Antigen of African swine fever virus, detection kit and application

Abstract

The invention provides an African swine fever virus antigen, a detection kit and application. The antigen of the African swine fever virus is an African swine fever virus C-type lectin protein, and the amino acid sequence of the African swine fever virus C-type lectin protein is as shown in SEQ ID NO. 1. In the antigens of the African swine fever virus (ASFV), the protective immunity of the African swine fever virus (ASFV) may be that the serotype specificity is inhibited by blood adsorption, and in the ASFV protein, the C-type lectin is a necessary protein for mediating the serological specificity of the HAI. Therefore, the C-type lectin is used as a target protein for ASFV serological diagnosis, and the antigen characteristic of the C-type lectin protein is utilized, so that an antibody ELISA detection method with application value is established, and research and development of a novel ASFV antibody ELISA detection method are facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an antigen, a detection kit and applications of African swine fever virus. Background Art

[0002] African swine fever virus (ASFV) can infect domestic pigs and wild boars, causing an acute, febrile, highly lethal hemorrhagic infectious disease, namely African swine fever (ASF).

[0003] Currently, detecting ASFV pathogenic genes or their antigens in pigs is an important means to determine whether the pig herd is infected, and serological detection methods can be used for ASF monitoring, screening and auxiliary diagnosis in pig herds.

[0004] In view of this, it is necessary to provide an antigen, a detection kit and an application of African swine fever virus. Summary of the invention

[0005] The main purpose of the present invention is to provide an African swine fever virus antigen, a detection kit and an application to solve the above-mentioned technical problems of African swine fever virus detection.

[0006] To achieve the above objectives, the present invention provides an African swine fever virus antigen, wherein the antigen is the African swine fever virus C-type lectin protein, and the amino acid sequence of the African swine fever virus C-type lectin protein is shown in SEQ ID NO.1.

[0007] According to an embodiment of the present application, the nucleotide sequence encoding the antigen is shown as SEQ ID NO.2.

[0008] The present invention provides an application of the above-mentioned African swine fever virus antigen in the preparation of a detection kit for African swine fever virus.

[0009] The present application also provides a detection kit for African swine fever virus, which includes an ELISA plate, a blocking solution, an enzyme-labeled secondary antibody, a color developing solution, a stop solution and an African swine fever virus antigen, wherein the African swine fever virus antigen is coated in the ELISA plate or is separated from the ELISA plate, and the antigen is the African swine fever virus C-type lectin protein, and the amino acid sequence of the African swine fever virus C-type lectin protein is shown in SEQ ID NO.1.

[0010] According to an embodiment of the present application, the antigen of the African swine fever virus is coated on an ELISA plate, and the coating concentration of the antigen of the African swine fever virus is 4 ng / μL.

[0011] According to an embodiment of the present application, the coating fluid used for the African swine fever virus antigen is CBS coating fluid.

[0012] According to an embodiment of the present application, the blocking solution is 1% BSA blocking solution.

[0013] According to an embodiment of the present application, the dilution ratio of the enzyme-labeled secondary antibody is 1:5000 to 1:8000.

[0014] The beneficial effects of the present invention include at least:

[0015] Among the above African swine fever virus antigens, since the protective immunity of African swine fever virus (ASFV) may be specific to the hemagglutination inhibition (HAI) serotype, and among ASFV proteins, C-type lectin is an essential protein that mediates the serological specificity of HAI. Therefore, C-type lectin is used as the target protein for ASFV serological diagnosis, and the antigenic characteristics of C-type lectin protein are used to establish an antibody ELISA detection method with application value, which will help the development of new ASFV antibody ELISA detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0017] Figure 1 This is a similarity analysis diagram of the nucleotide sequence of the domestic prevalent strain C-type lectin of Example 1 of the present invention;

[0018] Figure 2 This is a comparison diagram of the amino acid sequences of the domestically prevalent C-type lectin strains of Example 1 of the present invention;

[0019] Figure 3 This is the transmembrane structure analysis of the C-type lectin protein of the ASFV Chinese epidemic strain in Example 1 of the present invention;

[0020] Figure 4 This is a phylogenetic analysis diagram of the ASFV epidemic strains at home and abroad based on CD2v and C-type lectin in Example 1 of the present invention;

[0021] Figure 5 This is a graph of the detection rate of ASFV serum samples in Example 2 of the present invention;

[0022] Figure 6 This is the SDS-PAGE identification diagram of the C-type lectin protein in Example 1 of the present invention.

[0023] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with the implementation methods and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Furthermore, the technical solutions between the various embodiments of the present invention may be combined with each other, but this must be based on the fact that they can be implemented by ordinary technicians in the field. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0026] The present invention provides an African swine fever virus antigen, which is an African swine fever virus C-type lectin protein. The amino acid sequence of the African swine fever virus C-type lectin protein is shown in SEQ ID NO.1.

[0027] Serological diagnosis has become the most commonly used diagnostic technology due to its simple operation, relatively low cost and simple dedicated equipment. Antibody-based monitoring is very important for detecting pig infection, monitoring ASF epidemic areas and the invasion of low-virulence AS FV isolates. ELISA is the preferred serological detection method recommended by OIE. ASFV structural proteins with good antigenicity are often used as coating antigens to establish indirect ELISA detection methods. The cost of ELISA detection is lower than PCR, and it is suitable for large-scale screening of samples in a short period of time, and is assisted by other virological and serological tests. In the absence of vaccines, antibodies are clear indicators of ASFV infection and can be monitored for a long time. Serological determination also provides scientific data for vaccine evaluation. Serological testing mainly refers to antibody detection. Commonly used methods include ELISA, immunoblotting test (IB), indirect immunofluorescent assay (IFA), and indirect immunoperoxidase test (IPT). In the ELISA detection method, the basis is to determine the appropriate antigen.

[0028] C-type lectins (CTLs) refer to a class of proteins that require calcium ions (Ca2+) to participate in the selective binding of carbohydrates. The C-type lectin domain is usually composed of 110-130aa residues and has a conserved sequence. The entire fold consists of two α helices and 6 or 7 β strands, forming two antiparallel β folds, which recognize a variety of carbohydrate and non-carbohydrate ligands, such as lipids and proteins, through their lectin or lectin-like domains.

[0029] African swine fever virus C-type lectin (ASFV C-type lectin) has its own characteristics. The following C-type lectins all refer to ASFV C-type lectin. The African swine fever virus C-type lectin protein is encoded by the African swine fever virus EP153R gene. The EP153R ORF is located in the EcoRI E9 genomic fragment of the ASFV BA71V strain. It is a membrane protein encoding 153aa, with 8 possible N-glycosylation sites, 3 phosphorylation sites, 2 myristoylation sites, and an arginine-glycine-aspartic acid (RGD) motif associated with cell attachment. The intracellular N-terminal domain of the C-type lectin protein is homologous to the N-terminal domain of the CD44 molecule associated with T cell activation. Moreover, the EP153R gene is transcribed in both the early and late stages of the infection cycle, and has two different transcription start sites, one in the late transcription stage near the translation start codon, and the other in the early transcription stage near the translation start codon, a total of 164 nucleotides away from the translation start codon.

[0030] The amino acid sequence of SEQ ID NO.1 is:

[0031] MGEINKPICYQNDDKIFYCPKDWVGYNNVCYYFGNEEKNYNNASNYCKQLNSTLTNNNTILVNLTKTLNLTKTYNHESNYWVNYSLIKNESVLLRDSGYYKKQKHVSLLYICSKHHHHHH.

[0032] Among the above African swine fever virus antigens, since the protective immunity of African swine fever virus (ASFV) may be specific to the hemagglutination inhibition (HAI) serotype, and among ASFV proteins, C-type lectin is an essential protein that mediates the serological specificity of HAI. Therefore, C-type lectin is used as the target protein for ASFV serological diagnosis, and the antigenic characteristics of C-type lectin protein are used to establish an antibody ELISA detection method with application value, which will help the development of new ASFV antibody ELISA detection methods.

[0033] C-type lectin was selected as the target protein for ASFV serological diagnosis. The sequence conservation and important B cell antigen epitopes of C-type lectin protein were analyzed. The role of C-type lectin protein in serum grouping was studied through systematic evolutionary analysis. The antigenic characteristics of C-type lectin protein were revealed, and an antibody ELISA detection method with application value was established, which will help the development of new ASFV antibody ELISA detection methods.

[0034] In some embodiments, the nucleotide sequence encoding the antigen is shown as SEQ ID NO.2.

[0035] The nucleotide sequence of SEQ ID NO.2 is:

[0036] ATGGGCGAGATCAACAAGCCCATCTGCTACCAGAACGACGACAAGATCTTCTACTGCCCCAAGGACTGGGTGGGCTACAACAACGTGTGCTACTACTTCGGCAACGAGGAGAAGAACTACAACAACGCCAGCAACTACTGCAAGCAGCTGAACAGCACCCTGACCAACAACAACACCATCCT GGTGAACCTGACCAAGACCCTGAACCTGACCAAGACCTACAACCACGAGAGCAACTACTGGGTGAACTACAGCCTGATCAAGAACGAGAGCGTGCTGCTGAGGGACAGCGGCTACTACAAGAAGCAGAAGCACGTGAGCCTGCTGTACATCTGCAGCAAGCACCACCACCACCACCACTAG.

[0037] The present invention provides an application of the above-mentioned African swine fever virus antigen in the preparation of a detection kit for African swine fever virus.

[0038] The antigen of the African swine fever virus can be used in various detection kits, such as ELISA kits, illustratively, kits based on direct ELISA, kits based on indirect ELISA, kits based on sandwich ELISA, kits based on competitive ELISA, and kits based on competitive inhibition ELISA.

[0039] The present application also provides a detection kit for African swine fever virus, which includes an ELISA plate, a blocking solution, an enzyme-labeled secondary antibody, a color developing solution, a stop solution and an African swine fever virus antigen. The African swine fever virus antigen is coated in the ELISA plate or separated from the ELISA plate. The antigen is the African swine fever virus C-type lectin protein, and the amino acid sequence of the African swine fever virus C-type lectin protein is shown in SEQ ID NO.1.

[0040] The detection kit is a kit based on indirect ELISA. The antigen of African swine fever virus may be already coated in the wells of the ELISA plate. Alternatively, the antigen of African swine fever virus is in a state of being separately packaged, and when in use, such as when preparing to test the sample, the antigen of African swine fever virus is coated in the wells of the ELISA plate.

[0041] In some embodiments, the African swine fever virus antigen is coated on an ELISA plate, and the coating concentration of the African swine fever virus antigen is 4 ng / μL.

[0042] In some embodiments, the coating fluid used for the African swine fever virus antigen is CBS coating fluid.

[0043] In some embodiments, the blocking solution is 1% BSA blocking solution.

[0044] In some embodiments, the dilution ratio of the enzyme-labeled secondary antibody is 1:5000 to 1:8000.

[0045] The technical solution of the present application is described below in conjunction with specific embodiments.

[0046] Example 1

[0047] This example compares and analyzes the C-type lectin genome sequences of some domestic prevalent strains since the outbreak of ASF, and focuses on discussing the application potential of C-type lectin protein as a serological diagnostic target, as well as the role of C-type lectin protein in ASFV serogrouping and antigenic variation through linear B cell antigen epitope prediction analysis and phylogenetic analysis.

[0048] 1.1 Methods

[0049] 1.1.1 Amino acid and nucleotide sequence alignment

[0050] The EP153R gene sequences of ASFV epidemic strains in China were downloaded from NCBI (https: / / www.ncbi.nlm.nih.gov). The GenBank accession numbers of ASFV epidemic strains in China are: Anhui XCG Q (MK128995), ASFV-wbBS01 (MK645909), Pig / HLJ (MK333180), DB / LN (MK333181), GZ201801 (MT496893), Wuhan2019-1 (MN393476), Wuhan 2019-2 (MN393477), CAS19-01 (MN172368), InnerMongolia-AES01 (MK940252), and CADC_HN09 (MZ614662). Clustal W software was used to analyze the C-type sequences of the 10 ASFV strains collected in China. The lectin sequences were analyzed for nucleotide similarity, and Multalin (http: / / multalin.toulouse.inra.fr / multalin) was used for online analysis. The amino acid sequences of C-type lectin of 10 prevalent ASFV strains in China were compared to analyze the conservation of the sequences.

[0051] 1.1.2 Analysis of antigenic epitope regions of C-type lectin of ASFV epidemic strains in China

[0052] The Bepipred Linear Epitope Prediction 2.0 online tool in the IEDB (Immune Epitope Database, http: / / www.iedb.org / ) database was used to predict the B cell linear epitopes of ASFV C-type lectin. The threshold was set to 0.55, and the antigenicity of peptides greater than 7 amino acid residues was evaluated using the VaxiJenv2.0 (http: / / www.ddg-pharmfac.net / vaxijen / VaxiJen / Va xiJen.html) online tool.

[0053] 1.1.3 Evolutionary analysis of the C-type lectin protein sequence encoded by the EP153R gene

[0054] The amino acid sequences of C-type lectin and CD2v of ASFV strains at home and abroad were downloaded from the NCBI (https: / / www.ncbi.nlm.nih.gov) database. The amino acid sequences of C-type lectin and CD2v extracellular regions were analyzed using the TMHMM-2.0 (https: / / services.healthtech.dtu.dk / service.) online transmembrane region prediction tool. The amino acid sequences of ASFV CD2v and C-type lectin extracellular regions of 23 known serogroups were marked using MEGA 11.0 software, and compared with 56 sequences of unknown serogroups downloaded from the NCBI database through the Maximum Composite likelihood model (Boo tstrap, 1000 repetitions; Cut-off value was 50%), and the neighbor-joining (NJ) method was used to construct a phylogenetic tree to summarize the variation among ASFV strains.

[0055] 1.2 Results

[0056] 1.2.1 Collection of ASFV EP153R gene sequence information in China

[0057] The EP153R gene sequences of ASFV epidemic strains in China from 2018 to 2022 were downloaded from the NCBI database. The gene sequence lengths of the two Wuhan strains were 483 bp, and the gene sequence lengths of the remaining strains were all 477 bp (Table 1-1).

[0058] Table 1-1 Detailed information on the ASFV EP153R gene sequence in China

[0059]

[0060]

[0061] 1.2.2 Analysis of conservation of ASFV EP153R gene

[0062] After analyzing the representative ASFV C-type lectin nucleotide sequences in China, it was found that the C-type lectin nucleotide sequences of 10 Chinese ASFV strains had a similarity of 95%-100% ( Figure 1 ).

[0063] By comparing the amino acid sequences of C-type lectins of 10 ASFVs prevalent in China, it was found that the amino acid sequence similarity was 93%-100%. The third amino acid residue of the amino acid sequence of wbBS01 (MK645909) C-type lectin mutated from S to F, and the amino acid sequence of Wuhan 2019-1 (MN393476) and Wuhan 2019-2 (MN393477) C-type lectin had a QK insertion at 149-150aa. Figure 2 ). The blue and black parts of the epitope represent.

[0064] 1.2.3 Prediction of antigenic epitopes of C-type lectin of ASFV epidemic strains in China

[0065] A total of 7 potential linear B cell epitopes of ASFV C-type lectin were predicted using BepiPred 2.0 software. According to the antigenicity score of VaxiJen v2.0, 5 linear B cell epitopes with high antigenicity (Score>0.55) were screened out (Table 1-2). Among these 5 epitopes, epitopes B (KKYIGLINKKEGLKKK) and D (NEEKNYNN) were highly conserved and scored, 100% (0.7968) and 100% (0.6749), respectively. It is worth noting that the highly conserved epitope D is located in the extracellular region of the serum grouping of the C-type lectin protein.

[0066] Table 1-2 Prediction of linear B cell epitopes of ASFV C-type lectin using BepiPred 2.0

[0067]

[0068]

[0069] 1.2.4 Construction and analysis of the ASFV C-type lectin phylogenetic tree

[0070] The protein transmembrane region prediction tool based on the TMHMM method was used to analyze the transmembrane region of the C-type lectin protein. The results showed that 47-160aa was the extracellular region of the C-type lectin protein. Figure 3 ).

[0071] In order to study the association of ASFV CD2v and C-type lectin proteins with ASFV serogroup specificity and their potential role in serogrouping, 79 C-type lectin extracellular region amino acid sequences (10 domestic sequences and 69 foreign sequences) and CD2v extracellular region amino acid sequences of the corresponding strains (sequence numbers) were downloaded from the GenBank database for analysis, including 23 amino acid sequences (C-type lectin and CD2v extracellular region amino acid sequences) with determined serogroups. The phylogenetic tree was constructed using the neighbor-joining method using MEGA 11.0 software. Phylogenetic tree analysis showed that strains belonging to the same serogroup were located in the same branch and had the closest affinity ( Figure 4 ). A phylogenetic tree was constructed based on the extracellular region of C-type lectin. The amino acid homology within the same branch was 88%-100%, and strains of different serogroups were located in different branches. Based on the construction of a phylogenetic tree based on the extracellular regions of CD2v and C-type lectin, the Chinese ASFV epidemic strains Anhui XCGQ (MK128995), ASFV-wbBS01 (MK645909), Pig / HLJ (MK333180), DB / LN (MK333181), GZ201801 (MT496893), Wuhan 2019-1 (MN393476), Wuhan 2019-2 (MN393477), CAS19-01 (MN172368), InnerMongolia-AES01 (MK940252), and CADC_HN09 (MZ614662) are all located in the same branch with the SG8 of the determined serogroup, and the amino acid similarity within the branch is 99%.

[0072] In this example, 79 collected C-type lectin extracellular region amino acid sequences (10 domestic sequences and 69 foreign sequences) and CD2v extracellular region amino acid sequences with corresponding sequence numbers were analyzed, including 23 amino acid sequences of known serogroups (C-type lectin extracellular region amino acid sequences and CD2v amino acid sequences), and a phylogenetic tree was constructed. The results of phylogenetic analysis showed that among the ASFV prevalent strains in China, the phylogenetic groupings based on CD2v and C-type lectin were related to the serogroups, and the virus sequences from similar serogroups were often exactly the same or almost exactly the same, and belonged to the same branch in the evolutionary tree relative to isolates from different serogroups, with the closest affinity.

[0073] The above results indicate that ASFV C-type lectin protein can be used as a potential target for serological diagnosis. The use of standard P72 genotyping lacks serum specificity. The identification of ASFV isolates of non-specific serogroups will contribute to the study of ASFV strain diversity and antigenic variation, and provide a reference for the design, development and effective use of vaccines. However, the current understanding of C-type lectin protein is still very limited, and there is still a need to further understand its biological characteristics and its reliability as a serogrouping.

[0074] Example 2

[0075] In this example, the extracellular region of C-type lectin protein was used as the coating antigen to establish an IgG antibody ELISA detection method, and the target protein coating concentration, serum dilution multiple, enzyme-labeled antibody dilution multiple, serum antibody reaction time, substrate reaction time and other conditions were determined respectively. This method has good specificity and sensitivity, and the detection experiment time is short. The establishment of an indirect ELISA IgG antibody detection method based on C-type lectin protein expressed in a mammalian eukaryotic system has certain application reference value for the detection of African swine fever.

[0076] 2.1 Experimental Materials

[0077] 2.1.1 Configuration of main reagents

[0078] Proclin 300 liquid biological preservative: purchased from Beijing Solarbio Company.

[0079] ELISA test kit: ID-Vet, France.

[0080] 2.1.2 Main instruments and equipment

[0081] Table 2-1 Main instruments

[0082]

[0083] 2.2 Methods

[0084] 2.2.1 Operation steps of antibody ELISA detection method

[0085] The specific steps of the antibody ELISA method are as follows:

[0086] (1) Coating: Dilute the C-type lectin protein with filtered PBS to the working concentration. After dilution, place the plate on a shaker to mix the protein dilution evenly. Then mix the diluted target protein with a certain amount of carbonate buffer and coat it on the ELISA plate. The coating volume is 100 μL per well. Wrap it with tin foil and place it flat in a sealing bag to isolate it from light and air. Place it in a refrigerator at 4°C overnight for 12 hours for coating. Use an ELISA automatic plate washer and wash it 5 times with pre-prepared PBST (0.05% Tween), with a volume of 300 μL PBST washing solution per well each time.

[0087] (2) Blocking: Use filtered PBS to prepare BSA blocking solution. Note that BSA is sometimes difficult to dissolve and needs to be prepared in advance and mixed thoroughly on a shaker. Immediately after washing, add 200 μL of blocking solution to each well of the ELISA plate. Be careful not to let the ELISA plate dry. Incubate at room temperature (24°C) for 3 h. Use an ELISA automatic plate washer to wash 5 times with PBST (0.05% Tween), 300 μL of washing solution per well each time.

[0088] (3) Primary antibody (serum, i.e., test substance): After the serum is diluted, vortex mixed in a centrifuge tube and centrifuged briefly. 100 μL volume per well of the ELISA plate, incubated at room temperature (24°C), and washed 5 times with PBST (0.05% Tween) using an ELISA automatic plate washer, 300 μL washing solution per well each time.

[0089] (4) Enzyme-labeled secondary antibody (HRP-anti-porcine IgG) (KPL, USA, Cat. No. 5220-0363): After dilution, the enzyme-labeled secondary antibody was placed on a shaker and mixed thoroughly. 100 μL volume was placed in each well of the ELISA plate and incubated at room temperature (24°C). Use an ELISA automatic plate washer to wash 5 times with PBST (0.05% Tween), 300 μL washing solution per well each time.

[0090] (5) Color development and termination reaction: Divide the TMB substrate into the required volume, place it at room temperature (24°C) in advance to restore the temperature, and wrap it with tin foil to protect it from light. The volume of each well of the ELISA plate is 50 μL. Color development is carried out at room temperature (24°C). The reaction is terminated by adding 50 μL of concentrated sulfuric acid stop solution to each well. After adding the stop solution, it is necessary to immediately shake it by hand to fully terminate the reaction. Use a spectrophotometer to shake for 5 seconds and then measure the OD450 value to determine the result.

[0091] The preparation method of C-type lectin protein is as follows:

[0092] 1. Plasmid construction

[0093] The C-type lectin sequence of ASFV serotype 8 Pig / HLJ / 18 (GenBank number: QBH90545) was downloaded from the NCBI database, and its extracellular domain sequence was obtained after screening. After codon optimization, the sequence will be synthesized by the company and subcloned into the pCDNA3 series eukaryotic expression vector (for obtaining C-type lectin protein).

[0094] 2. Plasmid extraction

[0095] Take out the plasmid stored in the refrigerator at -20℃, and mix 1μL of the plasmid with 30μL of competent E. coli DH5α. After standing on ice for 30 minutes, put it in a 42℃ water bath for 60s of heat shock, and then put the mixture back on ice for 5 minutes. Then add 400μL of LB medium without antibiotics and culture it on a shaker at 37℃, 200rpm for 40 minutes. Take 20μL of the mixture and evenly spread it on the LB medium containing antibiotics, and place it upside down in a constant temperature incubator at 37℃. After 12 hours, add 5mL of LB containing antibiotics to the test tube, select a single colony from the LB medium and put it into the test tube, culture it on a shaker at 37℃, 200rpm for 14 hours, and continue to expand the culture scale as needed. Finally, use a commercial plasmid extraction kit for plasmid extraction.

[0096] 3. Expression and purification of C-type lectin protein

[0097] 2×10^6 293F cells in good growth condition were inoculated into a cell culture flask containing fresh SMM 293-TII medium, and the plasmid of C-type lectin extracellular domain was transfected into the cells, and cultured in a constant temperature shaker at 5% CO2, 37°C, and 140rpm for 4-5 days.

[0098] The total protein in the supernatant of the cells was obtained (the supernatant was collected by centrifugation), and after ultrafiltration through a 10 kDa membrane package, the total protein in the supernatant was mixed with Ni-NTA filler and incubated for 1 hour.

[0099] The total protein in the supernatant was purified by nickel column, and the following steps were performed in sequence:

[0100] Collect the flow-through liquid F;

[0101] Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) with a concentration of 20 mM imidazole was used to remove impurities and obtain a low-concentration imidazole washing solution W1;

[0102] Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 30 mM imidazole, pH 8.0) with a concentration of 30 mM imidazole was used to remove foreign proteins to obtain a high-concentration imidazole washing solution W2;

[0103] The target protein was eluted using Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole, pH 8.0) and collected in order to obtain eluates E1-E5 containing the target protein.

[0104] The collected liquid was subjected to SDS-PAGE electrophoresis.

[0105] Experimental results:

[0106] The protein solution before and after nickel column purification was identified by SDS-PAGE, referring to Figure 6 As shown: In the protein solution before purification, there are many impurity proteins, which makes the bands unclear.

[0107] After nickel column purification, the target protein (C-type lectin protein) was effectively enriched and purified, and its bands showed obvious enhancement and diffusion. In terms of molecular weight, it was observed that the target protein was glycosylated, so its bands were mainly distributed in the range of 40-55kDa and appeared as diffuse bands.

[0108] The following is Figure 6 The specific meaning of each symbol in:

[0109] M: protein standard 26616; S: ​​total protein in supernatant; F: flow-through; W1-W2: washing solutions with different imidazole concentrations; E1-E5 (E1, E2, E3, E4, E5): eluents.

[0110] 2.2.2 Determination of the optimal coating concentration of C-type lectin protein

[0111] The C-type lectin protein whose concentration was determined by the BCA method was diluted with PBS buffer to 1, 2, 4, and 6 ng / μL, respectively, with a volume of 100 μL per well, and coated on the ELISA plate longitudinally in sequence, wrapped in tin foil and placed horizontally in a sealing bag, and placed in a 4°C refrigerator for 12 hours for coating. When other experimental conditions remained unchanged, the OD450 value was measured, and the optimal coating concentration of C-type lectin protein was determined based on the ratio P / N of the average value of the duplicate wells of the antibody-positive serum and the antibody-negative serum. The ELISA plate was coated with the optimal C-type lectin protein concentration, wrapped in tin foil and sealed in a sealing bag and stored in a 4°C refrigerator for subsequent experiments.

[0112] 2.2.3 Determination of C-type lectin protein coating solution

[0113] PBS and CBS were used as coating solutions for C-type lectin protein, respectively. The OD450 value was measured when other experimental conditions remained unchanged. The optimal coating solution for C-type lectin protein was determined based on the ratio P / N of the average values ​​of the duplicate wells of antibody-positive serum and antibody-negative serum. The C-type lectin protein and the optimal coating solution were mixed and coated on the ELISA plate, which was then wrapped in tin foil and sealed in a sealing bag and stored in a 4°C refrigerator for subsequent experiments.

[0114] 2.2.4 Determination of blocking solution concentration

[0115] After diluting the BSA blocking solution at 0.5%, 1%, 1.5%, and 2%, add 200 μL per well to the ELISA plate, set up duplicate wells, wrap with tin foil and incubate at room temperature (24°C) to avoid light. Keep other experimental conditions unchanged, measure the OD450 value, and determine the optimal concentration of the blocking solution based on the ratio P / N of the average value of the duplicate wells of antibody-positive serum and antibody-negative serum.

[0116] 2.2.5 Determination of the optimal dilution factor of serum samples

[0117] Dilute the serum by 25, 50, and 100 times, vortex mix in a centrifuge tube, and centrifuge for a moment. Add 100 μL of volume to each well of the ELISA plate, set up duplicate wells, wrap with tin foil and incubate at room temperature (24°C) to avoid light. When other experimental conditions remain unchanged, measure the OD450 value, and determine the optimal dilution of serum antibodies based on the ratio P / N of the average value of the duplicate wells of antibody-positive serum and antibody-negative serum. 2.2.6 Determination of the optimal serum reaction time

[0118] The diluted serum was added to the ELISA plate and reacted at different reaction times: 15min, 30min, 45min, and 60min, and duplicate wells were set. They were wrapped in tin foil to avoid light and placed in a constant temperature incubator (24°C) one after another to ensure that they were taken out at the same time during washing, and other operations were performed together to reduce errors. When other experimental conditions remained unchanged, the OD450 value was measured, and the optimal reaction time of the serum was determined based on the ratio P / N of the average value of the duplicate wells of the antibody-positive serum and the antibody-negative serum.

[0119] 2.2.7 Determination of the optimal dilution factor of enzyme-labeled secondary antibody

[0120] After the enzyme-labeled secondary antibody was diluted at 1:5000, 1:6000, 1:7000, and 1:8000, 1:7000 was the best. It was placed on a shaker and mixed thoroughly. 100 μL was added to each well of the ELISA plate, and duplicate wells were set up. The plate was wrapped in tin foil and incubated at room temperature (24°C) to avoid light. When other experimental conditions remained unchanged, the OD450 value was measured, and the optimal dilution multiple of the secondary antibody was determined based on the ratio P / N of the average value of the duplicate wells of the antibody-positive serum and the antibody-negative serum.

[0121] 2.2.8 Determination of the optimal reaction time of secondary antibodies

[0122] The diluted secondary antibody was added to the ELISA plate, and the reaction was carried out at different reaction times: 15min, 30min, 45min, and 60min, and duplicate wells were set, wrapped in tin foil to avoid light and placed in a constant temperature incubator (24°C) one after another, ensuring that they were taken out at the same time during washing, and other operations were performed together to reduce errors. When other experimental conditions remained unchanged, the OD450 value was measured, and the optimal reaction time of the secondary antibody was determined based on the ratio P / N of the average value of the duplicate wells of the antibody-positive serum and the antibody-negative serum.

[0123] 2.2.9 Determination of TMB reaction time

[0124] TMB was divided into 15mL centrifuge tubes and wrapped with tin foil to avoid light, and placed in a constant temperature box to restore stability (24°C). According to the reaction time recommended in the instructions, the reaction time gradient was set: 5min, 10min, 15min, 50μL per well, and added to the ELISA enzyme labeling plate in a dark place. After ensuring the completion of the reaction, the plate was taken out and concentrated sulfuric acid was used to terminate the reaction to reduce errors. Other experimental conditions were kept unchanged, and the optimal action time of TMB was determined by calculating the ratio P / N of the average value of the duplicate wells of antibody-positive serum and antibody-negative serum.

[0125] 2.2.10 Determination of positive and negative critical values

[0126] The preliminarily established ASFV antibody ELISA method was used to detect 26 negative pig sera collected from a pig farm in Hunan during 2017. The OD450 value was determined, and the average value (AV) and standard deviation (SD) of the 26 antibody-negative sera were calculated. The positive and negative critical values ​​were calculated according to the statistical formula: average value (AV) + 3×standard deviation (SD).

[0127] 2.2.11 Specificity test

[0128] Three positive sera collected from a pig farm in Hunan in 2017 and tested by the French ID-Vet kit for classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and porcine epidemic diarrhea virus (PEDV) were selected from the laboratory, for a total of 15 sera. The established antibody ELISA method was used to set up the secondary wells and calculate the average value. The specificity of the method was determined by comparing it with the critical value.

[0129] 2.2.12 Repeatability test

[0130] 2.2.12.1 Variation within a batch

[0131] Ten pig serum samples (5 positive and 5 negative) provided by Hunan Provincial Animal Disease Prevention and Control Center were selected. The same ELISA plate was used in Hunan Provincial Animal Disease Prevention and Control Center. The established antibody ELISA method was used to perform 3 replicates for each serum. According to the OD450 value results, the average value (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) / average value (AV) × 100%.

[0132] 2.2.12.2 Batch Differences

[0133] Ten pig serum samples (5 antibody-positive and 5 antibody-negative) provided by Hunan Provincial Animal Disease Prevention and Control Center were selected and coated on ELISA plates in three batches every other day. The established antibody ELISA method was used to perform three replicates for each serum. According to the OD450 value results obtained in the experiment at Hunan Provincial Animal Disease Prevention and Control Center, the average value (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) / average value (AV) × 100%.

[0134] 2.2.13 Preliminary application of ELISA in clinical sample testing and compliance rate

[0135] 55 pig sera provided by Hunan Provincial Animal Disease Prevention and Control Center were selected, and a comparison test between the French ID-Vet kit and the established antibody ELISA kit was carried out at the Hunan Provincial Animal Disease Prevention and Control Center. The consistency rate between the established antibody ELISA method and the results of the French ID-Vet kit was calculated, including the positive consistency rate and the negative consistency rate.

[0136] 2.3 Results

[0137] 2.3.1 Determination of protein coating concentration

[0138] By changing the protein coating concentration and keeping other conditions unchanged, the results obtained in the microplate reader are shown in (Table 2-2), and the P / N value is calculated. When the data are compared, the P / N obtained is higher when the concentration is 4ng / μL.

[0139] Table 2-2 Determination of the optimal protein coating concentration

[0140]

[0141] 2.3.2 Determination of C-type lectin protein coating solution

[0142] Change the C-type lectin protein coating solution, keep other conditions unchanged, and get the results in the microplate reader as shown in (Table 2-3), and calculate the P / N value. After comprehensive comparison of each group of data, use CBS coating solution.

[0143] Table 2-3 Determination of protein coating solution

[0144]

[0145]

[0146] 2.3.3 Determination of blocking solution concentration

[0147] By changing the concentration of the blocking solution and keeping other conditions unchanged, the results obtained in the microplate reader are shown in (Table 2-4), and the P / N value is calculated. After a comprehensive comparison of the data of each group, the effect of using a blocking solution concentration of 1% by volume of BSA is better.

[0148] Table 2-4 Determination of BSA blocking solution concentration

[0149]

[0150] 2.3.4 Determination of the optimal dilution of serum samples

[0151] By changing the serum dilution multiple and keeping other conditions unchanged, the results obtained in the microplate reader are as shown in (Table 2-5), and the P / N value is calculated. Comparing the data, when the weak positive serum is diluted with 100-fold serum antibody, the P / N obtained is higher. Considering comprehensively, the 100-fold serum antibody dilution multiple is the best condition.

[0152] Table 2-5 Determination of the optimal dilution of serum antibodies

[0153]

[0154] 2.3.5 Determination of the optimal serum reaction time

[0155] By changing the serum reaction time and keeping other conditions unchanged, the results obtained in the microplate reader are shown in (Table 2-6), and the P / N value is calculated. When the data are compared, the P / N is the highest when the primary antibody action time is 15 minutes.

[0156] Table 2-6 Determination of the optimal reaction time of serum antibodies

[0157]

[0158] 2.3.6 Determination of the optimal dilution factor of enzyme-labeled secondary antibody

[0159] Changing the dilution multiple of the enzyme-labeled secondary antibody and keeping other conditions unchanged, the results obtained in the microplate reader are shown in (Table 2-7), and the P / N value is calculated. Comparing the data, the P / N obtained when the secondary antibody is diluted with a dilution multiple of 1 to 6000 is higher.

[0160] Table 2-7 Determination of the optimal dilution multiple of enzyme-labeled antibodies

[0161]

[0162] 2.3.7 Determination of the optimal reaction time of enzyme-labeled secondary antibody

[0163] By changing the reaction time of enzyme-labeled secondary antibody and keeping other conditions unchanged, the results obtained in the microplate reader are shown in (Table 2-8), and the P / N value is calculated. When the data are compared, the highest P / N is obtained when the secondary antibody reaction time is 30 minutes.

[0164] Table 2-8 Determination of the optimal reaction time of the secondary antibody

[0165]

[0166]

[0167] 2.3.8 Determination of the optimal duration of TMB action

[0168] By changing the TMB action time and keeping other conditions unchanged, the results obtained in the microplate reader are shown in (Table 2-9), and the P / N value is calculated. When the data are compared, the P / N obtained is higher when the TMB action time is 5 minutes.

[0169] Table 2-9 Determination of the optimal color development time

[0170]

[0171] 2.3.9 Determination of positive and negative critical values

[0172] 26 ASFV antibody-negative pig serum samples collected from a pig farm in Hunan in 2017 were selected and tested using the established antibody ELISA method. The results are shown in (Table 2-10). The average OD450 value (AV) of the 26 ASFV antibody-negative sera is 0.1287, and the standard deviation (SD) is 0.0565. The critical value is calculated as 0.2982 according to the formula: average value (AV) + standard deviation (SD) X3. If the OD450 value of the test sample is greater than 0.2982, it is judged as positive, and if it is less than 0.2982, it is judged as negative.

[0173] Table 2-10 Determination of positive and negative critical values

[0174]

[0175]

[0176] 2.3.10 Specificity test

[0177] There were 3 samples of positive sera for classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and porcine epidemic diarrhea virus (PEDV), totaling 15 samples, which were detected using the established antibody ELISA method and negative and positive controls were set up. The results are shown in (Table 2-11). The OD450 mean values ​​of the positive sera for classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and porcine epidemic diarrhea virus (PEDV) were all less than the critical value of 0.2982 and were judged to be negative, indicating that the established ELISA method has good specificity.

[0178] Table 2-11 Indirect ELISA specificity test results

[0179]

[0180] 2.3.11 Repeatability test

[0181] 2.3.11.1 Repeated testing within a batch

[0182] Ten pig serum samples (5 positive and 5 negative) were used, and each serum was repeated 3 times. The results obtained in the ELISA instrument are shown in (Table 2-12). The average value (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) / average value (AV) × 100%. The results showed that the intra-batch coefficient of variation was 0.8567%-7.1969%, which was less than 10%, indicating that the established antibody ELISA method had good intra-batch repeatability and stability.

[0183] Table 2-12 Indirect ELISA intra-batch repeat test

[0184]

[0185]

[0186] 2.3.11.2 Repeated testing between batches

[0187] Ten pig serum samples (5 positive and 5 negative) and three batches of ELISA plates were selected. Three replicates were made for each serum. The results obtained in the microplate reader are shown in (Table 2-13). The average value (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) / average value (AV) × 100%. The results showed that the inter-batch coefficient of variation was between 1.6743% and 6.2802%, which was less than 10%, indicating that the established antibody ELISA method had good inter-batch repeatability and stability.

[0188] Table 2-13 Indirect ELISA batch repetition test

[0189]

[0190] 2.3.12 Preliminary application of ELISA in clinical sample testing

[0191] The comprehensive coincidence rate between the C-type lectin indirect ELISA method established in this example and the French ID-Vet kit was 98.18% (54 / 55), of which the positive detection rate was 97.92% (47 / 48) and the negative coincidence rate was 100% (7 / 7) (see Figure 5 )

[0192] 2.4 Discussion

[0193] This example uses the C-type lectin protein successfully expressed in the laboratory eukaryotic mammal system to establish an IgG antibody ELISA detection method. First, the ELISA matrix titration test determined that the optimal reaction conditions were when the antigen coating concentration was 4ng / μL and the ASFV positive dilution was 1:100. The indirect ELISA method was established under this test condition, and the antibody positive critical value was determined to be 0.2982 using 26 ASFV negative sera. The specificity results showed that the ELISA method only reacted specifically with ASFV positive sera, indicating that the method had good specificity; the results of intra-batch and inter-batch repeatability tests showed that the coefficient of variation was less than 10%, with good repeatability and stability; the method was used to test the compliance rate of 55 ASFV clinical serum samples and ASFV commercial ELISA detection kits. The results showed that the method detected ASFV negative and positive sera and the commercial kit (ID-Vet-ELISA) with a compliance rate of 98.18%, indicating that the method can be used for ASFV antibody detection.

[0194] The study reported that six ASFV antigens (p12, p32, p54, pp62, C-type lectin and CD2v) were used to detect the antibody reactivity of serum samples of ASFV-infected pigs. It was found that all surviving pigs infected with ASFV had p32, p54 and pp62 antibodies, 91.3% of the surviving pigs had p12 antibodies, and only a small number of surviving pigs infected with ASF produced C-type lectin antibodies (34.8%) and CD2v antibodies (26.1%). Antibodies against p12, p32, p54 and pp62 were detected in fattening pigs and sows, while antibodies against C-type lectin and CD2v were mainly detected in sows, but not in fattening pigs. The ELISA test results may be related to many factors such as the C-type lectin protein sequence used (such as the extracellular domain, etc.), the expression system, the ASFV antibody sample, etc. The ELISA detection method and application prospects of C-type lectin as an antigen still need further in-depth research.

[0195] The above technical solutions of the present invention are only preferred embodiments of the present invention, and the patent scope of the present invention is not limited thereto. All equivalent structural changes made by using the contents of the present invention specification and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. An antigen of African swine fever virus, characterized in that: The antigen is the African swine fever virus C-type lectin protein, and the amino acid sequence of the African swine fever virus C-type lectin protein is shown in SEQ ID NO.

1.

2. The African swine fever virus antigen according to claim 1, characterized in that The nucleotide sequence encoding the antigen is shown in SEQ ID NO.

2.

3. Use of an African swine fever virus antigen as claimed in claim 1 in the preparation of a detection kit for African swine fever virus.

4. A detection kit for African swine fever virus, characterized in that: The detection kit includes an ELISA plate, a blocking solution, an enzyme-labeled secondary antibody, a color developing solution, a stop solution and an African swine fever virus antigen. The African swine fever virus antigen is coated in the ELISA plate or is separated from the ELISA plate. The antigen is the African swine fever virus C-type lectin protein, and the amino acid sequence of the African swine fever virus C-type lectin protein is shown in SEQ ID NO.

1.

5. The detection kit for African swine fever virus according to claim 4, characterized in that: The African swine fever virus antigen is coated on an ELISA plate, and the coating concentration of the African swine fever virus antigen is 4 ng / μL.

6. The detection kit for African swine fever virus according to claim 5, characterized in that: The coating liquid used for the African swine fever virus antigen is CBS coating liquid.

7. The detection kit for African swine fever virus according to claim 4, characterized in that: The blocking solution is 1% BSA blocking solution.

8. The African swine fever virus detection kit according to any one of claims 4 to 7, characterized in that: The dilution ratio of the enzyme-labeled secondary antibody is 1:5000 to 1:8000.

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