A coated protein, applications and kits for diagnosis of antibodies to african swine fever virus

Abstract

The application belongs to the field of biology, and discloses a coated protein, application and kit for diagnosing African swine fever virus antibody, wherein the coated protein comprises p22 protein and E199L protein; the amino acid sequence of the p22 protein is shown as SEQ ID NO:1, and the amino acid sequence of the E199L protein is shown as SEQ ID NO:3. The coated protein is composed of recombinant proteins of p22 and E199L, and both of the two kinds of recombinant proteins can react with ASF positive serum, and both have good reactivity. The detection kit based on the above coated protein has high detection precision, strong specificity and good repeatability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology, in particular to a coating protein, application and kit for African swine fever virus antibody diagnosis. BACKGROUND

[0002] African swine fever (ASFV) is an acute and highly contagious hemorrhagic infectious disease caused by African swine fever virus (ASFV) infection, and the disease process can be divided into acute, subacute, chronic and latent infection. Infection with acute ASF can cause high fever, ataxia, watery and bloody diarrhea, diffuse subcutaneous hemorrhage, necrotic dermatitis, severe hemorrhage of multiple organs and sudden death, and death occurs within 5-14 days, with a mortality rate of up to 100%. ASFV can infect pigs and wild boars of different breeds and different ages. At present, China classifies it as a class A animal infectious disease.

[0003] Due to the serious harm of ASFV to the pig industry, so far there is no commercial ASF vaccine or drug that can effectively prevent or treat the disease, and timely detection of pathogens and interruption of transmission routes is the most effective method to reduce the social and economic losses caused by ASF. Therefore, it is of great significance to develop a detection method with high sensitivity and accuracy for China's pig breeding industry.

[0004] The only effective strategy to control ASF at this stage is to isolate and eliminate infected animals. Therefore, high sensitivity and specificity diagnostic analysis is essential for rapid detection and discovery of ASFV infected pigs. At present, there are many ASFV antigen detection methods, including fluorescent quantitative PCR, RPA, microfluidic chip and other rapid detection methods, but antibody detection methods are relatively few, mainly focusing on ELISA antibody detection kits and colloidal gold test strips. As a conventional serological detection method, ELISA has the advantages of high sensitivity, simple operation, rapid detection and economy. Screening of ASFV antigens with good reactivity and establishment of effective ELISA detection methods are crucial for the prevention and control of ASF.

[0005] At present, the proteins coated in the African swine fever ELISA antibody detection kit mainly include P72, P30, P54 and PP62, among which P30 and P54 proteins are expressed and secreted in the early stage of ASFV infection, and P72 and PP62 proteins are expressed in the late stage of ASFV infection.

[0006] Currently, most commercial ELISA kits are based on P30 protein, which has a coincidence rate of only 90%, and the clinical detection effect is not good, and the accuracy needs to be further considered. Studies have shown that p22 protein, as a protein involved in early invasion of the virus into the body, has high antigenicity and can be used as an important antigen for antibody detection. Studies have shown that ASFV mainly escapes immune surveillance of the host by regulating the expression of pro-inflammatory molecules and cytokines, and related studies have shown that E199L may be involved in inflammatory responses after viral infection to escape immune defense of the host. According to reports, E199L antibodies can be detected within 7 days after ASFV infection, indicating that E199L can be used for early detection of ASFV.

[0007] The application of p22 in ELISA detection can be seen in the following literature:

[0008] Patent application with publication number CN118440160A, entitled "African swine fever virus p22 protein ELISA antibody detection kit", which uses two or more artificially synthesized p22 proteins to improve detection accuracy;

[0009] Patent application with publication number CN114966052A, entitled "Indirect ELISA detection kit based on African swine fever p30 and p22 proteins", which uses P30 and p22 proteins as antigens to improve detection accuracy. After ASFV positive serum is diluted 12800 times, the detection result is still positive, with high sensitivity.

[0010] The application of E199L in ELISA detection can be seen in the following literature:

[0011] Patent application with publication number CN115160411A, entitled "Screening, preparation and application of dominant antigen of African swine fever virus", which uses one or more of the following African swine fever virus antigens: optimized recombinant E199L, E184L, B475, E120R, A104R, A137R, K145R, CP312R, p12-p17-p10 and I73R-B169L.

[0012] In summary, p22 and E199L proteins play an important role in the early replication and immune response of ASFV and can be used as important immune antigens for ASFV antibody detection. In the previous work of this project, it was found that the detection effect of the detection kit based on p22 and / or E199L coating protein did not reach a satisfactory level. SUMMARY

[0013] The purpose of the present application is to provide a coating protein for African swine fever virus antibody diagnosis, which is composed of p22 and E199L recombinant proteins, both of which can react with ASF positive serum and have good reactivity, and the detection kit based on the coating protein has high detection accuracy, strong specificity and good repeatability.

[0014] Meanwhile, the present application also provides the application of the coating protein and the kit.

[0015] To achieve the above purpose, the present application provides the following technical solutions.

[0016] A coating protein for African swine fever virus antibody diagnosis, comprising p22 protein and E199L protein; the amino acid sequence of the p22 protein is shown as SEQ ID NO: 1, and the amino acid sequence of the E199L protein is shown as SEQ ID NO: 3.

[0017] In the coating protein, the nucleotide sequence of the p22 protein gene is shown as SEQ ID NO: 2, and the nucleotide sequence of the E199L protein gene is shown as SEQ ID NO: 4.

[0018] In the coating protein, the coating weight ratio of the p22 protein and the E199L protein is 2:1.

[0019] Meanwhile, the present application also discloses the use of the coating protein as described above for preparing a detection kit; the detection kit is used for detecting African swine fever virus antibody.

[0020] Finally, the present application also discloses an African swine fever virus antibody indirect ELISA detection kit, comprising the coating protein as described above.

[0021] In the African swine fever virus antibody indirect ELISA detection kit, it comprises a pre-coated plate containing p22 protein and E199L protein, serum diluent, antibody diluent, enzyme-labeled secondary antibody, substrate developing solution, termination solution and washing solution.

[0022] In the African swine fever virus antibody indirect ELISA detection kit, the blocking solution of the pre-coated plate is 2wt% TY protein protective agent and 2wt% trehalose PBS, the blocking time at 25℃ is 4h, and after drying in a 25℃ drying oven for 1h, it is stored at 4℃.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] The application is based on an indirect ELISA detection kit established by two proteins of p22 and E199L, both of which can react with ASF positive serum, and both have good reaction originality; the detection result is still positive when the ASFV positive serum is diluted by 12800 times, which can reach the same level of the kit on the market, and has high sensitivity; the ELISA method of the application only reacts with ASF positive serum, and has no cross reaction with positive serum of porcine circovirus disease, pseudorabies, porcine reproductive and respiratory syndrome, swine fever and porcine epidemic diarrhea virus, and has high specificity; the batch and batch variation coefficient (CV) is less than 5% when the positive serum is detected, which indicates that the kit has good repeatability; the kit can be stored at 4℃ for 1 year and at 37℃ for 15 days, and has good high-temperature stability and real-time stability. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is a map of plasmid pET-28a-p22;

[0026] Figure 2 It is a map of plasmid pET-28a-E199L;

[0027] Figure 3 It is a schematic diagram of amino acid sequence truncation of p22;

[0028] Figure 4 It is a schematic diagram of amino acid sequence truncation of E199L;

[0029] Figure 5A It is a reaction originality identification diagram of p22;

[0030] Figure 5B It is a reaction originality identification diagram of E199L;

[0031] Figure 6A It is a column chart of the best sample incubation time;

[0032] Figure 6B It is a column chart of the enzyme-labeled antibody incubation time;

[0033] Figure 6C It is a P / N diagram of color development time. DETAILED DESCRIPTION

[0034] The technical solutions of the application will be described clearly and completely in combination with the embodiments of the application. Obviously, the described embodiments are only some of the embodiments of the application, not all. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the application.

[0035] Example 1

[0036] 1. Construction and synthesis of recombinant positive plasmid

[0037] According to the gene sequence and amino acid sequence of the p22 and E199L encoding genes of the African swine fever virus Pig / HLJ / 2018, by analyzing the hydrophobicity and hydrophilicity, transmembrane region and signal peptide region of the target sequence, while ensuring the antigenicity and hydrophilicity, the hydrophobic region and signal peptide region are removed, according to the compatibility of codons, codon optimization is carried out, and BamHI and XhoI cloning sites are selected in the upstream and downstream, and the target genes encoding the p22 protein and E199L of the African swine fever virus are obtained.

[0038] In the nucleotide sequence optimization of the embodiment 1 of the application, the optimized sequence is analyzed for GC content, CAI, codon optimization, restriction sites and cis-acting elements, hairpin structure adjustment, model scoring, etc., and after preliminary theoretical expectation, the sequence synthesis is carried out by Suzhou Hongxun Biotechnology Co., Ltd. The comparison of the optimized sequence and the original sequence is shown in Table 1;

[0039] Table 1 Comparison of optimized sequence and original sequence

[0040]

[0041] According to the comparative analysis of the optimized sequence, the more stable the GC content change curve is, the minimum the hairpin structure is, which will theoretically make the ribosome translation efficiency the highest. The optimized sequence reduces the restriction sites and cis-acting elements. The comprehensive score of the optimized model is about 19%-27% of the original genotype. The higher the score of the gene model evaluation system is, the lower the expression level will be. And according to the comparison data, in theory, the expression level of the optimized gene in the host will be higher. The CAI is a quantity of codon adaptability index, ranging from 0 to 1.0, and theoretically, the closer the CAI index is to 1, the higher the expected expression level is, but at the same time, it does not mean that the protein can be expressed when the CAI is 1.

[0042] The positive cloned plasmid of p22 and E199L is double digested with restriction endonuclease BamHI and XhoI, respectively, and is connected with the same double enzyme treated pET-28 vector, and the ligation product is transformed into competent cells DH5α. Sequenced by Shengong Bioengineering (Shanghai) Co., Ltd., the two kinds of recombinant positive plasmids with correct sequence are named as pET-28a-p22 ( Figure 1 ) and pET-28a-E199L ( Figure 2 ).

[0043] The truncated p22 amino acid sequence is shown as SEQ ID NO: 1, and the schematic diagram of the truncation is shown in Figure 3;

[0044] The codon-optimized p22 nucleotide sequence is shown as SEQ ID NO: 2;

[0045] The truncated E199L amino acid sequence is shown as SEQ ID NO: 3, and the schematic diagram of truncation is shown in Figure 4 ;

[0046] The codon-optimized E199L nucleotide sequence is shown as SEQ ID NO: 4;

[0047] 2. Induction expression and protein purification of recombinant proteins p22 and E199L

[0048] The specific operation of induction expression is as follows:

[0049] The two recombinant plasmids pET28a-p22 and pET28a-E199L were transformed into competent cells BL21, respectively. Single colonies were picked from solid culture medium incubated overnight at 37°C, and then placed into LB liquid medium with Kana+ resistance and incubated overnight at 37°C and 200 rpm. 50 μL of bacterial solution of pET-28a-p22, pET-28a-E199L and pET-28a(+) were taken, respectively, and placed into test tubes containing 5 mL of LB liquid medium (Kana+). Another tube containing 5 mL of LB liquid medium (Kana+) without bacterial solution was set as negative control, and incubated at 37°C and 200 rpm for 3-4 h on a constant temperature shaker, so that the OD 600 of the two bacterial solutions was between 0.6 and 0.8. 500 μL of bacterial solution was taken from each tube as pre-induction control and stored at 4°C. Then, 0.1 mM IPTG solution was added to the remaining three tubes of bacterial solution, and incubated at 37°C and 200 rpm for 4 h. 500 μL of bacterial solution was taken from each tube. The three tubes of bacterial solution before induction and the three tubes of bacterial solution after induction were centrifuged (9000 rpm, 4°C) for 10 min at the same time, and the supernatant was discarded and the bacterial solution was retained. After resuspending the bacterial solution with 100 μL of PBS, centrifugation was performed at 9000 rpm for 10 min at 4°C, and the supernatant was discarded and the bacterial solution was retained. The above steps were repeated twice, and 6x SDS-PAGE Sample Buffer was added to the bacterial solution resuspended in the last time. The sample was boiled in boiling water for 10 min, and SDS-PAGE gel electrophoresis detection was performed.

[0050] The optimal expression conditions were determined by exploring the induction expression conditions (IPTG concentration, induction temperature, induction time). After IPTG induction expression of the target protein p22 and E199L, a single band of the target protein was obtained by nickel column purification, and the concentration of the two purified recombinant proteins was determined by BCA protein quantitative kit. The results are: pET28a-p22 is 0.45 mg / mL; pET-28a-E199L is 0.23 mg / mL.

[0051] 3. Reactogenicity identification of recombinant proteins p22 and E199L

[0052] Using purified p22 and E199L recombinant proteins as antigens, ASF positive serum (purchased from the Institute) as primary antibody, and goat anti-pig IgG HRP as secondary antibody, WB analysis was performed. The results show that the two proteins p22 and E199L are clear and single bands, and the expected size is consistent. It is shown that the two recombinant proteins p22 and E199L can react with ASF positive serum, and both have good reactogenicity. Figure 5A and Figure 5B , wherein Figure 5A is the reactogenicity identification diagram of P22, Figure 5B is the reactogenicity identification diagram of E199L).

[0053] 4. Optimization of ELISA method reaction conditions

[0054] The kit includes: pre-coated enzyme-labeled plate, 20x concentrated washing solution, sample diluent, enzyme-labeled antibody, TMB developing solution, stop solution, negative control, and positive control.

[0055] 4.1 Optimal protein coating concentration and serum dilution (P is the positive serum dilution of the Institute, and N is the SPF pig serum)

[0056] The optimal coating concentration of p22+E199L protein was determined by chessboard titration method as 2 μg / mL: 1 μg / mL, and the sample was diluted 100 times. The results are shown in Table 2.

[0057] Table 2 Optimal protein coating concentration and serum dilution screening

[0058]

[0059] 4.2 Determination of the optimal blocking solution

[0060] After determining the optimal protein coating concentration and optimal serum dilution, the above operation steps were repeated to determine the optimal blocking scheme. The P / N value and stability test results were compared. The results are shown in Table 3.

[0061] Table 3 Screening of different blocking conditions

[0062]

[0063] 4.3 Determination of the optimal sample incubation time, enzyme-labeled antibody incubation time and color development time

[0064] Under the above-mentioned optimal conditions, the above-mentioned operation steps were repeated to determine the optimal sample incubation time, enzyme-labeled antibody incubation time and color development time. The optimal color development conditions were determined by comparing P / N, and the results were as follows: Figure 6A to Figure 6C .

[0065] 4.4 Determination of the critical value

[0066] According to the above-mentioned optimal conditions, 500 negative sera (different brands of commercial kits) were selected for ELISA detection, and OD 450 was read, and S / P value was calculated to calculate the average value and standard deviation. The positive serum and SPF serum of the institute were diluted at different concentrations, and the antibody protectant was added as a positive control and a negative control. The results were calculated: S / P=(sample OD-NC) / (PC-NC). The results were determined: the calculated S / P value was greater than 0.3 as positive, and the S / P value was less than 0.3 as negative.

[0067] 4.5 Sensitivity test

[0068] The p22 and E199L indirect ELISA kits and two commercial kits were used to detect different dilution rates of the ASF positive serum purchased by the institute, and OD 450 was measured, and S / P value was calculated. The results were as shown in Table 4. According to the S / P value, after the serum was diluted 12800 times, the detection results of the p22 and E199L coated plates were still positive (Table 4). The comparison of the commercial kits could achieve the same level as the same type (indirect ELISA) commercial kit.

[0069] Table 4 Sensitivity detection of different schemes

[0070]

[0071] 4.6 Specificity test

[0072] The positive sera of porcine circovirus disease, pseudorabies, porcine reproductive and respiratory syndrome, swine fever and porcine epidemic diarrhea virus (China Veterinary Drug Inspection Institute) were subjected to ELISA cross-reaction test for specificity evaluation. The test results were as follows: the S / P values of the other five pig diseases were less than 0.3.

[0073] The results show that the ELISA kit established in the application only reacts with ASFV positive serum, and has no cross reaction with positive serum of porcine circovirus type 2, pseudorabies virus, porcine reproductive and respiratory syndrome, swine fever and porcine epidemic diarrhea disease. The method has good specificity, and the results are shown in Table 5.

[0074] Table 5 Specificity test

[0075]

[0076] 4.7 Reproducibility test

[0077] The positive serum (the positive serum gradient diluted 6400 times purchased by the center) is selected, and the batch and batch variation coefficients are less than 5%. The batch and batch variation coefficients show that the indirect ELISA kit established in the application has good reproducibility, and the results are shown in Table 6.

[0078] Table 6 Reproducibility test

[0079]

[0080] 4.8 Stability test

[0081] The prepared ASFV kit is selected for high temperature stability detection (positive-1 serum is serum diluted 1600 times purchased by the center, and positive-2 serum is serum diluted 3200 times purchased by the center). The same batch of kit is respectively stored at 4 DEG C and stored in a 37 DEG C constant temperature box after preparation. After 15 days of high temperature of the kit, the kit stored at 4 DEG C is compared. The OD value and S / P value are determined, the OD value is reduced by not more than 20%. The S / P value does not affect the analysis sensitivity determination.

[0082] The real-time stability detection of the kit stored at 4 DEG C for 3 months is compared. The degradation rate of OD value is not more than 20%. The S / P value does not affect the analysis sensitivity grade. Although the absolute value of negative control OD value decreases, the background value is very low, the value after the decrease is still much lower than the critical value, and the S / P value change rate is within the acceptable range, and the results are shown in Table 7.

[0083] Table 7 High temperature stability and real-time stability detection

[0084]

[0085] 4.8 Sample detection

[0086] 280 pig blood samples were detected by the above established indirect ELISA kit, 34 positive samples and 246 negative samples were detected. IDVET was used as a reference standard. The overall sample compliance of the p22 and E199L double protein indirect ELISA kit in this study compared with other finished kits (indirect method and competitive method) is shown in Tables 8, 9 and 10.

[0087] Table 8 Sample detection results of this embodiment

[0088]

[0089] Table 9 Sample detection results of commercial kit 1

[0090]

[0091] Table 10 Sample detection results of commercial kit 2

[0092]

[0093] Comparative Example 1 Expression of full-length sequence of E199L protein

[0094] The full-length sequence of E199L protein was expressed according to the reference. After optimizing the target sequence, the pET-28a (+) vector was connected for induced expression;

[0095] The full-length reference of E199L sequence after optimization is shown in SEQ ID NO. 5;

[0096] Table 11 is the full-length sequence alignment analysis result of E199L sequence in this comparative example;

[0097] Table 11 Full-length sequence alignment analysis result of E199L sequence

[0098]

[0099] Result: The sequence cannot be expressed.

[0100] Comparative Example 2

[0101] The truncated sequences of E199L and p22 were expressed in series, and the pET-28a (+) vector was connected for induced expression. The nucleotide sequence after series connection is shown in SEQ ID NO. 6. Table 12 is the full-length sequence alignment analysis result of E199L and p22 fusion expression sequence in this comparative example;

[0102] Table 12 Full-length sequence alignment analysis result of E199L and p22 fusion expression sequence

[0103]

[0104] Results: The target protein expressed by the sequence is too large, and the target protein cannot be purified.

[0105] Comparative Example 3

[0106] When the p22 protein is purified, an ELISA detection method is established for the p22 protein alone: ELISA condition optimization is performed for the p22 protein alone: sensitivity detection and clinical samples are performed.

[0107] Results: The analysis sensitivity is consistent with the results of mixed coating of p22 + E199L protein, and can also reach 12800-fold dilution. The coincidence rate of detection of clinical positive samples is low. The results are referred to Table 13;

[0108] Table 13 Coincidence rate detection results of single p22 protein coating

[0109]

[0110] Comparative Example 4

[0111] When the E199L protein is purified, an E199L detection method is established for the E199L protein: ELISA condition optimization is performed for the p22 protein alone: sensitivity detection and clinical samples are performed.

[0112] Results: The analysis sensitivity is consistent with the results of mixed coating of p22 + E199L protein, and can also reach 12800-fold dilution. The coincidence rate of detection of clinical positive samples is low. The results are referred to Table 14;

[0113] Table 14 Coincidence rate detection results of single E199L protein coating

[0114]

[0115] Comparative Example 5

[0116] According to the reference, the p22-2 nucleotide sequence (referring to SEQ ID NO. 7) is synthesized, the sequence is analyzed, the GC content is 49%, and the CAI analysis is 0.87. The GC content and CAI analysis of the sequence are lower than those of the synthesized p22 nucleotide sequence. The p22-2 sequence is connected to the pET-28a (+) vector to construct a recombinant plasmid for expression and purification. An ELISA detection method is established for the p22-2 protein alone: ELISA condition optimization is performed for the p22-2 protein alone: sensitivity detection and clinical samples are performed.

[0117] Results: The p22-2 target protein can be purified, the analysis sensitivity is consistent with the results of mixed coating of p22 + E199L protein, and can also reach 12800-fold dilution. The coincidence rate of detection of clinical positive and negative samples is low.

[0118] The results refer to Table 15;

[0119] Table 15 p22-2 protein coating coincidence rate detection results

[0120]

[0121] By comparison and summary, it can be known that the p22 and E199L proteins of the application mainly have the following aspects:

[0122] 1. The p22 and E199L protein sequences of the application are compared in different expression systems, and the p22 and E199L proteins are expressed and purified by constructing a prokaryotic expression system, which has high antigenicity and specificity.

[0123] 2. The p22 and E199L protein sequences of the application are subjected to hydrophilicity and antigenicity prediction, and the target sequence with good antigenicity and hydrophilicity is selected according to the expression characteristics of different expression systems, and is subjected to truncation expression, so as to have good antigenicity and hydrophilicity.

[0124] 3. After determining the p22 and E199L protein sequences, the codon preference of different expression systems is optimized, and in the case of not changing the amino acid sequence, the codon preference is optimized for the prokaryotic expression system, so that the success rate of p22 and E199L protein expression is greatly improved.

[0125] In addition, with reference to Comparative Example 1, when the sequence of E199L protein is designed, the sequence of E199L is used according to the sequence design in the reference, the optimized sequence is connected with the vector for induction expression. However, the target protein cannot be successfully expressed, and through amino acid sequence analysis, it is found that the protein region has a transmembrane region, which cannot be expressed. When the sequence of E199L is redesigned, the transmembrane region and antigenicity analysis are considered, in order to ensure that the target region has high antigenicity and is not interfered by foreign amino acid fragments, the transmembrane region (147-175AA) is removed. The results of antigenicity analysis show that the regions with low antigenicity (1-5AA) and (133-147AA) and the extracellular region (176-199AA) are removed, that is, the remaining amino acid sequence is SEQ ID NO: 3.

[0126] The base sequences of the p22 and E199L proteins of the application are the full-length sequences of the original strains p22 and E199L proteins, and through a large amount of biological software analysis, the base and amino acid sequences are analyzed, the transmembrane region and the antigenicity are analyzed, and the target sequence is selected based on the principles of good antigenicity and hydrophilicity priority, and then the final sequence is obtained by codon compatibility and codon optimization. After expression, the p22 and E199L proteins of the application are obtained.

[0127] The p22 and E199L proteins prepared by the prokaryotic expression system of the application are used as ELISA coating antigens, and have high specificity and sensitivity.

[0128] The p22 and E199L proteins of the application are used as coating antigens after protein purification, different blocking liquid combinations are screened, different blocking liquid combinations are compared, and it can be determined that the enzyme-labeled plate prepared by the screened combination is stable for 15 days in a high-temperature destruction experiment. It can be stored for one year at 4 DEG C.

[0129] The p22 and E199L proteins have good antigenicity and can be used as important antigens for ELISA detection of antibodies, but there is a certain missed detection in the p22 and E199L protein systems alone during sample detection. The p22 and E199L protein mixed coating is screened for the best concentration and ratio, so that the detection rate of positive samples in the clinic is the highest.

[0130] Comparing sequence 2, although the p22 and E199L after truncation expression can be expressed alone, through the connection of the peptide segment GGSG, the optimization of the codon base, but the p22 and E199L still form a multimer, which causes the target protein to be unable to be effectively expressed, which may be related to the conflict of the spatial structure of the two proteins.

[0131] It should be noted that the antigen used in the ASFV antibody detection kit of the application is an important antigen of ASFV during early replication and infection after the virus invades the body, and can be applied to important antibody detection of ASFV invasion and replication.

[0132] In summary, this shows that the advantages and disadvantages of the ASFV antibody detection kit are related to at least the following three factors:

[0133] 1. Comparison and screening of ASFV proteins with different functions and antigenicity;

[0134] 2. Truncation expression strategy and expression mode of p22 and E199L antigens of ASFV;

[0135] 3. System optimization scheme and optimization strategy of the ASFV antibody detection kit;

[0136] The three are closely related;

[0137] Meanwhile, compared with the method of obtaining nucleic acid sequences from samples in the prior art and then constructing plasmids, the application adopts gene synthesis technology after optimizing the expression sequence, so that the recombinant plasmid is quickly constructed by the target gene and the expression vector, and the cost required for constructing the recombinant expression plasmid is greatly reduced.

[0138] It will be apparent to those skilled in the art that the application is not limited to the details of the above-exemplified embodiments and that the present application can be implemented in other particular forms without departing from the spirit or essential characteristics of the present application. The embodiments should therefore be considered in all respects as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the above description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. No reference signs in the claims should be considered as limiting the scope of the claims with respect to the figures of the patent document.

Claims

1. A method for preparing an indirect ELISA test kit for diagnosis of antibodies against African swine fever virus, characterized by, The indirect ELISA detection kit comprises a pre-coated plate of p22 protein and E199L protein, serum diluent, antibody diluent, enzyme-labeled secondary antibody, substrate developing solution, termination solution and washing solution; The amino acid sequence of the p22 protein is shown as SEQ ID NO:1, and the amino acid sequence of the E199L protein is shown as SEQ ID NO:3; the nucleotide sequence of the p22 protein gene is shown as SEQ ID NO:2; the nucleotide sequence of the E199L protein gene is shown as SEQ ID NO:4; and the coating weight ratio of the p22 protein and the E199L protein is 2:1; The preparation method of the p22 protein and the E199L protein is as follows: The p22 protein gene and E199L protein gene were respectively bound with restriction endonuclease. BamH I and Xho The pET-28 vector, after double enzyme digestion, was ligated to obtain ligation products containing the p22 protein gene and the E199L protein gene. These ligation products were then transformed into competent DH5α cells and sequenced to identify the recombinant positive plasmids pET-28a-p22 and pET-28a-E199L. The recombinant positive plasmid pET-28a-p22 and the recombinant positive plasmid pET-28a-E199L are respectively transformed into competent cells BL In step 21, the positive monoclonal bacteria are selected for culture, induction of expression, purification, and obtaining of the p22 protein and the E199L protein.

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