African swine fever virus dominant antigen epitope tandem recombinant protein and application thereof

Abstract

The application discloses an ASFV dominant antigen epitope tandem recombinant protein, and an amino acid sequence of the ASFV dominant antigen epitope tandem recombinant protein is shown as SEQ ID NO:1, and belongs to the field of biology. The P54 and B602L proteins of the high immunogenicity region of the screened ASFV dominant antigen epitope are tandemly expressed according to the antibody induction efficiency in a pig group to obtain the recombinant protein. An ASFV indirect ELISA detection method is established by taking the recombinant protein as an antigen. The method has high accuracy, good specificity and high sensitivity, and has a large application prospect in the prevention and control of the ASF disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and relates to a recombinant protein, in particular, an African swine fever virus (ASFV) dominant antigen epitope tandem recombinant protein, and the application also relates to application of the recombinant protein in ASFV detection. BACKGROUND

[0002] The African swine fever virus (ASFV) is a double-stranded DNA virus, the virus shell is icosahedral symmetry, has a capsule, and has a large genome. The ASFV has a complex escape host immune mechanism, can cause acute, febrile, hemorrhagic and highly lethal infectious diseases in pigs, and is a great threat to the pig industry. The international animal health organization classifies the ASF as a statutory reported animal epidemic disease, and China classifies the African swine fever as a class animal epidemic disease. At present, there is no commercialized African swine fever vaccine available in China, and the prevention and control of ASFV mainly depends on biological safety management and pathogen monitoring.

[0003] With the continuous spread and variation of the ASFV, strains with long incubation period and causing no obvious clinical symptoms in pigs appear, which makes it more difficult to detect the ASFV nucleic acid, and the situation of missed detection often occurs, which brings great challenges to the prevention and control of the ASFV; therefore, the ASFV antibody detection with strong specificity and high sensitivity is crucial to the prevention and control of the ASFV. The p54 protein is a type I transmembrane protein, which spans the inner layer capsule of the virus particle, and the protein plays an important role in the generation process of the virus envelope precursor and the stability of the inner virus envelope of the mature virus particle. The B602L protein is a late-expressed non-structural protein of the ASFV, which is mainly located in the cytoplasm. Studies have shown that the B602L protein is considered as a molecular chaperone of the capsid protein p72, and a transient connection between the two proteins is also detected in the cells infected with the ASFV. Another study shows that the IgG FC fused B602L protein binds to and interacts with the FcRI receptor of the antigen presenting cell, and it is found that the IgG FC fused B602L protein can promote the expression of various cytokines at the mRNA level.

[0004] At present, the antibody detection methods on the domestic market are all based on a single antigen of the ASFV, lack reasonable design, and have the risk of missed detection. The inventors identified the ASFV antigen epitopes that can be recognized by different pigs, i.e. the public epitopes, through high-throughput antigen epitope analysis in the early stage. In the present application, the high immunogenicity segments located in the p54 and B602L proteins are selected, and a target antibody detection protein is designed by concatenating the segments, and the protein can detect the antibodies of the p54 and B602L. SUMMARY

[0005] The application provides an ASFV dominant antigen epitope tandem recombinant protein and a construction method thereof, and application of the protein in African swine fever virus detection.

[0006] To achieve the above-mentioned purpose, the application provides the following technical solutions.

[0007] The ASFV antigen epitope tandem recombinant protein comprises high immunogenic segments of p54 and B602L proteins shown in sequences of SEQ ID NO: 2-5, and the recombinant protein is named as ASFVSynG1, and the amino acid sequence is shown in SEQ ID NO: 1.

[0008] The recombinant protein provided by the application comprises high immunogenic segments of p54 and B602L proteins of ASFV, can react with ASFV antibodies, is suitable for detection of ASFV, and improves the detection range and accuracy, and the protein is simple to prepare, stable and easy to express.

[0009] The application further provides a construction method of the ASFVSynG1 recombinant protein, comprising the following steps.

[0010] 1) the amino acid sequences shown in SEQ ID NO: 2-5 are connected by flexible peptides to obtain a tandem sequence shown in SEQ ID NO: 1, and a TRX tag is connected to promote soluble expression of the recombinant protein;

[0011] 2) a target gene is cloned from an expression vector containing the tandem sequence, and the target fragment is connected with a pET28a vector to construct a recombinant plasmid containing the tandem sequence;

[0012] 3) the recombinant plasmid is expressed by using E. coli BL21, and the target protein is extracted and purified from the recombinant bacteria.

[0013] The application further provides application of the recombinant protein in preparation of an African swine fever virus diagnostic kit and an African swine fever virus diagnostic kit.

[0014] The kit is an indirect ELISA kit for detecting African swine fever virus antibodies, and the kit contains the ASFVSynG1 recombinant protein, an enzyme-labeled plate, a washing solution, a diluent, a blocking solution, an enzyme-labeled secondary antibody, a color developing solution and a termination solution.

[0015] The application further provides a method for detecting African swine fever virus without diagnostic purposes, comprising the following steps.

[0016] 1) coating an enzyme-labeled plate with the ASFVSynG1 recombinant protein;

[0017] 2) blocking the enzyme-labeled plate with a blocking solution;

[0018] 3) After dilution, the serum sample to be tested is added to the enzyme-labeled plate for incubation of the primary antibody;

[0019] 4) After dilution, the enzyme-labeled secondary antibody is added to the enzyme-labeled plate for incubation of the enzyme-labeled secondary antibody;

[0020] 5) Color developing solution and termination solution are added, and the OD450 value is measured by an enzyme-labeled instrument and the result is determined.

[0021] Preferably, the coating concentration of the recombinant protein is 0.25 μg / mL, and the dilution of the serum sample to be tested is 1:400.

[0022] Preferably, the blocking solution is 5% skimmed milk powder.

[0023] Preferably, the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-swine IgG.

[0024] The result is determined according to the following standard: if the OD450 value of the serum sample to be tested is greater than 0.251, it is determined to be positive, and if the OD450 value of the serum sample to be tested is less than 0.251, it is determined to be negative.

[0025] The beneficial effects of the present application are:

[0026] The present application obtains an African swine fever antigen epitope tandem recombinant protein ASFVSynG1 by connecting the dominant antigen epitopes of ASFV key proteins, and verifies that the tandem recombinant protein ASFVSynG1 can be specifically combined with ASFV antibodies and can be used as a candidate antigen for ASFV detection. The ELISA method established in the present application can be used for disease diagnosis and laboratory screening identification of ASF, and has the advantages of high accuracy, strong specificity, high sensitivity, wide detection range, etc. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 : PCR amplification band of ASFVSynG1 (containing TRX tag).

[0028] Figure 2 : Electrophoresis result of enzyme digestion identification of ASFVSynG1 protein expression plasmid.

[0029] Figure 3 : SDS-PAGE verification of purified ASFVSynG1 protein.

[0030] Figure 4 : Western-blot verification of specific binding of expressed ASFVSynG1 protein and HIS monoclonal antibody. M: Marker, 1: pET28a vector control, 2: ASFVSynG1.

[0031] Figure 5 : Western-blot verification that the expressed ASFV SynG1 protein specifically binds to non-virulent positive serum. M: Marker, 1: pET28a vector control, 2: ASFV SynG1.

[0032] Figure 6 : Specificity test results of the constructed indirect ELISA method.

[0033] Figure 7 : Sensitivity test results of the constructed indirect ELISA method. DETAILED DESCRIPTION

[0034] The application will be further described in detail below with reference to examples. The following examples are only used to illustrate the application and should not be regarded as limiting the scope of the application.

[0035] According to the dominant antigen epitopes of African swine fever screened by the previous research group, the applicant selected the high immunogenicity segments of p54 and B602L protein and optimized the design, and connected them through a flexible peptide. The selected high immunogenicity antigen epitopes are shown in the sequence of Table 1.

[0036] Table 1. Antigen epitope sequences contained in ASFV SynG1

[0037]

[0038] Finally, the optimized antigen epitope sequences are connected in series with a flexible peptide to obtain the following amino acid sequence: EEEDIQFINPYQDQQWVEVTPQPGTSKPAGATTASVGKPVTGRPATNRPATNKPVTGGSGGSDNPVTDRLVMATGGPAAAPAAASAPAHPAEPYTTVTTQNTASQTMSAIENLRQRNTGGSGSMAEFNIDELLKNVLEDPSTEISEETLKQLYQRTNPYKQFKNDSRVAFCSFTNLREQGSGGSTHTTKTLLSELITLVDTLKQETNDVPSESVVNTILSIADSCKTQTQKSKEAKTTID (SEQ ID NO: 1)

[0039] Example 1. Design and preparation of ASFV antibody detection target

[0040] 1. Design and preparation of ASFV SynG1 protein

[0041] The nucleotide sequence of the tandem protein was synthesized by E. coli codon optimization and added TRX tag protein. The primer sequence for amplifying the nucleotide sequence of the target protein was designed, which included protective bases, enzyme cutting sites and nucleotide sequences combined with the template. The enzyme cutting site was Nhe I and Xho I. The designed primer was sent to the company for synthesis. The synthesized strain containing the target fragment was used as a template to amplify the gene fragment of ASFVSynG1 by PCR and add a TRX tag. The amplification results are shown in Figure 1 As shown in the figure, the target band is clear and obvious, and the band size meets the expectation. After gene amplification, agarose gel electrophoresis was performed, the DNA of the fragment was recovered by OMEGA gel recovery kit, and the DNA concentration was measured. QuickCut TM Nhe I / QuickCut TM Xho I (Takara) The DNA of the gene fragment was double digested, and the DNA was recovered by agarose gel electrophoresis. The concentration was measured by spectrophotometer and stored at -20℃ for standby.

[0042] 2. Construction of pET28-ASFVSynG1 expression plasmid

[0043] The extracted pET28a plasmid vector was double digested by QuickCut TM Nhe I / QuickCut TM Xho I. After enzyme digestion, agarose gel electrophoresis was performed, and the DNA of the vector was recovered by OMEGA gel recovery kit. The digested DNA fragment of the target protein was connected with the pET28a plasmid vector in vitro by seamless cloning enzyme. The ligation product was added to the DH5α competent cells, placed on ice for 30 minutes, heated at 42℃ for 90 seconds, and then placed on ice for 2 minutes. 500 μl of LB liquid medium was added to the competent cells, and incubated at 37℃ for 45 minutes. After incubation, centrifugation was performed at 5000 rpm for 3 minutes, 500 μl of supernatant was discarded, and the remaining 100 μl of supernatant was resuspended. After resuspension, the bacterial solution was inoculated on an LB solid plate containing 100 μg / ml kanamycin, and the plate was inverted and cultured in a constant temperature incubator at 37℃ overnight. The next day, a single colony was picked into 10 ml of LB liquid medium containing 100 μg / ml kanamycin, and the plasmid was extracted and identified after 12 hours of culture. The identification results are shown in Figure 2 As shown in the figure, the plasmid was digested, and there were two target bands, one band was the digested vector, and the other band was the digested target protein gene fragment, which met the expectation, proving that the expression plasmid was successfully connected. The biological company was sent for sequencing.

[0044] 3. Protein expression and purification

[0045] The sequencing correct plasmid is taken 10 ng with a pipette and added to the expression competent BL21 (DE3), placed in ice bath for 30 minutes, 42°C heat shock for 45 seconds, after heat shock, the competent is placed on ice, ice bath for 2 minutes again, 500 μl of LB liquid medium is added to the competent, placed in 37°C shaker 180 rpm for 45 minutes, after incubation, centrifuged at 5000 rpm for 3 minutes, 500 μl supernatant is discarded, the remaining 100 μl supernatant is resuspended after sedimentation, the bacterial liquid is inoculated on solid LB plate containing 50 μg / ml kanamycin and 34 μg / mL chloramphenicol, 37°C constant temperature incubator culture for 12 hours, a single colony is picked up to a bacterial bottle containing 10 mL medium, and kanamycin and chloramphenicol are added to make the final concentration 500 μg / ml and 34 μg / mL, about 8 hours of shaking culture, according to 1:100 transfer to 1L of LB liquid medium, and put into 37°C shaker 200 rpm, shake bacteria to OD value about 0.6, the shaking speed is adjusted to 170 rpm, and 1 ml of IPTG is added to make the final concentration 1 mmol / L, and the expression is induced for 5 hours. After 5 hours, the cultured bacteria are poured into a centrifuge bottle, centrifuged at 6000 rpm for 10 min, the supernatant is discarded and the precipitate is collected, the bacterial precipitate is washed twice with PBS. Resuspend the bacterial precipitate in 200 ml of binding buffer (300 mM NaCl, 20 mM Tris-HCl, concentrated hydrochloric acid to pH 8.0, 10 mM imidazole), use a high-pressure disrupter to break the protein at low temperature for about half an hour, collect the broken protein after the liquid is clear, centrifuge at 30000 g for 20 min to collect the protein supernatant, and filter the supernatant with a 0.22 μm filter, then purify the filtered protein supernatant through a His-tag protein purification pre-packed column. After the protein liquid is drained, change the washing buffer (300 mM NaCl, 20 mM Tris-HCl, concentrated hydrochloric acid to pH 8.0, 20 mM imidazole) to wash the impurities, and change the elution buffer (300 mM NaCl, 20 mM Tris-HCl, concentrated hydrochloric acid to pH 8.0, 400 mM imidazole) to elute the target protein combined with the His column after about 30 minutes of washing. Concentrate the eluted target protein by ultrafiltration with a 30 kDa ultrafiltration tube, first wash the ultrafiltration tube twice with ultrapure water, then add the protein to the ultrafiltration tube, centrifuge at 4000 rpm at 4°C, and the centrifugation time is determined according to the amount of protein. After each centrifugation, add a certain volume of GE buffer (20 mM Tris, 100 mM NaCl, adjusted to pH 8.0 with concentrated hydrochloric acid) to replace the high concentration of salt and imidazole in the protein. After about 4-5 times of centrifugation and replacement, the volume of the centrifuged protein is about one tenth of the volume before replacement. The protein is sucked out of the ultrafiltration tube into a 1.5 mL centrifuge tube, mixed well, and then aliquoted into PCR tubes, 40 μL per tube, and stored at -80°C for standby use.

[0046] 4. SDS-PAGE and Western blot verification of purified protein

[0047] 4.1 SDS-PAGE verification

[0048] After the protein was purified, 10 μL was taken out and added to a 1.5 ml EP tube containing 70 μL PBS, then 20 μL of 5x protein loading buffer was added, vortexed and mixed, and boiled at 100°C for 10 min. The prepared SDS-PAGE gel was taken out. First, run the concentrated gel at 80V, then switch the voltage to 120V to run the separation gel. After the protein gel was run, it was directly placed in a coomassie brilliant blue staining solution containing 2.5 g / L for 2 h, then transferred to a decolorizing solution (10% glacial acetic acid, 5% ethanol) for decolorization. After decolorization, the results were observed by gel imaging system, as shown in Figure 3 .

[0049] 4.2 Western blot verification

[0050] After running the concentrated gel at 80V and the separation gel at 120V, the protein gel was cut and transferred to a PVDF membrane at 65V for 55 min. The membrane was washed with TBST for 3 times, each time for 5 min, 5% skim milk powder was blocked at room temperature for 2 h, the membrane was washed with TBST for 3 times, each time for 5 min, mouse anti-His-Tag monoclonal antibody was used as the primary antibody and incubated at room temperature for 2 h (company: Abclonal, product number: AE003, the antibody was diluted with TBST at 1:10000), the membrane was washed with TBST for 5 times, each time for 5 min, HRP-labeled goat anti-mouse IgG was used as the secondary antibody and incubated for 45 min (company: Abbkine, product number: A21010, diluted with TBST at 1:8000), the membrane was washed with TBST for 5 times, each time for 5 min, ECL developing solution A:B was mixed in equal proportions and used for chemiluminescence imaging under light protection (company: Biosharp, product number: BL520A), the results of the purified protein are shown in Figure 4 , and it was found that the size of the purified target protein was consistent with the predicted results.

[0051] After running the concentrated gel at 80V, switch the voltage to 120V to run the separation gel, cut the gel block, transfer the protein to the PVDF membrane at 65V for 55min. Wash the membrane with TBST for 3 times, each time interval 5min, block with 5% skim milk at room temperature for 2h, wash the membrane with TBST for 3 times, each time interval 5min, incubate with the positive serum as the first antibody at room temperature for 2h (the serum dilution ratio is 1:20000), wash the membrane with TBST for 5 times, each time interval 5min, incubate with the HRP labeled goat anti-pig IgG as the second antibody for 45min (company: Solarbid, product number: SE137, dilute with TBST at 1:8000), wash the membrane with TBST for 5 times, each time interval 5min, mix equal volume of ECL developing solution A and B, and use the chemiluminescence instrument to develop the color under the dark condition (company: Biosharp, product number: BL520A). The results of the purified protein are shown in Figure 5 Figure 1, and it is found that the purified target protein can specifically bind to the African swine fever positive serum, which can be used as a target for the detection and diagnosis of African swine fever antibodies.

[0052] Example 2 Establishment of antibody indirect ELISA method for ASFV SynG1 antigen epitope tandem protein

[0053] 1. Screening of indirect ELISA detection conditions

[0054] 1.1 Chessboard titration method to determine the optimal concentration of coated antigen protein and the optimal serum dilution

[0055] Table 2 result analysis shows that when the concentration of coated antigen is 0.25μg / ml and the serum dilution is 1:400, the OD value of the positive serum is close to 1 and the P / N ratio is the highest (9.339).

[0056] Table 2 Optimal conditions of protein coating concentration and detection sample dilution

[0057]

[0058] 1.2 Screening of antigen incubation time

[0059] Change the antigen incubation time, keep other conditions unchanged, detect the OD value and calculate the P / N value. Table 3 results show that when the antigen incubation time is 37℃, 60min, the P / N value is the highest (8.535).

[0060] Table 3 Screening of antigen incubation time

[0061]

[0062] 1.3 Determination of blocking solution

[0063] Change the different blocking solution, keep other conditions unchanged, detect OD value and calculate P / N value. Table 5 results show that when the blocking solution is 5% skimmed milk powder, its P / N value is the highest (10.513).

[0064] Table 5 Determination of blocking solution

[0065]

[0066] 1.4 Incubation time of blocking solution screening

[0067] Change the incubation time of blocking solution, keep other conditions unchanged, detect OD value and calculate P / N value. Table 4 results show that when the incubation time of blocking solution is 37℃, 60min, its P / N value is the highest (9.891).

[0068] Table 4 Incubation time of blocking solution screening

[0069]

[0070] 1.5 Determination of serum reaction time

[0071] Change the different serum reaction time, keep other conditions unchanged, detect OD value and calculate P / N value. Table 6 results show that when the serum reaction time is 37℃, 60min, its P / N value is the highest (8.833).

[0072] Table 6 Determination of serum reaction time

[0073]

[0074] 1.6 Determination of enzyme-labeled secondary antibody incubation conditions

[0075] Change the different enzyme-labeled secondary antibody incubation conditions, keep other conditions unchanged, detect OD value and calculate P / N value. Table 7 results show that when the enzyme-labeled secondary antibody incubation conditions are 37℃, 60min, its P / N value is the highest (7.439).

[0076] Table 7 Incubation conditions of enzyme-labeled secondary antibody screening

[0077]

[0078] 1.7 Determination of enzyme-labeled secondary antibody dilution

[0079] Change the different enzyme-labeled secondary antibody dilution, keep other conditions unchanged, detect OD value and calculate P / N value. Table 8 results show that when the enzyme-labeled secondary antibody dilution is 1:5000, its P / N value is the highest (9.74).

[0080] Table 8 Determination of HRP-labeled secondary antibody dilution

[0081]

[0082] 1.8 Determination of incubation time of TMB developing solution

[0083] The incubation time of TMB developing solution was changed, and other conditions were kept unchanged, the OD value was detected and the P / N value was calculated. The results in Table 9 showed that when the incubation time of TMB developing solution was 7.5 min, the P / N value was the highest (9.859).

[0084] Table 9 Screening of incubation time of TMB developing solution

[0085]

[0086] 2. Determination of critical value

[0087] Seventy-one negative pig serum samples collected before 2018 were selected and detected by the established indirect ELISA method, and the results are shown in Table 10. The average OD450nm value of the 71 ASFV negative serum samples was 0.131, the standard deviation (SD) was 0.040, and according to the formula: critical value = average OD450nm value + 3 x standard deviation = 0.251. 450

[0088] Table 10 OD values of 71 samples 450

[0089]

[0090] 3. Establishment of indirect ELISA detection method

[0091] According to the above experiments, the finally determined detection method is as follows:

[0092] (1) Coating antigen: dilute the purified antigen ASFVSynG1 to 0.25 μg / mL with coating buffer, add 100 μL to each well of the 96-well plate, incubate at 37°C for 1 h for coating, discard the liquid in the well, and wash with PBST.

[0093] (2) Blocking: use 5% skimmed milk powder in PBST as blocking solution, 200 μL per well, incubate at 37°C for 1 h. Discard the liquid in the well and pat dry, and wash with PBST.

[0094] (3) Incubate serum: dilute the serum to 1:400 (the diluent is 1% BSA in PBST), add 100 μL to each well, incubate at 37°C for 1 h, discard the liquid in the well and pat dry, and wash with PBST.

[0095] ​​(4) Incubation with enzyme-labeled secondary antibody: Dilute HRP-labeled goat anti-pig IgG to 1:5000, add 100 μL to each well, incubate at 37°C for 1 h, shake off the liquid in the well and pat dry, then wash with PBST.

[0096] (5) TMB color development: Add 100 μL of TMB color development solution to each well, and develop the color at room temperature in the dark for 7.5 min.

[0097] (6) Termination of reaction: Add 100 μL of 2M H2SO4 stop solution to each well.

[0098] (7) Result determination: The OD450nm value is measured on the microplate reader, and the result is determined based on the positive / negative threshold OD. 450 The judgment result is that when OD 450 A value ≥ 0.251 indicates a positive result. 450 A value less than 0.251 indicates a negative result.

[0099] HRP-labeled goat anti-pig IgG was obtained by volumetric dilution of its stock solution at a ratio of 1:5000; PBST washing buffer was 0.01 mol / L phosphate buffer containing 0.05% Tween 20 at pH 7.4; dilution buffer was PBST washing buffer containing 1% BSA; stop solution was 2 mol / L sulfuric acid solution. Positive control serum was immune serum containing tandem recombinant protein; negative control serum was specific pathogen-free porcine serum.

[0100] 4. Repeatability test

[0101] Six porcine serum samples were selected, and each serum sample was used in triplicate. The results obtained by the ELISA reader are shown in the table. The mean (AV) and standard deviation (SD) were calculated, and the coefficient of variation was calculated according to the formula: coefficient of variation CV (%) = standard deviation (SD) / mean (AV) × 100%. The results showed that the intra-assay coefficient of variation was 1%-6.8%, and the inter-assay coefficient of variation was 2.1%-9.3%, which was less than 10%, indicating that the established antibody ELISA method has good repeatability and stability.

[0102] Table 11 Inter-batch and intra-batch repeatability tests

[0103]

[0104] 5. Specificity test

[0105] An established ELISA method for African swine fever antibodies was used to detect positive sera for other common swine viral diseases (including CSFV, PRRSV, PRV, PEDV, and PCV2), with negative and positive controls set up. Figure 6 As shown, the following are positive serum OD values ​​for several swine viral diseases. 450The values are all less than the positive-negative critical value 0.251, and it is judged as negative, which indicates that the established ELISA method has good specificity.

[0106] 6. Sensitivity test

[0107] The positive serum and the negative serum of African swine fever were diluted by times through the established ELISA method, and as shown in Table 2, the serum was positive when diluted to 1:6400, and was negative when diluted to 1:12800. Figure 7

[0108] 7. Detection of ELISA clinical samples

[0109] The clinical samples were detected by the established ELISA method and a domestic kit, respectively. Among them, 100 pig sera were detected, 14 positive sera were detected by the commercial kit, and 20 positive sera were detected by the method. 86 negative sera were detected by the commercial kit, and 80 negative sera were detected by the method, and the total coincidence rate was 94.00%.

[0110] Table 12 Coincidence rate test results of the commercial kit

[0111]

[0112] In summary, the ASFVSynG1 recombinant protein provided by the application can be specifically combined with the positive serum of African swine fever, and can be used as a candidate target for the diagnosis of African swine fever, and is used for the detection of African swine fever antibody. The provided ELSIA method has good specificity and high sensitivity, and the serological detection method based on the conserved epitope is expected to provide a wider diagnostic range.​

Claims

1. A tandem recombinant protein of African swine fever virus dominant antigen epitopes, the amino acid sequence of which is shown as SEQ ID NO:

1.

2. A method for preparing the tandem recombinant protein of dominant antigen epitope of African swine fever virus according to claim 1, characterized in that The method comprises the following steps: 1) concatenating the amino acid sequences shown as SEQ ID NOs: 2-5 with flexible peptides to obtain a tandem sequence shown as SEQ ID NO: 1; 2) cloning a target gene from an expression vector containing the tandem sequence and connecting the target fragment with a pET28a vector to construct a recombinant plasmid containing the tandem sequence; 3) expressing the recombinant plasmid with Escherichia coli BL21 and extracting and purifying the target protein from the recombinant bacteria. 3.Use of the recombinant protein of claim 1 in the preparation of an African swine fever virus diagnostic kit. 4.An African swine fever virus diagnostic kit, the kit containing the recombinant protein of claim 1. 5.The African swine fever virus diagnostic kit of claim 4, which is an indirect ELISA kit for diagnosing African swine fever virus.

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