An african swine fever virus antigen epitope polypeptide and an elisa antibody detection kit

Abstract

The application discloses an African swine fever virus antigen epitope polypeptide, which is composed of an African swine fever virus p72 protein antigen epitope polypeptide and a pp62 protein antigen epitope polypeptide, the p72 protein antigen epitope polypeptide sequence is NKSYGKPDPEPTLSQI, position 37-52 aa, and the pp62 protein antigen epitope polypeptide sequence is DFLYTAIPEEKVGGNE, position 450-465 aa. The application also provides an African swine fever virus synthetic peptide ELISA antibody detection kit, which comprises an enzyme-linked reaction plate coated with the antigen epitope polypeptide. The antigen epitope of the African swine fever virus p72 and pp62 proteins is accurately analyzed by using a bioinformatics method, and a peptide segment suitable for ELISA detection is screened out, and the peptide segment has the advantages of high sensitivity and strong specificity. The African swine fever virus p72 and pp62 protein antigen epitope polypeptide is used as a coating antigen to prepare the kit, and the kit can effectively detect whether an animal to be detected is infected with the African swine fever virus, the kit has the advantages of strong specificity, high sensitivity and good repeatability, and is favorable for the establishment of an African swine fever virus prevention and control system in China.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology. Background Technology

[0002] African swine fever (ASF) is an acute, febrile, and highly contagious disease of pigs (including domestic and wild pigs) caused by the African swine fever virus (ASFV). Clinically, it is characterized by high fever, reticuloendothelial hemorrhage, and high mortality, causing severe losses to the global pig industry. The World Organisation for Animal Health (WOAH) lists it as a notifiable animal disease, and my country classifies it as a Category A animal infectious disease. Due to the complex infection mechanism of ASFV, there is currently no effective ASF vaccine for prevention and treatment. Therefore, establishing accurate diagnostic and detection technologies is crucial for ASF control. However, the nonspecific clinical symptoms caused by ASF are difficult to distinguish from common swine diseases such as classical swine fever. Diagnostic methods recommended by the OIE, such as virus isolation, antigen identification (erythrocyte adsorption assay, fluorescent antibody assay, and PCR), and serological tests (enzyme-linked immunosorbent assay, indirect fluorescent antibody assay, immunoperoxidase assay, and immunoblotting), all require skilled technicians to operate under laboratory conditions and with expensive equipment. This makes it impossible to make timely and accurate diagnoses of ASF in resource-limited and poorly equipped grassroots and remote areas, making it difficult to effectively control the epidemic.

[0003] ASFV is an enveloped double-stranded DNA virus belonging to the family African swine feverviridae and the genus African swine fevervirus. The virus particle has an average diameter of approximately 200 nm, and its genome is about 170-194 kb in length, encoding at least 125 viral proteins, including more than 34 structural proteins. Among the major structural proteins, the p72 protein, encoded by the B646L gene, is the main capsid protein of ASFV, participating in the adsorption and invasion process of ASFV. It possesses important antigenicity and immunogenicity and is often used as a diagnostic antigen for ASFV. The p72 protein is suitable for late-stage detection, and can be detected for the first time as early as 9 days after infection. The pp62 protein is a polymeric precursor of the p35 and p15 proteins, participating in the assembly of the viral core and forming in the early stages of viral infection. The pp62 antigen has high sensitivity and specificity, and its detection effect is superior to p30 and p54.

[0004] Currently, in China, ASFV is mainly detected qualitatively using quantitative real-time PCR. However, false negatives can occur when sampling blood from pigs infected with naturally attenuated strains or transient infections, making it difficult to effectively and promptly identify infected pigs. Therefore, there is an urgent clinical need for a rapid, accurate, easy-to-operate, and convenient on-site ASF antibody detection technology. Summary of the Invention

[0005] The present invention aims to provide an African swine fever virus antigenic epitope polypeptide, and another objective of the present invention is to provide an ELISA antibody detection kit using the antigenic epitope polypeptide as the coating antigen.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] An African swine fever virus antigenic epitope polypeptide is provided, which is composed of an African swine fever virus p72 protein antigenic epitope polypeptide and a pp62 protein antigenic epitope polypeptide. The sequence of the p72 protein antigenic epitope polypeptide is NKSYGKPDPEPTLSQI, located at positions 37-52aa; the sequence of the pp62 protein antigenic epitope polypeptide is DFLYTAIPEEKVGGNE, located at positions 450-465aa.

[0008] Antigenic epitope peptides were prepared using bioinformatics methods and artificial chemical synthesis techniques.

[0009] An ELISA antibody detection kit for synthetic peptides of African swine fever virus, the kit comprising an enzyme-linked reaction plate coated with an antigenic epitope peptide.

[0010] Method for coating antigenic epitope peptides on ELISA plates: Dissolve p72 and pp62 protein antigenic epitope peptides in coating buffer at a certain mass ratio (antigenic epitope peptide concentration 1200-1800 ng / mL), add 100 μL per well (antigenic epitope peptide coating amount 120-180 ng / well), let stand, add blocking buffer, seal, dry, and store in a sealed container; the mass ratio of p72 to pp62 protein antigenic epitope peptides is (1-2):1.

[0011] The kit also includes one or more of the following: enzyme-labeled secondary antibody, sample diluent, washing solution, chromogenic solution, stop solution, positive control serum, and negative control serum.

[0012] The coating solution is a carbonate solution with a pH of 9.6; the blocking solution is a phosphate buffer containing 10 mg / mL bovine serum albumin, and the amount of blocking solution added is 300 μL / well; the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-pig IgG antibody; the enzyme-linked reaction plate is a detachable 96-well microplate; the chromogenic solution includes chromogenic solution A and chromogenic solution B.

[0013] The colorimetric solution A is a citrate-phosphate buffer containing 0.6 mg / mL hydrogen peroxide urea; the colorimetric solution B is a 0.2 mg / mL tetramethylbenzidine solution; and the stop solution is a 2 mol / L sulfuric acid solution.

[0014] The sample diluent was a phosphate buffer containing 5 mg / mL casein; the washing buffer was a phosphate buffer containing 0.8–1.2% Tween-20 by volume percentage.

[0015] The phosphate buffer solution has a molar concentration of 0.01 mol / L and a pH of 7.4.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1) This invention uses bioinformatics methods to accurately analyze the antigenic epitopes of African swine fever virus p72 and pp62 proteins, and screens out peptides suitable for ELISA detection. These peptides have the advantages of high sensitivity and strong specificity.

[0018] 2) This invention utilizes African swine fever virus p72 and pp62 protein antigenic epitope peptides as coating antigens to prepare a kit that can effectively detect whether the tested animals are infected with African swine fever virus. Experiments have shown that the kit has high specificity, high sensitivity and good repeatability, which is conducive to the establishment of my country's African swine fever virus prevention and control system. Attached Figure Description

[0019] Figure 1 Results of p72 protein antigenic epitope analysis;

[0020] Figure 2 The results are from the analysis of pp62 protein antigenic epitopes. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the methods in the embodiments are conventional methods.

[0022] Example 1: Bioinformatics Prediction of Preferred Antigenic Epitopes of ASFV p72 and pp62 Proteins 1.1 Materials

[0023] ASFV p72 protein (B646L) sequence: The amino acid sequence of p72 (YP_009927206.1) from the ASFVpig / HLJ / 2018 strain (NCBI accession number: 59226937) was obtained from GenBank in NCBI. The specific amino acid sequence is as follows:

[0024] MASGGAFCLIANDGKADKIILAQDLLNSRISNIKNVNKSYGKPDPEPTLS

[0025] QIEETHLVHFNAHFKPYVPVGFEYNKVRPHTGTPTLGNKLTFGIPQYGDFFHD

[0026] MVGHHILGACHSSWQDAPIQGTSQMGAHGQLQTFPRNGYDWDNQTPLEGA

[0027] VYTLVDPFGRPIVPGTKNAYRNLVYYCEYPGERLYENVRFDVNGNSLDEYSS

[0028] DVTTLVRKFCIPGDKMTGYKHLVGQEVSVEGTSGPLLCNIHDLHKPHQSKPIL

[0029] TDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDATYLDIRRNVHY

[0030] SCNGPQTPKYYQPPLALWIKLRFWFNENVNLAIPSVSIPFGERFITIKLASQKDL

[0031] VNEFPGLFVRQSRFIAGRPSRRNIRFKPWFIPGVINEISLTNNELYINNLFVTPEI

[0032] HNLFVKRVRFSLIRVHKTQVTHTNNNHHDEKLMSALKWPIEYMFIGLKPTWN

[0033] ISDQNPHQHRDWHKFGHVVNAIMQPTHHAEISFQDRDTALPDACSSISDISPV

[0034] TYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGGNAIKTPDDPGAMMITF

[0035] ALKPREEYQPSGHINVSRAREFYISWDTDYVGSITTADLVVSASAINFLLLQNG

[0036] SAVLRYST.

[0037] pp62 protein (CP530R) sequence: The amino acid sequence of pp62 (QBH90582.1) from the ASFVpig / HLJ / 2018 strain (NCBI accession number: AHN94308.1) was obtained from GenBank in NCBI. The specific amino acid sequence is as follows:

[0038] >AHN94308.1 pp62[African swine fever virus]

[0039] MPSNMKQFCKISVWLQQHDPDLLEIINNLCMLGNLSAAKYKHGVTFIYPKQAKIRDEIKKHAYSNDPSQAIKTLESLILPFYIPTPAEFTGEIGSYTGVKLEVEKTEANKVILKNGEAVLVPAADFKPFPDR RLAVWIMESGSMPLEGPPYKRKKEGGGNDPPVPKHISPYTPRTRIAIEVEKAFDDCMRQNWCSVNNPYLAKSVSLLSFLSLNHPTEFIKVLPLIDFDPLVTFYLLLEPYKTHGDDFLIPETILFGPTGWNGTD LYQSAMLEFKKFFTQITRQTFMDIADSATKEVDVPICYSDPETVHSYANHVRTEILHHNAVNKVTTPNLVVQAYNELEQTNTIRHYGPIFPESTINALRFWKKLWQDEQRFVIHGLHRTLMDQPTYETSEFA EIVRNLRFSRPGNNYINELNITSPAMYGDKHTTGDIAPNDRFAMLVAFINSTDFLYTAIPEEKVGGNETQTSSLTDLVPTRLHSFLNHNLSKLKILNRAQQTVRNILSNDCLNQLKHYVKHTGKNEILKLLQE 1.2 Method

[0040] Bioinformatics tools, including online software BepiPred 3.0 Server (http: / / www.cbs.dtu.dk / services / BepiPred / ), IEDB Analysis Resource (http: / / tools.iedb.org / bcell / ), and the ABCpred method (https: / / webs.iiitd.edu.in / raghava / abcpred / ABC_submission.html), were used to predict and analyze the major antigenic epitopes of the p72 and pp62 proteins in the Pig / HLJ / 2018 strain sequence (MK333180.1) published in GenBank. Their hydrophilicity, surface accessibility, antigenicity, and flexibility were evaluated. The predicted B-cell antigenic epitopes were compared with those established by training epitope data from the Bcipep database.

[0041] 1.3 Results

[0042] The predictions show that the ASFV p72 protein has 10 candidate B-cell linear antigenic epitopes, and their sequences, start positions, and scores are shown in Table 1; the pp62 protein has 10 candidate B-cell linear antigenic epitopes, and their sequences, start positions, and scores are shown in Table 2.

[0043] Table 1. Candidate linear antigenic epitopes of ASFV p72 protein on B cells

[0044]

[0045]

[0046] Table 2. ASFV pp62 protein candidate B-cell linear antigen epitopes

[0047] 1 DFLYTAIPEEKVGGNE 450 0.93 2 PETILFGPTGWNGTDL 251 0.93 3 HISPYTPRTRIAIEVE 167 0.93 4 FFTQITRQTFMDIADS 277 0.92 5 PICYSDPETVHSYANH 300 0.91 6 PFYIPTPAEFTGEIGS 80 0.89 7 AMYGDKHTTGDIAPND 422 0.89 8 MESGSMPLEGPPYKRK 139 0.89 9 QAKIRDEIKKHAYSND 51 0.88 10 HGVTFIYPKQAKIRDE 42 0.88

[0048] This invention utilizes online software tools for comprehensive analysis, as shown in Tables 1-2, and... Figure 1-2 Analysis and screening of common sequence segments from multiple approaches identified the optimal peptide, ultimately determining two antigenic epitopes for p72 and pp62 proteins. Among these, p72 protein (NKSYGKPDPEPTLSQI, 37-52 aa) and pp62 protein (DFLYTAIPEEKVGGNE, 450-465 aa) are antigenic epitopes discovered for the first time in this invention. These epitopes were purified using existing conventional synthetic methods or the solid-phase synthesis method of Jier Biochemical (Shanghai) Co., Ltd., and used as the coating antigenic epitope peptides for the kit of this invention.

[0049] Example 2 Assembly of African Swine Fever Virus Synthetic Peptide ELISA Antibody Detection Kit

[0050] An African swine fever virus antigenic epitope polypeptide is provided, which is composed of an African swine fever virus p72 protein antigenic epitope polypeptide and a pp62 protein antigenic epitope polypeptide. The sequence of the p72 protein antigenic epitope polypeptide is NKSYGKPDPEPTLSQI, located at positions 37-52aa; the sequence of the pp62 protein antigenic epitope polypeptide is DFLYTAIPEEKVGGNE, located at positions 450-465aa.

[0051] An ELISA antibody detection kit for African swine fever virus synthetic peptides, comprising an enzyme-linked reaction plate coated with antigenic epitope peptides, an enzyme-labeled secondary antibody, sample diluent, washing buffer, chromogenic solution, stop solution, positive control serum, and negative control serum, wherein the enzyme-linked reaction plate is a 96-well polystyrene enzyme-linked reaction plate.

[0052] Method for coating antigenic epitope peptides onto ELISA plates: Dissolve p72 and pp62 protein antigenic epitope peptides at a mass ratio of 2:1 in a pH 9.6 carbonate solution (antigenic epitope peptide concentration 1500 ng / mL), and add 100 μL per well of a removable 96-well polystyrene ELISA plate (antigenic epitope peptide coating amount 150 ng / well). Incubate at 4°C for 8–12 hours to allow the antigenic epitope peptides to fully bind to the ELISA plate. Then, add 300 μL / well of PBS buffer containing 10 mg / mL bovine serum albumin (BSA) at a molar concentration of 0.01 mol / L and a pH of 7.4. Block at 37°C for 2–3 hours, spin dry, and store at 4°C after the ELISA plate has dried.

[0053] The enzyme-labeled secondary antibody was horseradish peroxidase-labeled goat anti-pig IgG antibody.

[0054] The colorimetric solution includes colorimetric solution A and colorimetric solution B. Colorimetric solution A is a citrate-phosphate buffer containing 0.6 mg / mL hydrogen peroxide urea, and colorimetric solution B is a tetramethylbenzidine solution containing 0.2 mg / mL. The ratio of the two solutions used is 1:1.

[0055] The stop solution is a 2 mol / L sulfuric acid solution.

[0056] The sample dilution solution was a phosphate buffer solution with a molar concentration of 0.01 mol / L containing 5 mg / mL casein and a pH of 7.4.

[0057] The washing solution was a phosphate buffer with a volume percentage concentration of 0.8–1.2% Tween-20, a molar concentration of 0.01 mol / L, and a pH of 7.4.

[0058] Positive control serum: Purchased from China Veterinary Microbial Culture Collection Center (catalog number Z287), used as the positive control serum for the kit (1 tube, 1.5 mL / tube).

[0059] Negative control serum: Specific pathogen-free (SPF) porcine serum.

[0060] Example 3: Reagent Kit Sensitivity Test

[0061] Sensitivity testing was performed on the kit from Example 2 by collecting 10 samples of swine serum from a pig farm as test samples.

[0062] Dilute the detergent with deionized water 20 times.

[0063] Before use, the serum samples to be tested, negative control serum, and positive control serum should be diluted with sample diluent at a ratio of 1:20.

[0064] 1) Before use, remove the kit from the refrigerated environment and place it at room temperature for 30 minutes to allow it to return to room temperature.

[0065] 2) Remove the ELISA plate strips, set up 2 wells each for negative and positive controls, and the rest for sample wells;

[0066] 3) Add 50 μl of negative and positive control serum to each negative and positive control well, and add 50 μl of diluted test sample serum to each sample well;

[0067] 4) Mix well and place at 37°C for 15 minutes;

[0068] 5) Discard the liquid in the well, shake dry, fill each well with diluted washing solution, let stand for 30 seconds, discard the washing solution, repeat washing 5 times, and pat dry.

[0069] 6) Add 50 μl of enzyme-labeled secondary antibody to each well and incubate at 37°C for 15 minutes;

[0070] 7) Discard the liquid in the hole, shake dry, and wash each hole 5 times using the washing method in step 5), then pat dry.

[0071] 8) Add 50 μl of colorimetric reagent A to the negative control well, positive control well, and sample well, then add 50 μl of colorimetric reagent B, gently shake to mix, and react at 37°C in the dark for 15 minutes.

[0072] 9) Add 50 μl of stop solution to each well to stop the reaction (the blue color will immediately turn yellow). Measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0073] Determination of the validity of test results: Average OD of positive control wells 450nm Value ≥1.00; mean OD of negative control 450nm Value ≤ 0.30, otherwise invalid.

[0074] Sample well OD 450nm A value ≥0.6 indicates a positive African swine fever antibody test; otherwise, it indicates a negative result.

[0075] Sensitivity test results: A total of 9 positive samples of the pig serum to be tested were detected. The sensitivity of the kit to 10 samples of pig serum to be tested was 90%.

[0076] Example 4 Specificity Test

[0077] The specificity of the kit in Example 2 was tested. The kit from Example 2 of this invention was used to test 10 normal antibody-detecting swine serum samples retained before May 2018 and control serum samples from existing commercial kits. The testing method was the same as in Example 2. Two positive control sera for classical swine fever (from the IDEXX test kit), two positive control sera for porcine respiratory and reproductive syndrome (from the IDEXX test kit), two positive control sera for pseudorabies GB (from the IDEXX test kit), two positive control sera for porcine foot-and-mouth disease type O (from Luoyang Laipusheng), two positive control sera for porcine circovirus type 2 (from Beijing Yisenbao), and two positive control sera for African swine fever (from Jinno Diagnostics, positive), for a total of 22 samples, were tested. The detection method was the same as in Example 3.

[0078] Table 3. Specificity detection results of the African swine fever virus synthetic peptide ELISA antibody detection kit

[0079]

[0080] As shown in Table 3, the kit of Example 2 of this invention was used to test normal antibody-containing swine serum retained before May 2018, as well as two positive control sera for classical swine fever, two positive control sera for porcine respiratory and reproductive syndrome, two positive control sera for pseudorabies GB, two positive control sera for porcine foot-and-mouth disease type O, and two positive control sera for porcine circovirus type 2 from the commercial kit. All test results were negative. The test results for the two positive sera for African swine fever were positive, indicating that the specificity of the kit of this invention is 100.0%.

[0081] Example 4: Comparison test between the kit of the present invention and a commercially available antibody kit.

[0082] The African swine fever virus synthetic peptide ELISA antibody detection kit prepared in Example 2 and the imported ASFV indirect ELISA antibody detection kit (imported kit) were used to detect 20 samples of swine serum. The results are shown in Table 4.

[0083] Table 4. Results of the Compliance Rate Test

[0084]

[0085] Table 4 shows that the African swine fever virus synthetic peptide ELISA antibody detection kit of the present invention has a sensitivity of 80.0% for 20 samples of swine serum, while the imported kit has a sensitivity of 90.0%. Results from both kits were consistent for 15 samples. Therefore, the concordance rate between the present invention's kit and the imported kit is 75.0%.

Claims

1. A polypeptide composition containing African swine fever virus antigenic epitopes, characterized in that, The antigenic epitope polypeptide composition consists of African swine fever virus p72 protein antigenic epitope polypeptide and pp62 protein antigenic epitope polypeptide. The sequence of the p72 protein antigenic epitope polypeptide is NKSYGKPDPEPTLSQI, located at positions 37-52aa; the sequence of the pp62 protein antigenic epitope polypeptide is DFLYTAIPEEKVGGNE, located at positions 450-465aa.

2. An ELISA antibody detection kit for African swine fever virus synthetic peptides, characterized in that, The kit comprises an enzyme-linked reaction plate coated with the antigen epitope polypeptide composition of claim 1.

3. The African swine fever virus synthetic peptide ELISA antibody detection kit as described in claim 2, characterized in that, Method for coating an enzyme-linked reaction (ELISA) plate with an antigenic epitope peptide composition: Dissolve p72 protein antigenic epitope peptide and pp62 protein antigenic epitope peptide in a coating solution at a certain mass ratio, add the solution to the wells of the ELISA plate, with a coating amount of 120-180 ng / well, let stand, add blocking solution, seal, dry, and store in a sealed container; the mass ratio of p72 protein antigenic epitope peptide to pp62 protein antigenic epitope peptide is (1-2):

1.

4. The African swine fever virus synthetic peptide ELISA antibody detection kit as described in claim 3, characterized in that, The kit also includes one or more of the following: enzyme-labeled secondary antibody, sample diluent, washing solution, chromogenic solution, stop solution, positive control serum, and negative control serum.

5. The African swine fever virus synthetic peptide ELISA antibody detection kit as described in claim 4, characterized in that, The coating solution is a carbonate solution with a pH of 9.6; the blocking solution is a phosphate buffer containing 10 mg / mL bovine serum albumin, and the amount of blocking solution added is 300 μL / well; the enzyme-labeled secondary antibody is horseradish peroxidase-labeled goat anti-pig IgG antibody; the enzyme-linked reaction plate is a detachable 96-well microplate; the chromogenic solution includes chromogenic solution A and chromogenic solution B.

6. The African swine fever virus synthetic peptide ELISA antibody detection kit as described in claim 5, characterized in that, The colorimetric solution A is a citrate-phosphate buffer containing 0.6 mg / mL hydrogen peroxide urea; the colorimetric solution B is a 0.2 mg / mL tetramethylbenzidine solution; and the stop solution is a 2 mol / L sulfuric acid solution.

7. The African swine fever virus synthetic peptide ELISA antibody detection kit as described in claim 6, characterized in that, The sample diluent was a phosphate buffer containing 5 mg / mL casein; the washing buffer was a phosphate buffer containing 0.8–1.2% Tween-20 by volume percentage.

8. The African swine fever virus synthetic peptide ELISA antibody detection kit as described in claim 7, characterized in that, The phosphate buffer solution has a molar concentration of 0.01 mol / L and a pH of 7.4.

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