, then filter through a 70 μm filter and collect the filtrate into a 50 ml centrifuge tube, then centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend the cell pellet with ACK red blood cell lysate, incubate at room temperature for 2 min, then wash the cells with 1640 medium, then add 20 ml of 1640 medium to resuspend the cells, pipette and mix well and count.

[0073] 3. Cell fusion

[0074] 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 splenocytes at a ratio of 1:1 - 1:3, then centrifuge at 1500 rpm for 10 min, discard the supernatant, tap the cells to make a paste, then place in a 37 °C water bath, add 1 ml of preheated 50% PEG solution drop by drop, then slowly add 40 ml of preheated 1640 medium, then centrifuge at 1500 rpm for 10 min, discard the supernatant, add 10 ml of HAT medium and pipette up and down several times, then add HAT medium to about 90 ml, mix well and pipette into the feeder cell culture plate, 2 drops per well, and culture.

[0075] 4. Screening of positive wells of hybridoma cells

[0076] Four days after cell fusion, replace half of the HAT medium. After about 7 - 10 days, the hybridoma cell clusters grow to a certain size. About 200 μl of the medium is aspirated one day before detection, and 200 μl of fresh HT medium is added again.

[0077] On the day of detection, aspirate the culture medium of the hybridoma cell clusters as the test antibody, use the positive control as the positive control, and the serum of non-immunized mice as the negative control to detect the titer (the method for detecting the antibody titer is shown in Example 7, the only difference is replacing the antibody diluent with the test antibody or control), and screen positive clones.

[0078] 5. Subcloning of positive hybridoma cells

[0079] For the first subcloning, use HT medium; select positive clone wells for the first subcloning, observe the cell status and the size of cell clusters under a microscope; gently pipette and mix the cells to be subcloned under sterile conditions, avoiding blowing out air bubbles; aspirate 10 μl for counting, add 10 μl of 0.04% trypan blue solution to the counted cells, mix well, count the cells in 8 large grids, and calculate the cell concentration; according to the cell counting result, by the method of limiting dilution, aspirate 100 - 150 cells and add them to 9.5 ml of medium, mix well, and then pipette and add the mixture to a feeder cell culture plate that has been cultured for 1 day.

[0080] About 5 days after subcloning, count the hybridomas in each well under an inverted microscope. When the hybridoma cells grow to an appropriate size, perform detection again and screen for positive monoclonal cells for the second subcloning, and change the medium to 1640 complete medium; continue subcloning 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 Transfer the identified positive hybridoma cells for expansion culture to a 24 - well cell culture plate and culture until the confluence reaches about 80%.

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

[0084] ③ After completing step ②, repeatedly pipette the inner wall of the culture flask with the medium, then transfer 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 well, transfer the cell suspension to a cryopreservation tube, transfer to a programmable freezing box, let it stand at - 80 °C for 24 h, and then transfer to liquid nitrogen for long - term storage.

[0085] Based on the above steps, multiple hybridoma cell lines secreting the target monoclonal antibody (i.e., monoclonal antibody binding to African swine fever virus P30 protein) were obtained, and one of the hybridoma cell lines was named BY013 hybridoma cell.

[0086] Example 3. Preparation and identification of monoclonal antibody

[0087] I. Obtaining of monoclonal antibody supernatant

[0088] Culture hybridoma cell BY013 with 1640 complete medium (culture time is 2 - 3 days), then collect the supernatant, which is the solution containing monoclonal antibody BY013, abbreviated as BY013 solution.

[0089] II. Identification of monoclonal antibody subtype

[0090] Take the BY013 solution and use the monoclonal antibody isotyping kit according to the instructions. Monoclonal antibody isotyping kit (Isotyping Kit for Mouse Monoclonal Antibody): Beijing Sino Biological Science and Technology Co., Ltd., product catalog number SEK003.

[0091] See the results Figure 2 Monoclonal antibody BY013 is of IgG1 subtype.

[0092] 3. Identification of Antigen Epitopes Recognized by Monoclonal Antibodies

[0093] 1. Design and preparation of peptides

[0094] Based on the amino acid sequence of the ASFV P30 protein, polypeptides were designed as shown in Table 1. The polypeptide names are consistent with the position interval of the polypeptide corresponding to SEQ ID NO: 5 (e.g., polypeptide 74-95 is the polypeptide represented by positions 74-95 in SEQ ID NO: 5). Each polypeptide in Table 1 was prepared separately.

[0095] Table 1

[0096]

[0097] 2. Preparation of coating

[0098] The polypeptides prepared in step 1 were coupled to ovalbumin (OVA) to obtain coatings.

[0099] 3. ELISA test

[0100] 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.

[0101] 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.

[0102] 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 3 times with PBST solution, and pat dry.

[0103] ③ Take the 96-well plate prepared in step ②, add BY013 solution (100 μl / well), incubate at room temperature for 1 hour, discard the supernatant, wash three times with PBST solution, and pat dry.

[0104] ④ Take the 96-well plate after completing step ③, add the 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 after completing step ④, add the TMB chromogenic solution (100 μl / well), and react in the dark for 5 - 10 minutes.

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

[0107] The results are shown in Figure 3 . The monoclonal antibody BY013 reacts with polypeptide 74 - 95.

[0108] IV. Verification of the effect of the monoclonal antibody

[0109] The test cells are respectively: PAM cells or PAM cells infected with the ASFV virus HLJ / 18 strain.

[0110] After lysing the test cells, extract the total protein, and then perform Western blot. Primary antibody working solution: Dilute the BY013 solution with PBS buffer to an antibody concentration of 1 μg / ml. Secondary antibody working solution: Enzyme-labeled secondary antibody working solution.

[0111] The results are shown in Figure 4 (PAM represents the total protein of PAM cells, and ASFV + PAM represents the total protein of PAM cells infected with the ASFV virus HLJ / 18 strain). The Western blot results show that the BY013 solution can recognize the P30 protein in PAM cells infected with the ASFV virus.

[0112] Example 4. Obtaining the variable region sequences of the antibody

[0113] Take the BY013 hybridoma cells, perform cell lysis, then extract total RNA and reverse transcribe to obtain cDNA. Using cDNA as a template, perform PCR amplification with primers specific for the antibody variable region, then recover the PCR amplification product and sequence it to obtain the gene sequence of the heavy chain variable region of the antibody and the gene sequence of the light chain variable region of the antibody, and further obtain the amino acid sequence of the heavy chain variable region of the antibody and the amino acid sequence of the light chain variable region of the antibody.

[0114] The amino acid sequence of the heavy chain variable region of the antibody is shown as positions 22 - 141 in SEQ ID NO: 1 (the three CDRs are in turn: GYAFPYYL, INPGSGGT, AASGYSGKFYFDY). The amino acid sequence of the light chain variable region of the antibody is shown as positions 23 - 134 in SEQ ID NO: 3 (the three CDRs are in turn: QSLLNSGTRKNY, WAS, KQSYYLWT).

[0115] The coding sequence of the heavy chain variable region of the antibody is shown as positions 64 - 423 in SEQ ID NO: 2. The coding sequence of the light chain variable region of the antibody is shown as positions 67 - 402 in SEQ ID NO: 4.

[0116] Example 5: Preparation of genetically engineered antibody

[0117] 1. Preparation of recombinant expression vector

[0118] Replace the small fragment between the EcoRI and NotI restriction enzyme recognition sites of the PTT3 vector with the double-stranded DNA molecule shown in SEQ ID NO: 2, keeping other sequences unchanged, to obtain the heavy chain expression plasmid. Replace the small fragment between the EcoRI and NotI restriction enzyme recognition sites of the PTT3 vector with the double-stranded DNA molecule shown in SEQ ID NO: 4, keeping other sequences unchanged, to obtain the light chain expression plasmid. The recombinant plasmids have been verified by sequencing.

[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, the amino acid residues at positions 1 - 21 form a signal peptide (which is excised when secreted extracellularly), the amino acid residues shown at positions 22 - 141 form the heavy chain variable region, and the amino acid residues at positions 142 - 471 form the heavy chain constant region.

[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, the amino acid residues at positions 1 to 22 form a signal peptide (which is excised when secreted extracellularly), the amino acid residues at positions 23 - 134 form the light chain variable region, and the amino acid residues at positions 135 - 241 form the light chain constant region.

[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 Co., product catalog number P4000) into 90 mL of Milli-Q ultrapure water, stir evenly until completely dissolved, adjust the pH to 6.9 - 7.1, make up the volume to 100 mL with Milli-Q ultrapure water, then filter through a 0.22 μm filter membrane and collect the filtrate.

[0124] Preparation method of transfection complex (dosage for 1 L of cell suspension): Add 500 μg of heavy chain expression plasmid and 500 μg of light chain expression plasmid into 5 mL of Hi-exp medium, pipette and mix well, which is liquid phase A; Add 3 mL of PEI transfection reagent solution into 5 mL of Hi-exp medium, pipette and mix well, which is liquid phase B; Add liquid phase B into liquid phase A, pipette and mix well, then place at room temperature for 5 min.

[0125] 3. Preparation of cell suspension

[0126] Take the cultured 293F cells, after cell counting, centrifuge at 800 rpm for 5 minutes, discard the supernatant, resuspend with Hi-exp medium to make the cell content 1×10 6 cells / mL.

[0127] 4. Dropwise add the transfection complex into 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 filter membrane and collect the filtrate.

[0128] 5. Use an affinity chromatography column (column volume is 10 mL; packing is protein At Beads LX, Changzhou Tiandi Renhe Biotechnology Co., product catalog number SA08501L), first equilibrate the chromatography column with PBS buffer, then load the filtrate obtained in step 4 (loading volume is 950 mL), then wash with PBS buffer for 10 - 15 column volumes, and then elute with glycine buffer (pH 2.5 - 3.0, 0.1 M) and collect the solution after passing through the column. Mix 10 volumes of the solution after passing through the column with 1 volume of neutralization buffer (i.e., Tris-HCl buffer with pH 9.0, 1 M) to obtain a mixed solution.

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

[0130] The SDS-PAGE electrophoresis pattern of the genetically engineered antibody solution is shown in Figure 5 .

[0131] Example 6, Specific Identification of Genetically Engineered Antibody

[0132] Test coating antigens: Each of the coating antigens prepared in Step 2 of Step 3 of Example 3 or the target protein prepared in Example 1, respectively. Take the test coating antigen and dilute it with PBS buffer to a protein concentration of 1 μg / ml to obtain 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 the coating solution (100 μl / well), incubate at 4 °C for 12 hours, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0134] ② Take the 96-well plate that has completed Step ①, add the 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 that has completed Step ②, add the antibody dilution solution (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 that has completed Step ③, add the working solution of enzyme-labeled secondary antibody (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 that has completed Step ④, add the TMB chromogenic solution (100 μl / well), and react in the dark for 5 - 10 minutes.

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

[0139] The results are shown in Figure 6 . The genetically engineered antibody prepared in Example 5 reacts with polypeptide 74 - 95.

[0140] Example 7, Titer Identification of Genetically Engineered Antibody

[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 the antibody dilution solution: 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 2-fold serial dilution with PBS buffer.

[0142] 1. Take a 96-well plate, add the coating solution (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 that has completed step 1, add the blocking solution (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 that has completed step 2, add the antibody dilution solution (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 that has completed step 3, add the working solution of enzyme-labeled secondary antibody (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 that has completed step 4, add the TMB chromogenic solution (100 μl / well), and react in the dark for 5 - 10 minutes.

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

[0148] The results are shown in Figure 7 .

[0149] The above has detailed the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In general, according to the principle of the present invention, this application intends to cover any changes, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made with conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended 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 118-130 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 22-141 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 (a1) or (a2): (a1) the protein shown at positions 22-471 in SEQ ID NO: 1; (a2) the protein shown in SEQ ID NO: 1; The light chain is (b1) or (b2) as follows: (b1) the protein shown at positions 23 to 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), (e2) or (e3): (e1) for binding to the African swine fever virus P30 protein fragment; (e2) for binding to African swine fever virus P30 protein; (e3) for binding to 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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