African swine fever virus p30 protein antibody and application thereof

Abstract

The invention discloses an African swine fever virus p30 protein antibody and application thereof. When the African swine fever virus p30 protein antibody is used for a competitive ELISA method, a serum sample with the p30 antibody after African swine fever virus infection can be rapidly and accurately detected, the detection time is shortened, the detection steps are simplified, the African swine fever virus p30 protein antibody has the characteristics of rapidness and high-throughput detection, and a reliable immunological technology is provided for rapidly and efficiently detecting African swine fever virus infection.

Description

Technical Field

[0001] The invention relates to an African swine fever virus p30 protein antibody and application thereof, belonging to the field of biotechnology. Background Art

[0002] African swine fever (ASF) is an acute, hemorrhagic, highly contagious disease caused by the African swine fever virus (ASFV) infecting domestic pigs and various wild boars (such as African wild boars, European wild boars, etc.). There is currently no effective treatment or vaccine for African swine fever, and the prevention and control of the disease is still mainly based on prevention and quarantine purification. In view of the serious harm of ASF to pig production and related industries, the prevention and control of African swine fever is particularly important for the pig farming industry at home and abroad.

[0003] At present, no effective commercial vaccine has been produced at home and abroad, making the prevention and control of ASF difficult. ASF prevention and control still needs to rely on strict hygiene requirements and perfect biosafety management, so reliable early diagnosis technology and epidemiological monitoring are crucial for ASF prevention and control and purification. With the continuous emergence of domestic resistant pigs, long-term infected pigs with subclinical symptoms, and weakly toxic gene mutation deletion strains, single pathogen detection or serological detection can no longer meet the needs of monitoring ASFV infected pigs. Antibody detection combined with nucleic acid detection has become the main means of ASFV prevention and control in my country under the new situation. p30 in ASFV is an early expression protein that can be detected 2 hours after infection and lasts throughout the infection cycle. Compared with other proteins, it has a higher antibody detection efficiency and greatly improves the ability to distinguish between negative and positive sera. Therefore, it is urgent to develop an accurate and rapid ASFVp30 antibody detection method that can be used in China. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide an African swine fever virus p30 antibody fragment, antibody, and application thereof for detecting African swine fever virus.

[0005] Technical solution: The present invention provides an African swine fever virus p30 protein antibody fragment, which includes light chain and heavy chain variable regions, the amino acid sequences of the three complementary determining regions in the light chain variable region are respectively shown as SEQ ID NO.1-3, and the amino acid sequences of the three complementary determining regions in the heavy chain variable region are respectively shown as SEQ ID NO.4-6.

[0006] The present invention also provides an African swine fever virus p30 protein antibody, which comprises a light chain and a heavy chain, the amino acid sequence of the light chain is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.9.

[0007] The heavy and light chains are linked by disulfide bonds.

[0008] The nucleotide sequence encoding the light chain is shown in SEQ ID NO.8, and the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO.10.

[0009] The present invention also provides the use of the above-mentioned African swine fever virus p30 protein antibody fragment and African swine fever virus p30 antibody in detecting African swine fever virus.

[0010] The present invention also provides a kit for detecting African swine fever virus, wherein the kit comprises the above-mentioned African swine fever virus p30 protein antibody fragment or the above-mentioned African swine fever virus p30 protein antibody.

[0011] Furthermore, the kit also includes a solid phase carrier coated with African swine fever virus p30 protein, an African swine fever virus positive control, a negative control, a coating solution, a blocking solution, a diluent, PBST, a TMB colorimetric solution, and a reaction termination solution.

[0012] The present invention also provides a method for using the above kit, comprising the following steps:

[0013] (1) diluting the African swine fever virus p30 protein with a coating solution and adding it to each well of an ELISA plate for coating; then washing with a washing solution, adding a blocking solution for blocking, and then washing with a washing solution; then patting the plate dry to obtain an ELISA plate coated with the African swine fever virus p30 protein;

[0014] (2) respectively mixing the diluted African swine fever virus negative serum, positive serum and test serum with the diluted HRP-labeled African swine fever virus p30 protein antibody according to any one of claims 2 to 3, adding them to the ELISA plate in step (1), and incubating;

[0015] (3) Add colorimetric solution, incubate, add stop solution, and read OD 450 value.

[0016] Furthermore, the coating concentration of the African swine fever virus p30 protein is 2 μg / mL, and the concentration of the African swine fever virus p30 protein antibody is 90 ng / mL.

[0017] Furthermore, the African swine fever virus p30 protein is an African swine fever virus p30 protein carrying a His or GST tag.

[0018] Furthermore, the method for judging the test results is to calculate the inhibition rate PI, and it is judged as positive when PI≥50.6%; it is judged as negative when PI<50.6%.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following outstanding significant advantages: the African swine fever virus p30 protein antibody of the present invention is used in the competitive ELISA method to quickly and accurately detect serum samples containing p30 antibodies after African swine fever virus infection, shortening the detection time, simplifying the detection steps, and having the characteristics of rapid and high-throughput detection, providing a reliable immunological technology for rapid and efficient detection of African swine fever virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 To express, purify and identify the recombinant proteins His-p30 and GST-p30. Figure 1 A is the expression, purification and identification of His-p30 protein, lane M is 180kDa Prestained Protein Marker, lane 1 is BL21(DE3)(pColdI) empty vector bacteria control, lane 2 is the lysis supernatant after induction of the recombinant expression bacteria BL21(DE3)(pCold-p30), lane 3 is the lysis precipitate after induction of the recombinant expression bacteria BL21(DE3)(pCold-p30), and lane 4 is the purified His-p30 protein. Figure 1 B is the expression, purification and identification of GST-p30 protein, lane M is 180kDa Prestained Protein Marker, lane 1 is BL21 (DE3) (pGEX-6p-1) empty vector bacteria control, lane 2 is the lysis supernatant after induction of the recombinant expression bacteria BL21 (DE3) (pGEX-6p-1-p30), lane 3 is the lysis precipitate after induction of the recombinant expression bacteria BL21 (DE3) (pGEX-6p-1-p30), and lane 4 is the purified GST-p30 protein.

[0021] Figure 2 This is the reactivity identification of monoclonal antibody 4E8 and African swine fever virus p30 protein. Lane M is 180kDa Prestained Protein Marker, lane 1 is recombinant protein His-p30, and lane 2 is recombinant protein GST-p30.

[0022] Figure 3 This is the affinity analysis of monoclonal antibody 4E8 and African swine fever virus p30 protein.

[0023] Figure 4 To determine the optimal working conditions for competitive ELISA method.

[0024] Figure 5 This is the ROC curve analysis.

[0025] Figure 6 Results for competition ELISA specificity analysis.

[0026] Figure 7 The results are analyzed for the detection limit of competition ELISA. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0028] In order to make the invention purpose, technical scheme and beneficial technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the method, steps or conditions of the present invention are within the scope of the present invention.

[0029] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques are well described in the literature, see Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0030] Example 1: Obtaining hybridoma cell lines

[0031] 1. Preparation of recombinant African swine fever proteins His-p30 and GST-p30

[0032] (1) Construction of recombinant plasmids pCold-p30 and pGEX-6p-1-p30

[0033] The sequence of the African swine fever virus p30 encoding gene published by GenBank (accession number MW828829.1) was selected. After optimization according to the codon preference of Escherichia coli, XhoI and XbaI restriction sites were added at both ends of the sequence for connection with the pCold I vector (Takara, catalog number 3361); or SmaI and NotI restriction sites were added at both ends of the sequence for connection with the pGEX-6p-1 vector (Solarbio, catalog number P0300). The sequences after adding restriction sites were commissioned to Nanjing GenScript Biotechnology Co., Ltd. for synthesis and cloned into expression vectors respectively to form recombinant expression plasmids pCold-p30 and pGEX-6p-1-p30.

[0034] (2) Expression and purification of target protein

[0035] The recombinant expression plasmids pCold-p30 and pGEX-6p-1-p30 were transformed into Escherichia coli BL21 (DE3), respectively, to obtain expression bacteria BL21 (DE3) (pCold-p30) and BL21 (DE3) (pGEX-6p-1-p30) that can express recombinant p30 protein carrying His and GST tags. The expression and purification process of His-p30 recombinant protein is as follows: a single colony of BL21 (DE3) (pCold-p30) is inoculated into 5 mL of liquid LB (containing ampicillin) and cultured at 37°C with shaking overnight; the next day, the mother liquor is inoculated into fresh LB liquid culture medium (containing ampicillin) at a ratio of 1:100, cultured at 37°C with shaking until OD600 is about 0.6, IPTG with a final concentration of 0.5 mM is added, and the culture is induced with shaking at 15°C for 24 hours; finally, the recombinant protein His-p30 is purified using a His tag protein purification kit (Sangon Biotechnology Co., Ltd., catalog number: C600332), and identified by SDS-PAGE electrophoresis. The expression and purification process of the GST-p30 recombinant protein was as follows: a single colony of BL21 (DE3) (pGEX-6p-1-p30) was inoculated into 5 mL of liquid LB (containing ampicillin) and cultured overnight at 37°C with shaking; the next day, the mother liquor was inoculated into fresh LB liquid culture medium (containing ampicillin) at a ratio of 1:100, cultured at 37°C with shaking until OD600 was about 0.6, and IPTG with a final concentration of 0.5 mM was added for induction culture at 25°C with shaking for 8 h; then, the recombinant protein GST-p30 was purified using a GST tag protein purification kit (GenScript Biotech Co., Ltd., catalog number: L00207), and identified by SDS-PAGE electrophoresis.

[0036] The results are as follows Figure 1 As shown, at 35KDa ( Figure 1 A, lane 4) and 56 kDa ( Figure 1B, target bands of expected sizes appeared in lane 4), which are His-p30 and GST-p30 proteins respectively.

[0037] 2. Animal Immunization

[0038] Six-week-old female BALB / c mice were purchased from the Comparative Medicine Center of Yangzhou University. The specific immunization procedure was as follows: the His-p30 protein of African swine fever virus was mixed with Freund's complete adjuvant in a ratio of 1:1 and fully emulsified, and then three mice were subcutaneously injected at multiple points (neck back and abdomen). 14 days after the first immunization, the second immunization was performed, and both immunizations were 200 μL / mouse (containing 100 μg of recombinant p30 protein). 14 days after the second immunization, blood was collected from mice to test the serum antibody titer, and mice with higher titers were selected for intraperitoneal injection of 100 μg of purified recombinant His-p30 protein without adjuvant for booster immunization.

[0039] 3. Cell Fusion

[0040] Three days after the booster immunization, blood was collected from the mice, and the serum was collected and stored at -20°C for positive control in subsequent screening. The mice were killed according to the biosafety method, disinfected by alcohol immersion, and spleen cells were fused with myeloma cells SP2 / 0 in the logarithmic growth phase at a cell ratio of 1:5 under the action of polyethylene glycol PEG50%. ICR mouse peritoneal macrophages were used as feeder cells, and the fused cells and feeder cells were suspended and mixed with HAT medium and spread in a 96-well plate and cultured in a 37°C cell culture incubator. HAT medium was added after 5 days, and HT medium was used for culture after 9 days. When the single cell cluster grew to about 90% of the 96-well plate, the cell supernatant was collected for indirect ELISA detection.

[0041] 4. Establishment of indirect ELISA detection method and screening of positive clones

[0042] The indirect ELISA method was used to screen positive cell clones. The specific method is as follows: GST-p30 was used to coat the ELISA plate according to the optimal coating concentration (1 μg / mL) of the antigen determined by the array test, 100 μL / well, overnight at 4°C; washed 3 times with PBST, added PBS blocking solution containing 1% BSA, 200 μL per well, and incubated at 37°C for 2 hours; after blocking, washed 3 times with PBST, and added hybridoma cell supernatant; negative and positive control groups were set up at the same time, with SP2 / 0 cell supernatant as negative control and immune mouse polyclonal antibody serum as positive control, 100 μL / well, in a 37°C water bath for 2 hours; washed 5 times with PBST; added 1:5000 diluted horseradish peroxidase HRP-labeled goat anti-mouse IgG, 100 μL / well, in a 37°C water bath for 1 hour; after washing 7 times, TMB was added for color development for 10 minutes, and after color development was terminated, the OD was detected by an enzyme reader. 450 The experimental results are determined according to the following formula: OD450 Cell well / OD 450 Negative wells ≥ 2.1 were judged as positive wells. The screened positive clone was named 4E8.

[0043] 5. Cloning of positive hybridoma cells

[0044] The positive cell clone 4E8 was subcloned three times by limiting dilution method and preserved.

[0045] Example 2: Preparation of monoclonal antibodies against African swine fever virus p30 protein

[0046] In vivo ascites induction method: 9-12 week old healthy BALB / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin. Seven days later, hybridoma cells 4E8 cultured to logarithmic phase and diluted with PBS were intraperitoneally inoculated with 2×10 5 After 7 days, the ascites was collected, the supernatant was collected by centrifugation and stored at -70°C.

[0047] The prepared ascites was purified using Protein A affinity chromatography, labeled with HRP, and stored at -70°C.

[0048] Example 3: Monoclonal Antibody Characterization Detection

[0049] 1. Identification of mAb subclasses

[0050] The monoclonal antibody subclass kit (Beijing Biolong BF16001) was used to identify the monoclonal antibody subclass. The hybridoma cell 4E8 culture supernatant prepared in Example 1 was added to the ELISA plate pre-coated with 1 μg / mL recombinant GST-p30 protein, 100 μL / well, and incubated at 37°C for 2 hours; washed 3 times with PBST; sheep anti-mouse IgA, IgG1, IgG2a, IgG2b, IgG3 and IgM diluted 1:1000 with PBS were added, 100 μL / well, placed at room temperature for 30 minutes, washed 3 times with PBST; HRP-rabbit anti-sheep IgG enzyme-labeled antibody diluted 1:5000 with PBS was added, 100 μL / well, incubated at room temperature for 15 minutes; washed 5 times with PBST; TMB color development solution was added, 100 μL / well, and placed at room temperature for 5 minutes; 0.5M H2SO4 was added to terminate the reaction, 50 μL / well, and the OD was detected by an enzyme reader. 450 , according to OD 450 Determine the subclass of the monoclonal antibody.

[0051] The results showed that the subclass of monoclonal antibody 4E8 was IgG1.

[0052] After identification, the results showed that the amino acid sequence of the complementary determining region 1 (CDR1) of the light chain variable region of the monoclonal antibody was as shown in SEQ ID NO.1, specifically: KASQDVSTAVA.

[0053] The amino acid sequence of the complementary determining region 2 (CDR2) of the light chain variable region is shown in SEQ ID NO.2, specifically: WASTRHT.

[0054] The amino acid sequence of the complementarity determining region 3 (CDR3) of the light chain variable region is shown in SEQ ID NO.3, specifically: QQHYSTPFT.

[0055] The amino acid sequence of the heavy chain variable region complementarity determining region 1 (CDR1) is shown in SEQ ID NO.4, specifically: DYYMS.

[0056] The amino acid sequence of the heavy chain variable region complementarity determining region 2 (CDR2) is shown in SEQ ID NO.5, specifically: FIRNKANGYTTKYGASVKG.

[0057] The amino acid sequence of the heavy chain variable region complementarity determining region 3 (CDR3) is shown in SEQ ID NO.6, specifically: EAAYYGYDAYFDY.

[0058] The full-length amino acid sequence of the light chain is shown in SEQ ID NO.7: MESQIQAFVFVFLWLSGVNGDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQ QKPGQSPRLLINWASTRHTGVPDRFTGSGSGTDFTLTISSVQTEDLALYYCQQHYSTPF TFGGGTKLEIK.

[0059] The nucleotide sequence encoding the light chain is shown in SEQ ID NO.8: ATGGAGTCACAGATTCAGGCATTTGTATTCGTGTTTCTCTGGTTGTCTGGTGTTAACGGAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACAAAAACCAGGACAATCTCCTAGACTACTGATTAACTGGGCTTCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTCACCATCAGCAGTGTGCAGACTGAAGACCTGGCACTTTATTACTGTCAGCAACATTATAGCACTCCGTTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA。

[0060] The full-length amino acid sequence of the heavy chain is shown in SEQ ID NO.9: MRLWLNWIFLVTLLNGIQCEVKLVESGGGLVQPGDSLRLSCATSGFTFTDYYMSWV RQPPGKALEWLGFIRNKANGYTTKYGASVKGRFTISRDNSQDILYLQMNSLRAEDSA TYYCAREAAYYGYDAYFDYWGQGTTLTVSS。

[0061] Nucleotide sequence encoding the heavy chain such as SEQ ID NO.10 shows: ATGAGGTTGTGGCTGAACTGGATTTTCCTTGTAACACTTTTAAATGGTATCCAGTGTGAGGTGAAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGGATTCTCTGAGACTCTCCTGTGCAACTTCTGGGTTCACCTTCACTGATTACTACATGAGCTGGGTCCGCCAGCCTCCAGGAAAGGCACTTGAGTGGTTGGGTTTTATTA GAAACAAAGCTAATGGTTACACAACAAAATACGGTGCATCTGTGAAGGGTCGGTTCACCATCTCCAGAGATAATTCCCAAGACATCCTCTATCTTCAAATGAACTCCCTGAGAGCTGAGGACAGTGCCACTTATTACTGTGCAAGAGAGGCGGCCTACTATGGGTACGACGCCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0062] 2. Determination of monoclonal antibody ascites titer

[0063] The GST-p30 protein was coated at a concentration of 1 μg / mL on a 96-well ELISA plate using a coating buffer, overnight at 4°C; washed 3 times with PBST, 200 μL of blocking solution was added to each well, and incubated at 37°C for 2 h; washed 3 times with PBST, the monoclonal antibody ascites supernatant prepared in Example 2 was added with a multiple dilution, and SP2 / 0 ascites was diluted with the same multiple as a negative control, and incubated at 37°C for 2 h; washed 5 times with PBST; HRP-sheep anti-mouse IgG diluted 1:5000 was added, 100 μL / well, and incubated at 37°C for 1 h; after washing with PBST, TMB colorimetric solution was added for color development, 100 μL / well, and incubated at 37°C for 10 min; 0.5 M H2SO4 was added to terminate the reaction, and OD was detected by an enzyme marker. 450 The value was determined by taking P / N ≥ 2.1 as the criterion to determine the monoclonal antibody ascites titer.

[0064] The results showed that the titer of monoclonal antibody 4E8 was 1:32768000, which had a very high titer.

[0065] 3. Identification of monoclonal antibody immunoreactivity

[0066] The recombinant proteins of African swine fever virus His-p30 and GST-p30 were added to the loading buffer, and the electrophoresis gel was transferred to the constant temperature metal bath for 10 minutes for SDS-PAGE electrophoresis. Then, the electrophoresis gel was transferred to the nitrocellulose membrane using a fast transfer instrument for 10 minutes, and the protein was transferred to the nitrocellulose membrane; PBST containing 5% skim milk powder was used for blocking, and the membrane was blocked at room temperature for 1 hour, and washed with PBST for 3 times; 1:1000 diluted monoclonal antibody 4E8 was added, and the membrane was incubated at 4℃ overnight, and washed with PBST for 5 times; 1:5000 diluted HRP-goat anti-mouse enzyme-labeled antibody was added, and the membrane was exposed to room temperature for 1 hour, and washed with PBST for 7 times; ECL color development was performed for 1 minute, and the membrane was photographed using an ultra-sensitive multifunctional imager.

[0067] The results are as follows Figure 2 As shown, 4E8 monoclonal antibody reacted well with African swine fever virus recombinant His-p30 (lane 1) and GST-p30 (lane 2) proteins.

[0068] 4. Monoclonal antibody affinity identification

[0069] The GST-p30 protein was coated on a 96-well ELISA plate at 2, 1, 0.5 and 0.25 μg / mL using coating buffer, overnight at 4°C; washed 3 times with PBST, 200 μL of blocking solution was added to each well, and incubated at 37°C for 2 h; washed 3 times with PBST, purified monoclonal antibody ascites diluted from 1:500 was added, and incubated at 37°C for 2 h; washed 5 times with PBST; HRP-sheep anti-mouse IgG diluted 1:5000 was added, 100 μL / well, and incubated at 37°C for 1 h; after washing with PBST, TMB colorimetric solution was added for color development, 100 μL / well, and incubated at 37°C for 10 min; 0.5 M H2SO4 was added to terminate the reaction, and OD was detected by microplate reader. 450 The affinity constant (K) of the antibody was calculated according to the formula: K = (n-1) / 2 (nAb'-Ab), and the affinity of the monoclonal antibody was determined.

[0070] The results are as follows Figure 3 As shown in Figure 2, 4E8 monoclonal antibody stably binds to the recombinant p30 protein of African swine fever virus, with the highest affinity reaching 2.88×10 10 M -1 .

[0071] Example 4: Establishment of the optimal conditions for ELISA reaction and establishment of a diagnostic method

[0072] The optimal coating concentration of African swine fever virus GST-p30 protein was determined to be 2 μg / mL by square array experiment, and the optimal reaction concentration of enzyme-labeled antibody 4E8 was 90 ng / mL (dilution multiple was 1:35000). The optimal blocking solution, optimal blocking time, optimal serum incubation time, optimal serum dilution multiple, and optimal substrate action time were optimized.

[0073] The optimization results are shown in Figure 4 , based on the optimal results, the final operating procedure was established as follows:

[0074] (1) The African swine fever virus GST-p30 protein was diluted to 2 μg / mL with a coating solution (carbonate buffer (CBS)), added to an ELISA plate, 100 μL / well, and coated at 37°C for 2 h; washed three times with PBST, and PBS containing 2% bovine serum albumin (BSA) was used as the optimal blocking solution, and the coated ELISA plate was blocked at 37°C for 2.5 h, washed three times with PBST, and patted dry;

[0075] (2) Dilute the test serum with diluent (2% bovine serum albumin (BSA) in PBS) at a ratio of 1:12, then mix with 90 ng / mL horseradish peroxidase-labeled antibody 4E8 at a ratio of 1:1, take 100 μL and add it to the wells in step (1), incubate for 2 h, and wash 5 times with PBST;

[0076] (3) TMB was added for 10 min; 0.5 M H2SO4 was added to terminate the reaction, and the OD value was measured by microplate reader. 450 Take a reading.

[0077] Serum samples were collected from the pig slaughterhouse, and 50 African swine fever-negative sera and 50 African swine fever-positive sera with clear backgrounds verified by the commercial kit African Swine Fever Competition Competitive ELISA test kit (ID-Vet) were selected for detection using the competitive ELISA method established above, and their PI values ​​were analyzed (PI = (1-OD of serum sample) 450 Value / OD of blank control 450 )×100%), and GraphPadPrism software was used to draw ROC curves and calculate Youden index, sensitivity, and specificity. Figure 5 As shown, when the cut-off is 50.6%, the sensitivity and specificity of this method are as high as 98.3% and 98.3% respectively. Therefore, the judgment criteria are PI ≥ 50.6% for positive and PI < 50.6% for negative.

[0078] Example 5: Competitive ELISA method specific detection

[0079] The competitive ELISA method established in Example 4 was used to detect positive serum of common pathogenic microorganisms such as PDCoV (porcine deltacoronavirus), PEDV (porcine epidemic diarrhea), PRRSV (porcine blue ear virus), PCV2 (porcine circovirus type 2), etc. Based on the judgment threshold, the method was tested for cross-reaction. The results are shown in FIG. Figure 6 As shown, only the PI of the African swine fever virus-positive serum was higher than the threshold, and the PIs of the other sera were all lower than the threshold, indicating that the established competitive ELISA method had good specificity.

[0080] Example 6: Determination of detection limit of competitive ELISA method

[0081] The monoclonal antibody with an original concentration of 2 mg / mL was diluted in a gradient of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, 1:5120, and 1:10240, and the PI value was determined by the competitive ELISA method established in Example 4, and a reaction curve was drawn to calculate its detection limit. Drawing the reaction curve ( Figure 7 ), calculated by the formula, when the antibody concentration is lower than 477ng / mL, the test result is negative, otherwise it is positive.

[0082] Example 7: Preliminary application of African swine fever virus p30 protein monoclonal antibody competitive ELISA

[0083] 30 positive sera and 50 negative sera of African swine fever verified by the commercialized African Swine Fever Competition Competitive ELISA test kit (ID-Vet) were taken; the above 80 sera were tested using the competitive ELISA method established in Example 4. The results showed that the test results of 75 samples were consistent with the background, and the overall compliance rate was 93.75% (Table 1).

[0084] Table 1 Competitive ELISA detection of pig serum samples

[0085]

[0086] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. An African swine fever virus p30 protein antibody fragment, characterized in that: The antibody fragment includes light chain and heavy chain variable regions, the amino acid sequences of the three complementary determining regions in the light chain variable region are shown as SEQ ID NO.1-3, and the amino acid sequences of the three complementary determining regions in the heavy chain variable region are shown as SEQ ID NO.4-6.

2. An antibody against African swine fever virus p30 protein, characterized in that: The antibody comprises a light chain and a heavy chain, the amino acid sequence of the light chain is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

9.

3. The African swine fever virus p30 protein antibody according to claim 2, characterized in that: The nucleotide sequence encoding the light chain is shown in SEQ ID NO.8, and the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO.

10.

4. Use of the African swine fever virus p30 protein antibody fragment according to claim 1 and the African swine fever virus p30 antibody according to any one of claims 2 to 3 in detecting African swine fever virus.

5. A kit for detecting African swine fever virus, characterized in that: The kit comprises the African swine fever virus p30 protein antibody fragment according to claim 1 or the African swine fever virus p30 protein antibody according to any one of claims 2 to 3.

6. The African swine fever virus detection kit according to claim 5, characterized in that: The kit also comprises a solid phase carrier coated with African swine fever virus p30 protein, an African swine fever virus positive control, a negative control, a coating solution, a blocking solution, a diluent, PBST, a TMB color developing solution, and a reaction termination solution.

7. The method for using the kit according to any one of claims 5 to 6, characterized in that: The following steps are involved: (1) diluting the African swine fever virus p30 protein with a coating solution and adding it to each well of an ELISA plate for coating; then washing with a washing solution, adding a blocking solution for blocking, and then washing with a washing solution; then patting the plate dry to obtain an ELISA plate coated with the African swine fever virus p30 protein; (2) respectively mixing the diluted African swine fever virus negative serum, positive serum and test serum with the diluted HRP-labeled African swine fever virus p30 protein antibody according to any one of claims 2 to 3, adding them to the ELISA plate in step (1), and incubating; (3) Add colorimetric solution, incubate, add stop solution, and read OD 450 value.

8. The method according to claim 7, characterized in that The coating concentration of the African swine fever virus p30 protein is 2 μg / mL, and the concentration of the African swine fever virus p30 protein antibody is 90 ng / mL.

9. The method according to claim 7, characterized in that: The African swine fever virus p30 protein is an African swine fever virus p30 protein carrying a His or GST tag.

10. The method according to claim 7, characterized in that The method for judging the test results is to calculate the inhibition rate PI. When PI ≥ 50.6%, it is judged as positive; when PI < 50.6%, it is judged as negative.

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