African swine fever virus p30 protein antibody and its application

Abstract

The present invention discloses an antibody against the p30 protein of African swine fever virus and its application. The antibody against the p30 protein of the present invention can be used in a competitive ELISA method to quickly and accurately detect serum samples containing p30 antibodies after infection with African swine fever virus. This method shortens detection time, simplifies detection steps, and offers the advantages of rapid and high-throughput detection, providing a reliable immunological technique for the rapid and efficient detection of African swine fever virus infection.

Description

Technical Field

[0001] The present 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, and highly contagious disease caused by the African swine fever virus (ASFV) infecting domestic pigs and various wild boar species, such as African wild boars and European wild boars. Currently, there are no effective treatments or vaccines for ASF, and prevention and control of the disease still primarily rely on quarantine and decontamination. Given the serious impact of ASF on pig production and related industries, the prevention and control of ASF is of particular importance to both the domestic and international pig farming industries.

[0003] Currently, no effective commercial vaccine has been produced domestically or internationally, making ASF prevention and control difficult. ASF prevention and control still relies on strict hygiene requirements and comprehensive biosafety management. Therefore, reliable early diagnostic technologies and epidemiological monitoring are crucial for ASF control and elimination. With the continuous emergence of resistant pigs, chronically infected pigs with subclinical symptoms, and attenuated genetic mutation deletion strains in China, single pathogen detection or serological testing is no longer sufficient for monitoring ASFV-infected pigs. Antibody detection combined with nucleic acid testing has become the primary means of ASFV prevention and control in my country under the new circumstances. p30 is an early expressed protein in ASFV, detectable as early as 2 hours after infection and persisting throughout the infection cycle. Compared with other proteins, it has a higher antibody detection efficiency and significantly improves the ability to distinguish between negative and positive sera. Therefore, there is an urgent need to develop an accurate and rapid ASFV p30 antibody detection method that can be used domestically. 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 shown in SEQ ID NO.1-3, and the amino acid sequences of the three complementary determining regions in the heavy chain variable region are shown in SEQ ID NO.4-6.

[0006] The present invention also provides an African swine fever virus p30 protein antibody, which includes 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, which contains 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 color development 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, 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 when PI ≥ 50.6%, it is judged as positive; when PI < 50.6%, it is judged as negative.

[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 the 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 the 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 image shows the reactivity of monoclonal antibody 4E8 with African swine fever virus p30 protein. Lane M is 180 kDa prestained protein marker, lane 1 is recombinant protein His-p30, and lane 2 is recombinant protein GST-p30.

[0022] Figure 3 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 The results are for competition ELISA specificity analysis.

[0026] Figure 7 The results are from the competitive ELISA detection limit analysis. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0028] In order to make the purpose of the invention, technical solutions and beneficial technical effects of the present invention more clear, the present invention is further described in detail below with reference to the embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence 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 molecular biology, biochemistry, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art. These techniques are well described in the literature, for example, in 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 in GenBank (accession number MW828829.1) was selected. After optimization based on the codon preference of Escherichia coli, XhoI and XbaI restriction sites were added at both ends of the sequence for ligation with the pCold I vector (Takara, Catalog No. 3361); alternatively, SmaI and NotI restriction sites were added at both ends of the sequence for ligation with the pGEX-6p-1 vector (Solarbio, Catalog No. P0300). The sequences with added 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 the His-p30 recombinant protein was as follows: a single BL21(DE3)(pCold-p30) colony was inoculated into 5 mL of liquid LB (containing ampicillin) and cultured with shaking at 37°C overnight; the next day, the mother liquor was inoculated into fresh LB liquid medium (containing ampicillin) at a ratio of 1:100, and cultured with shaking at 37°C to an OD600 of approximately 0.6. IPTG was added to a final concentration of 0.5 mM and induced with shaking at 15°C for 24 h; finally, the recombinant His-p30 protein was purified using a His-tag protein purification kit (Sangon Biotech Co., Ltd., Catalog No. C600332) and identified by SDS-PAGE electrophoresis. The expression and purification process of GST-p30 recombinant protein was as follows: a single BL21 (DE3) (pGEX-6p-1-p30) colony was inoculated into 5 mL of liquid LB (containing ampicillin) and cultured with shaking at 37°C overnight; the next day, the mother liquor was inoculated into fresh LB liquid culture medium (containing ampicillin) at a ratio of 1:100, cultured with shaking at 37°C until the OD600 was approximately 0.6, and IPTG was added at a final concentration of 0.5 mM and induced with shaking at 25°C 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 were His-p30 and GST-p30 proteins, respectively.

[0037] 2. Animal immunization

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

[0039] 3. Cell Fusion

[0040] Three days after the booster immunization, the mice were bled and the serum was collected and stored at -20°C for use as a positive control for subsequent screening. The mice were killed according to biosafety methods, 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 50%. ICR mouse peritoneal macrophages were used as feeder cells, and the fused cells and feeder cells were suspended and mixed with HAT medium and plated 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. The single cell clusters grew to about 90% of the 96-well plate, and the cell supernatant was collected for indirect ELISA detection.

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

[0042] Positive cell clones were screened using the indirect ELISA method. 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, 4°C overnight; 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; at the same time, negative and positive control groups were set up, with SP2 / 0 cell supernatant as the negative control and immune mouse polyclonal antibody serum as the positive control, 100 μL / well, 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, 37°C water bath for 1 hour; after washing 7 times, TMB was added for color development for 10 minutes. After color development was terminated, the OD was detected by microplate reader. 450 The experimental results are determined according to the following formula: OD450 Cell well / OD 450 Negative wells with a p-value of ≥2.1 were considered positive. The positive clone screened was named 4E8.

[0043] 5. Cloning of Positive Hybridoma Cells

[0044] The positive cell clone 4E8 was subcloned three times using the 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 and 2×10 cells diluted with PBS and cultured to logarithmic phase were inoculated intraperitoneally. 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

[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 enzyme-labeled 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 PBS; added goat anti-mouse IgA, IgG1, IgG2a, IgG2b, IgG3 and IgM diluted 1:1000 with PBS, 100 μL / well, respectively, and placed at room temperature for 30 minutes, washed 3 times with PBST; added HRP-rabbit anti-goat IgG enzyme-labeled antibody diluted 1:5000 with PBS, 100 μL / well, and incubated at room temperature for 15 minutes; washed 5 times with PBST; added TMB color development solution, 100 μL / well, and placed at room temperature for 5 minutes; added 0.5M H2SO4 to stop the reaction, 50 μL / well, and detected the OD with 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 complementarity 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 complementarity 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 complementarity determining region 2 (CDR2) of the heavy chain variable region is shown in SEQ ID NO.5, specifically: FIRNKANGYTTKYGASVKG.

[0057] The amino acid sequence of the complementarity determining region 3 (CDR3) of the heavy chain variable region 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 using coating buffer on a 96-well ELISA plate at 4°C overnight; the plate was washed three times with PBST, 200 μL of blocking solution was added to each well, and the plate was incubated at 37°C for 2 h; the plate was washed three times with PBST, the monoclonal antibody ascites supernatant prepared in Example 2 was added with a dilution ratio, and SP2 / 0 ascites was diluted with the same dilution ratio as a negative control, and the plate was incubated at 37°C for 2 h; the plate was washed five times with PBST; 100 μL / well of 1:5000 diluted HRP-goat anti-mouse IgG was added and the plate was incubated at 37°C for 1 h; after washing with PBST, TMB color development solution was added for color development at 100 μL / well and the plate was incubated at 37°C for 10 min; 0.5 M H2SO4 was added to terminate the reaction, and the OD was measured using a microplate reader. 450 The titer of monoclonal antibody in ascites was determined with P / N≥2.1 as the judgment standard.

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

[0065] 3. Identification of Monoclonal Antibody Immunoreactivity

[0066] The recombinant African swine fever virus proteins His-p30 and GST-p30 were added to the loading buffer and incubated in a constant-temperature metal bath for 10 minutes before SDS-PAGE electrophoresis. The gel was then transferred to a nitrocellulose membrane using a rapid transfer apparatus for 10 minutes. The membrane was blocked with PBST containing 5% skim milk powder and incubated at room temperature for 1 hour with shaking. The membrane was then washed three times with PBST. The membrane was then incubated with monoclonal antibody 4E8 at a 1:1000 dilution, incubated at 4°C with shaking overnight, and washed five times with PBST. The membrane was then incubated with HRP-goat anti-mouse enzyme-linked antibody at a 1:5000 dilution, incubated at room temperature for 1 hour, and washed seven times with PBST. ECL staining was performed for 1 minute, and images were taken using an ultrasensitive multifunctional imager.

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

[0068] 4. Monoclonal antibody affinity identification

[0069] GST-p30 protein was coated on a 96-well ELISA plate at 2, 1, 0.5, and 0.25 μg / mL using coating buffer and incubated at 4°C overnight. The plates were washed three times with PBST, 200 μL of blocking buffer was added to each well, and the plates were incubated at 37°C for 2 h. The plates were washed three times with PBST, purified monoclonal antibody ascites diluted from 1:500 was added, and the plates were incubated at 37°C for 2 h. The plates were washed five times with PBST, 100 μL / well of HRP-goat anti-mouse IgG diluted 1:5000 was added, and the plates were incubated at 37°C for 1 h. After washing with PBST, TMB colorimetric solution was added for color development at 100 μL / well, and the plates were incubated at 37°C for 10 min. The reaction was terminated by adding 0.5 M H2SO4, and the OD was measured using a 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 a maximum affinity of 2.88×10 10 M -1 .

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

[0072] Array experiments determined that the optimal coating concentration of African swine fever virus GST-p30 protein was 2 μg / mL, and the optimal reaction concentration of enzyme-labeled antibody 4E8 was 90 ng / mL (dilution factor of 1:35,000). The optimal blocking solution, blocking time, serum incubation time, serum dilution factor, and substrate reaction time were optimized.

[0073] Optimization results can be found in Figure 4 , based on the optimal results, the final operating procedure was established as follows:

[0074] (1) Dilute the African swine fever virus GST-p30 protein to 2 μg / mL with a coating solution (carbonate buffer (CBS)) and add it to the ELISA plate at 100 μL / well. Coat the plate at 37°C for 2 h. Wash the plate three times with PBST. Use PBS containing 2% bovine serum albumin (BSA) as the optimal blocking solution and block the coated plate at 37°C for 2.5 h. Wash the plate three times with PBST and pat dry.

[0075] (2) Dilute the serum to be tested 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) Add TMB to develop color for 10 min; add 0.5 M H2SO4 to terminate the reaction, and read the OD value on the 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 and tested using the competitive ELISA method established above, and their PI values ​​were analyzed (PI = (1-OD value of serum sample) 450 Value / OD of blank control 450 )×100%), and GraphPadPrism software was used to draw the ROC curve and calculate the Youden index, sensitivity, and specificity. Figure 5 As shown in the figure, when the cut-off is 50.6%, the sensitivity and specificity of this method are the highest at 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 delta coronavirus), PEDV (porcine epidemic diarrhea), PRRSV (porcine blue ear virus), and PCV2 (porcine circovirus type 2). Based on the judgment threshold, the method was tested for cross-reaction. The results are shown in Figure 4. Figure 6 As shown in the figure, 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. 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. 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] Thirty sera that were positive for African swine fever and 50 sera that were negative for African swine fever, tested using the commercial African Swine Fever Competition Competitive ELISA test kit (ID-Vet), were tested using the competitive ELISA method established in Example 4. The results showed that all 75 samples matched the background, with an overall compliance rate of 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 skilled in 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 one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to 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 CDR1-3 in the light chain variable region are shown as SEQ ID NOs.1-3, respectively, and the amino acid sequences of the three complementary determining regions CDR1-3 in the heavy chain variable region are shown as SEQ ID NOs.4-6, respectively.

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 or the African swine fever virus p30 protein antibody according to any one of claims 2 to 3 in the preparation of a kit for 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 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 color developing solution and a reaction termination solution.

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