African swine fever virus p30 protein monoclonal antibody and preparation method and application thereof

Abstract

The present invention belongs to biotechnology, and specifically relates to monoclonal antibodies against p30 protein of African swine fever virus and its preparation method and application. The main technology includes using recombinant p30 protein prepared by prokaryotic expression as antigen, immunizing BALB / c mice, and then using hybridoma technology, through cell fusion, indirect ELISA method screening, limited dilution method subcloning, and hybridoma continuous passage culture, to obtain a hybridoma cell line that can stably secrete monoclonal antibodies against p30 protein of African swine fever virus. The present invention also discloses the sequence of the prepared monoclonal antibody immunogen, the subtype of the monoclonal antibody, the CDR sequence, the accession number of the hybridoma cell line, and the application of the monoclonal antibody in Western Blot experiment, indirect immunofluorescence experiment, neutralization experiment and blocking ELISA method for detecting African swine fever virus antibodies in pig serum.

Description

Technical Field

[0001] The present invention belongs to biotechnology, and specifically relates to a monoclonal antibody against p30 protein of African swine fever virus and a preparation method and application thereof. Background Art

[0002] African swine fever (ASF) is an acute, febrile, highly contagious disease of pigs caused by infection with African swine fever virus (ASFV). Clinically, it is characterized by high fever, bleeding in the skin and internal organs, ataxia and loss of appetite, with a mortality rate close to 100%. Clinical manifestations include fever, cyanosis of the skin, bleeding in the lymph nodes, kidneys, and gastrointestinal mucosa.

[0003] African swine fever virus belongs to the Asfarviridae family and the Asfivirus genus, and is the only member of this family. The ASFV genome size is 170~190 kb. It is a large double-stranded DNA virus with an envelope that replicates in the cytoplasm and encodes 160~175 genes. The diameter of the virus particle is 175-215 nm and has icosahedral symmetry. The B646L gene encoding the VP72 protein was used to analyze ASFV isolates from different regions, and the ASFV epidemic strains can be divided into 22 major genotypes, among which the main genotype II is prevalent in China.

[0004] The structural protein p30 of African swine fever virus is encoded by the viral gene CP204L. It plays an important role in the process of virus entry into host cells and is more abundant in the early stage of viral infection. In the study of protective immune response of the body, the p30 protein has a certain inhibitory effect on the internalization of the virus and is associated with the induction of neutralizing antibodies during infection. Therefore, the p30 protein is the main antigenic region for the detection of African swine fever antibodies. At present, the detection of African swine fever virus is mainly based on polymerase chain reaction (PCR), which detects the genomic DNA of African swine fever virus, but there is a lack of detection methods for African swine fever virus antibodies. Summary of the invention

[0005] Based on the highly pathogenic ASFV-SY18 strain of genotype II that first broke out in China, the purpose of the present invention is to provide an anti-p30 monoclonal antibody and the establishment of an African swine fever virus antibody blocking ELISA method.

[0006] A monoclonal antibody against the p30 protein of African swine fever virus, characterized in that the monoclonal antibody is produced by a hybridoma cell with a deposit number of CCTCC NO: C2024103.

[0007] The monoclonal antibody is characterized in that the heavy chain variable region of the monoclonal antibody includes VH-FR1, VH-CDR1, VH-FR2, VH-CDR2, VH-FR3, VH-CDR3 and VH-FR4, and its amino acid sequence is shown in SEQ ID NO.2-SEQ ID NO.8; the light chain variable region includes VL-FR1, VL-CDR1, VL-FR2, VL-CDR2, VL-FR3, VL-CDR3 and VL-FR4, and its amino acid sequence is shown in SEQ ID NO.9-SEQ ID NO.15, which encodes the monoclonal antibody against African swine fever virus p30 protein according to any one of claims 1 to 3.

[0008] The heavy chain of this monoclonal antibody is of IgG1 type and the light chain is of κ type.

[0009] The method for preparing the monoclonal antibody is characterized in that it is prepared by the following steps: using African swine fever virus p30 recombinant protein as an antigen, inoculating BALB / c mice, taking spleen cells of the immunized mice and fusing them with myeloma cells, obtaining hybridoma cells after subclone screening and indirect ELISA identification, and the monoclonal antibody is produced by the hybridoma cells.

[0010] More specifically:

[0011] The accession number of the hybridoma cell line is CCTCC NO: C2024103. The hybridoma cell line p30-6E12 is named Hybridoma cell line p30-6E12, and the deposit time is September 30, 2024. The deposit unit is China Center for Type Culture Collection, and the deposit address is Wuhan University, Wuhan, China.

[0012] The African swine fever virus p30 recombinant protein selected a 9-189 aa amino acid fragment with good antigenicity based on the results of antigenicity analysis, and its N-terminal to C-terminal sequence is SEQ ID NO.1.

[0013] p30 recombinant protein:

[0014] MKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKX.

[0015] The method is further characterized in that the monoclonal antibody is obtained by injecting the hybridoma cells into BALB / c mice, collecting ascites 7 to 14 days after the injection, and purifying it with a kit.

[0016] A fragmented monoclonal antibody F(ab')2 against African swine fever virus p30 protein, characterized in that the F(ab')2 is obtained by digesting the monoclonal antibody with pepsin.

[0017] A method for detecting African swine fever virus, characterized in that the detection method is achieved through the monoclonal antibody or the F(ab')2.

[0018] An ELISA kit for detecting African swine fever virus, characterized in that the kit comprises the monoclonal antibody or the F(ab')2.

[0019] Specifically:

[0020] The invention provides a hybridoma cell, which is a hybridoma cell 6E12 whose deposit number is CCTCC NO: C2024103.

[0021] The present invention provides a monoclonal antibody against African swine fever virus p30 protein, which is secreted by hybridoma cell 6E12 with a deposit number of CCTCC NO: C2024103. The hybridoma cell provided by the present invention can be stably passaged and continuously secrete the anti-p30 monoclonal antibody.

[0022] The antibody provided by the present invention can specifically recognize the p30 protein of African swine fever virus in terms of biological function, and can be used for Western Blot and indirect immunofluorescence experiments.

[0023] The present invention also provides subtypes of the monoclonal antibody against the p30 protein of the African swine fever virus: the heavy chain is of IgG1 type, the light chain is of κ type, and the antibody variable region sequences are also provided. The heavy chain sequences are VH-FR1, VH-CDR1, VH-FR2, VH-CDR2, VH-FR3, VH-CDR3 and VH-FR4, and the light chain sequences are VL-FR1, VL-CDR1, VL-FR2, VL-CDR2, VL-FR3, VL-CDR3 and VL-FR4.

[0024] VH-FR1(SEQ NO.2): VKVVESGGGLVQPGTSLRLSC ATS

[0025] VH-CDR1(SEQ NO.3): GFTFTDYY

[0026] VH-FR2(SEQ N0.4): M S W V R Q P P G E A L E W L G F

[0027] VH-CDR2(SEQ NO.5): I R N K P A G Y T T

[0028] VH-FR3(SEQ NO.6): E Y S A S V K G R F T I S R D N S Q S I L Y L Q L NT L R T E D S A T Y Y C

[0029] VH-CDR3(SEQ NO.7): A R Y V Y Y A Y D G Y F D V

[0030] VH-FR4(SEQ NO.8): W G A G T T V

[0031] VL-FR1(SEQ NO.9): D I V I T Q S P A S L A V S L G Q R A T I S Y R A S

[0032] VL-CDR1(SEQ NO.10): K S V S T S G Y S Y

[0033] VL-FR2(SEQ NO.11): M H W N Q Q K P G Q P P R L L I Y

[0034] VL-CDR2(SEQ NO.12) : L V S

[0035] VL-FR3(SEQ NO.13): N L E S G V P A R F S G S G S G T D F T L N I H PV E E E D A A T Y Y C

[0036] VL-CDR3(SEQ NO.14): Q H I R E L T R

[0037] VL-FR4(SEQ NO.15): S E G G Q S W K

[0038] The present invention provides a fragmented monoclonal antibody p30-F(ab')2 against African swine fever virus p30 protein. The antibody can still specifically identify the African swine fever virus p30 protein and can also be used for Western Blot and indirect immunofluorescence experiments.

[0039] The invention also provides anti-African swine fever virus p30 protein monoclonal antibodies and fragmented monoclonal antibodies for establishing an African swine fever virus antibody detection blocking ELISA method.

[0040] The principle of the African swine fever virus antibody detection blocking ELISA method is: first, the antigen is coated, then the serum sample to be tested is incubated, and then the monoclonal antibody and the enzyme-labeled secondary antibody are incubated. The antibody (polyclonal antibody) in the sample to be tested (serum sample) will preferentially bind to the coated antigen to block the binding of the antigen with the monoclonal antibody of the present invention, thereby achieving an inhibitory effect, and the antibody level in the serum is determined by calculating the blocking rate to determine whether it is positive. This blocking ELISA is more specific and sensitive than the general indirect ELISA.

[0041] Detection formula: blocking rate = (negative serum OD 450 nm value - serum OD 450 value) / negative serum OD 450 Value × 100%‌ Beneficial Effects

[0042] The invention provides a novel monoclonal antibody and fragmented antibody against African swine fever p30 protein, the sequence of which has not been reported in the literature. The monoclonal antibody and fragmented antibody are used to detect African swine fever virus, and have good specificity in Western Blot and indirect immunofluorescence experiments; the 6E12 hybridoma cell line is stable in passage; the monoclonal antibody and fragmented antibody against African swine fever virus p30 protein provided by the invention can also be used to detect African swine fever virus antibodies in the serum of pigs infected with African swine fever virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for use in the embodiments will be briefly introduced below. The following drawings only show certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0044] Figure 1This is an SDS-PAGE diagram for the expression verification of African swine fever virus p30 recombinant protein (where M: protein marker; 1: induced pET-28a empty vector; 2: uninduced; 3: induced whole bacteria; 4: supernatant after induction; 5: precipitate after induction). The CP204L gene of the African swine fever virus type II strain was cloned into the pET-28a prokaryotic expression vector, transferred into BL21 competent cells, and induced for expression with 1mM IPTG.

[0045] Figure 2 SDS-PAGE image for the purification verification of African swine fever virus p30 recombinant protein (M: protein marker; 1: unpurified protein; 2: flow-through; 3, 4, 5: purified protein). The African swine fever virus p30 recombinant protein expressed in inclusion bodies was purified using an affinity chromatography column.

[0046] Figure 3 The results of serum antibody titer determination after mice were immunized with African swine fever virus p30 protein. BALB / c mice were immunized with purified p30 recombinant protein, and serum was collected from mice 14 days after the second immunization for serum antibody titer determination.

[0047] Figure 4 SDS-PAGE image for purification verification of anti-p30 monoclonal antibody (M: protein marker; 1: unpurified antibody; 2: flow-through; 3-11: purified antibody). Mouse ascites was purified using a Protein A / G purification column to remove other contents of the mouse ascites. The purified antibody contained only heavy and light chain structures.

[0048] Figure 5 SDS-PAGE diagram of fragmentation verification of anti-p30 monoclonal antibody (M: protein marker; 1: full-length antibody; 2: antibody after pepsin cleavage)

[0049] Figure 6 Western-Blot identification of anti-p30 protein monoclonal antibodies. PAM cells were infected with African swine fever virus for 36 h, and an uninfected group was set up at the same time. After harvesting the cell samples, 6E12 and 6E12-F(ab')2 were used as primary antibodies to verify their binding ability to p30 protein.

[0050] Figure 7 Indirect immunofluorescence assay for the identification of anti-p30 protein monoclonal antibody 6E12. PAM cells were infected with African swine fever virus for 36 h, and an uninfected group was set up at the same time. Then 6E12 and 6E12-F(ab')2 were used as primary antibodies to determine their reactivity in PAM cells infected with African swine fever virus.

[0051] Figure 8To identify the neutralizing effect of anti-p30 monoclonal antibodies and fragment antibodies. After pre-incubation of antibodies with African swine fever virus for 1 h at different ratios, the mixture of antibodies and African swine fever virus was infected with PAM cells for 36 h, and an uninfected group and an unmixed antibody group were set up at the same time. Then, monoclonal antibodies against African swine fever virus p54 and p72 proteins were used as primary antibodies to determine the neutralizing effect of antibodies on African swine fever virus infection.

[0052] Fig. 9 The results of specificity test of the blocking ELISA method for detecting antibodies to African swine fever virus based on monoclonal antibodies (A) and fragment monoclonal antibodies (B) against p30 protein of African swine fever virus. The blocking ELISA method was used to detect the positive sera of porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV) and pseudorabies virus (PRV) to determine its specificity. DETAILED DESCRIPTION

[0053] The following is a further detailed description of the claims of the present disclosure in conjunction with experiments and examples, but does not constitute any limitation to the present disclosure. Any limited modifications made within the scope of protection of the claims of the present disclosure are still within the scope of protection of the claims of the present disclosure. If no specific conditions are specified in the implementation method, the conventional conditions or the conditions recommended by the manufacturer shall be followed. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0054] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure.

[0055] Test materials

[0056] African swine fever virus CHINA / 2018 / AnhuiXCGQ strain was from the National African Swine Fever Reference Laboratory, Chinese Center for Animal Health and Epidemiology (Qingdao, Shandong);

[0057] PAM cells (porcine alveolar macrophages) were prepared according to the literature DDX19A Senses Viral RNA and Mediates NLRP3-Dependent Inflammasome Activation;

[0058] Example 1 Expression of African swine fever virus p30 recombinant protein

[0059] The pET-28a-CP204L prokaryotic expression plasmid with successful enzyme digestion and correct sequencing results was transferred into the competent cells of E. coli BL21. After plating, the cells were placed in a constant temperature incubator at 37°C overnight. After a single colony grew out, a single colony was picked and placed in LB medium containing kanamycin. The cells were cultured in a shaking incubator at 37°C and 250 rpm for 16 h. Then 0.5 mL of the bacterial solution was added to 50 mL of LB medium, placed in a shaking incubator, and cultured at 37°C and 250 rpm. After about 2 h, the OD was measured. 600 Value, when OD 600 When the value is between 0.6 and 0.8, take 2 mL of bacterial solution as the uninduced negative control, and then add IPTG with a final concentration of 1 mM to induce expression at 37℃250 rpm for 4 h. Then take 1 mL of bacterial solution, centrifuge at 12000 rpm for 1 min, collect the bacteria after centrifugation, resuspend in PBS, and ultrasonically lyse the bacteria on ice, and add an equal amount of 2×SDS loading buffer; take another 40 mL of bacterial solution, collect the precipitate after centrifugation, resuspend in PBS and ultrasonically break it, centrifuge at 4℃12000 rpm for 10 min, collect the supernatant and precipitate respectively, resuspend the precipitate with an equal volume of PBS as the supernatant, take 100 mL and add the corresponding volume of 2×SDS loading buffer as the protein samples of the supernatant and precipitate, and cook the sample in a 96℃ sample cooker for 10 min. Then prepare a polyacrylamide gel with a separation gel concentration of 12%, and identify whether p30 is expressed and the solubility of the expressed protein by SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining ( Figure 1 ).

[0060] like Figure 1 As shown, after induction, the p30 recombinant protein can express a band of about 35 kDa, and the target recombinant protein is mainly expressed in the form of inclusion bodies.

[0061] Example 2 Purification of African swine fever virus p30 recombinant protein

[0062] The obtained inclusion bodies were denatured with urea, filtered and added to the Ni-NTA affinity chromatography column. The protein was incubated at 4°C for 2 h to allow the protein to fully bind to the affinity chromatography column. The purification column was washed with binding buffer twice the column volume to wash away the unbound protein, and the flow-through sample was collected. Different concentrations of elution buffer were added to the purification column, and the eluted protein was collected. The purified protein was subjected to SDS-PAGE detection. The results showed that a higher purity of p30 protein ( Figure 2 ).

[0063] Figure 2 The figure shows the SDS-PAGE results after the p30 protein was purified by nickel column.

[0064] Example 3 Determination of serum titer after immunization of mice with African swine fever virus p30 recombinant protein

[0065] The blood was collected 2 weeks after the second immunization by tail amputation of mice. The collected blood was placed in a 37°C incubator for 1 hour, then placed at 4°C for 3-5 hours, centrifuged at 5000 rpm for 10 minutes, and the upper clear liquid was taken as serum, which was placed at -20°C for later use. The purified p30 recombinant protein in Example 2 was coated at a concentration of 2 mg / mL at 4°C overnight, and the serum to be tested was diluted from 1:1000. At the same time, a negative serum control was made for indirect ELISA detection. The titer of serum from mice immunized with p30 recombinant protein is as follows: Figure 3 shown.

[0066] like Figure 3 As shown, after immunization with p30 recombinant protein, the five mice all had high serum antibody titers, reaching the fusion requirements. Mouse No. 1 was selected for the third immunization, and subsequent cell fusion was performed one week later.

[0067] Example 4 Preparation of anti-p30 monoclonal antibody

[0068] The spleen cells of mouse No. 1 were mixed with myeloma cells SP2 / 0 in a ratio of 10:1, and PEG2000 was added to allow for full fusion. The fused cells were cultured in HAT selection medium for 10 days. The p30 protein purified in Example 2 was used as the coating antigen, and the hybridoma cell supernatant was used as the antibody, and the antibody content in the supernatant was detected by indirect ELISA. The cells in the positive wells were aspirated, and subclone screening was performed 4 times by the limiting dilution method, and finally a monoclonal hybridoma cell line that can secrete anti-p30 antibodies was obtained, which was named cell line 6E12. The cell line was continuously passaged 20 times, and the cell supernatant was collected every 5 generations, and the antibody content in the supernatant was detected by the indirect ELISA method in Example 3. The results of indirect ELISA are shown in Table 1. The OD value of the antibody secreted by the cell line is 450 There was no significant change in the values, proving that the properties of the 6E12 cell line were stable after continuous passage.

[0069]

[0070] Take 1×10 6 6E12 hybridoma cells were injected into the peritoneal cavity of BALB / c mice. Ten days later, the ascites of the mice were removed and purified using a Protein A / G purification column. The resulting monoclonal antibody was named 6E12. Figure 4 shown.

[0071] Example 5 Fragmentation of anti-p30 monoclonal antibody - F(ab')2

[0072] The anti-p30 monoclonal antibody 6E12 was mixed with pepsin at a ratio of 10:1, placed in a 37°C incubator for enzyme digestion for 24 hours, and then verified by SDS-PAGE;

[0073] Results: Figure 5 As shown, F(ab')2 is a band with a molecular weight of about 100 kDa in lane 2.

[0074] Example 6 Western Blot Identification of Anti-p30 Monoclonal Antibodies and Fragment Antibodies

[0075] PAM cells were cultured at 2 × 10 6 / well was added to a 6-well plate and cultured overnight in a cell culture incubator at 37°C and 5% CO2. When the cells attached to the wall and grew into a monolayer, they were inoculated with 1 MOI of African swine fever virus CHINA / 2018 / AnhuiXCGQ strain, and an uninfected group was set up at the same time and placed in a 37°C, 5% CO2 incubator for 36 hours. The cells were washed three times with PBS to remove the mixture of virus and culture medium in the supernatant. After discarding the supernatant, 300 mL / well of 2×SDS sample buffer was added, and Western Blot detection was performed after heating in a 98°C metal bath for 10 min. The results are shown in Figure 6 middle.

[0076] result: Figure 6 It was shown that the prepared anti-p30 protein monoclonal antibody and F(ab')2 antibody can specifically bind to the p30 protein in virus-infected cells, and can be used in Western Blot experiments with good specificity.

[0077] Example 7 Identification of anti-p30 monoclonal antibodies and fragment antibodies by indirect immunofluorescence assay

[0078] PAM cells were cultured at 2 × 10 6 / well was added to a 6-well plate with a slide, and placed in a cell culture incubator at 37°C and 5% CO2 for overnight culture. When the cells adhered to the wall and grew into a monolayer, the African swine fever virus CHINA / 2018 / AnhuiXCGQ strain was inoculated with 1 MOI, and an uninfected group was set up at the same time, and incubated at 37°C and 5% CO2 for 36 hours. The cells were washed with PBS and then fixed with paraformaldehyde fixative at 37°C for 30 min. The fixative was discarded, and after washing with PBS three times, a PBS blocking solution containing 3% BSA was added, and the cells were blocked at 37°C for 30 min. The blocking solution was discarded, and the purified 6E12 monoclonal antibody and 6E12-F(ab')2 antibody were added at a final concentration of 10 mg / mL, respectively, and incubated at 37°C for 1 h. The primary antibody was recovered, and the cells were washed with PBS three times, each time on a shaker for 10 min. FITC-labeled goat anti-mouse IgG was added at a dilution of 1:500 and incubated at 37°C for 30 min. Discard the secondary antibody, wash the cells 3 times with PBS, and shake on a shaker for 10 min each time. Add DAPI solution for visualization of cell nuclei, seal the slides and observe under a fluorescence microscope.

[0079] Results: Figure 7 As shown in the figure, in ASFV-infected cells, the prepared anti-p30 monoclonal antibody and 6E12-F(ab')2 antibody can bind to ASFV-infected cells and produce specific fluorescence, and the fluorescence is basically distributed in the cytoplasm, while no specific fluorescence was found in the cells of the uninfected group. This result shows that both 6E12 antibody and 6E12-F(ab')2 antibody can be used for immunofluorescence experiments and have good specificity.

[0080] Example 8 Identification of anti-p30 monoclonal antibody subtypes

[0081] The obtained p30 protein monoclonal antibody was subtyped using a mouse monoclonal antibody subtype identification kit. The identification results showed that the heavy chain of the anti-p30 protein monoclonal antibody was IgG1 type and the light chain was κ chain (Table 2).

[0082]

[0083] The amino acid sequences of the heavy chain variable regions are VH-FR1, VH-CDR1, VH-FR2, VH-CDR2, VH-FR3, VH-CDR3 and VH-FR4, and the sequences of the light chain variable regions are VL-FR1, VL-CDR1, VL-FR2, VL-CDR2, VL-FR3, VL-CDR3 and VL-FR4.

[0084] VH-FR1(SEQ NO.2): V K V V E S G G G L V Q P G T S L R L S C A T S

[0085] VH-CDR1(SEQ NO.3): G F T F T D Y Y

[0086] VH-FR2(SEQ N0.4): M S W V R Q P P G E A L E W L G F

[0087] VH-CDR2(SEQ NO.5): I R N K P A G Y T T

[0088] VH-FR3(SEQ NO.6): E Y S A S V K G R F T I S R D N S Q S I L Y L Q L NT L R T E D S A T Y Y C

[0089] VH-CDR3(SEQ NO.7): A R Y V Y Y A Y D G Y F D V

[0090] VH-FR4(SEQ NO.8): W G A G T T V

[0091] VL-FR1(SEQ NO.9): D I V I T Q S P A S L A V S L G Q R A T I S Y R A S

[0092] VL-CDR1(SEQ NO.10): K S V S T S G Y S Y

[0093] VL-FR2(SEQ NO.11): M H W N Q Q K P G Q P P R L L I Y

[0094] VL-CDR2(SEQ NO.12) : L V S

[0095] VL-FR3(SEQ NO.13): N L E S G V P A R F S G S G S G T D F T L N I H PV E E E D A A T Y Y C

[0096] VL-CDR3(SEQ NO.14): Q H I R E L T R

[0097] VL-FR4 (SEQ NO. 15): SEGGQSWK.

[0098] In the following examples, 6E12 and 6E12-F(ab')2 represent anti-p30 monoclonal antibody and fragment antibody, respectively.

[0099] Example 9 Identification of Neutralizing Activity of Anti-p30 Monoclonal Antibodies and Fragment Antibodies

[0100] PAM cells were cultured at 4 × 10 5 / well was added to a 12-well plate and cultured overnight in a cell culture incubator at 37°C and 5% CO2. The purified monoclonal antibody in Example 4 was mixed with 1 MOI of African swine fever virus CHINA / 2018 / AnhuiXCGQ strain in different proportions and pre-incubated in a 37°C incubator for 1 h. The virus-antibody mixture was then added to the PAM cells that had grown into a monolayer and placed in a 37°C, 5% CO2 incubator for 36 hours. The cell supernatant was discarded, the cells were washed with PBS to remove the virus, the antibody and culture medium mixture, PBS was discarded, 200 mL / well of 2×SDS sample buffer was added, and Western Blot was detected after heating in a 98°C metal bath for 10 min. The results are shown in Figure 8 middle.

[0101] Results: Figure 8 As shown, both 6E12 and 6E12-F(ab')2 have neutralizing activity, and 6E12-F(ab')2 is more effective in neutralizing African swine fever virus.

[0102] Example 10 Preliminary establishment of ELISA antibody detection method based on anti-p30 monoclonal antibody and fragment antibody

[0103] The purified p30 recombinant protein in Example 2 was coated on the ELISA plate at 4°C overnight, and the coating solution was discarded and blocked with 3% skim milk at 37°C for 1 h. Then, 10-fold diluted African swine fever negative and positive sera to be tested were added, incubated for 1 h, and then 600 mg / mL p30-6E12 monoclonal antibody (1:16000-1:128000 dilution) or 600 mg / mL p30-6E12-F(ab')2 monoclonal antibody (1:16000-1:128000 dilution) and enzyme-labeled secondary antibody were added, and OD was measured after color development. 450 The values ​​of

[0104]

[0105]

[0106] Results: Tables 3 and 4 show that compared with the control group, both the p30-6E12 monoclonal antibody and the 6E12-F(ab')2 fragment antibody can be used in the blocking ELISA experiment for African swine fever virus antibody detection, with a blocking rate of about 50%.

[0107] Example 11 Evaluation of specificity of anti-p30 monoclonal antibodies and fragment antibodies

[0108] The ELISA detection method of Example 10 was used, and the positive pig serum of PEDV (porcine epidemic diarrhea virus), PRRSV (porcine reproductive and respiratory syndrome virus), PRV (pseudorabies virus), and ASFV (African swine fever virus) was used as the primary antibody for detection, and the detection conditions were all parallel. The test results are as follows Fig. 9 As shown, it can be seen that all serum test results except ASFV positive serum are negative.

[0109] The results showed that the blocking ELISA based on anti-p30 monoclonal antibodies and fragment antibodies had good specificity for the diagnosis of ASFV and had no cross-reaction with other porcine viruses.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A monoclonal antibody against p30 protein of African swine fever virus, characterized in that: The monoclonal antibody is produced by a hybridoma cell line with a deposit number of CCTCC NO: C2024103.

2. The monoclonal antibody according to claim 1, characterized in that The heavy chain variable regions of the monoclonal antibody are VH-FR1, VH-CDR1, VH-FR2, VH-CDR2, VH-FR3, VH-CDR3 and VH-FR4, and their amino acid sequences are shown in SEQ ID NO.2 to SEQ ID NO.8, respectively; the light chain variable regions are VL-FR1, VL-CDR1, VL-FR2, VL-CDR2, VL-FR3, VL-CDR3 and VL-FR4, and their amino acid sequences are shown in SEQ ID NO.9 to SEQ ID NO.15, respectively.

3. The monoclonal antibody according to claim 2, characterized in that The heavy chain of this monoclonal antibody is of IgG1 type and the light chain is of κ type.

4. A fragmented monoclonal antibody F(ab')2 against African swine fever virus p30 protein, characterized in that: The F(ab')2 is obtained by digesting the monoclonal antibody according to any one of claims 1 to 3 with pepsin.

5. Use of the monoclonal antibody according to any one of claims 1 to 3 or the F(ab')2 according to claim 4 in the preparation of a reagent for detecting African swine fever virus.

6. An ELISA kit for detecting African swine fever virus, characterized in that: The kit comprises the monoclonal antibody according to any one of claims 1 to 3 or the F(ab')2 according to claim 4.

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