African swine fever virus antigen peptide combination, ELISA kit and application

Abstract

The invention relates to the technical field of biology, in particular to an African swine fever virus antigen peptide combination, an ELISA kit and application, and the antigen peptide combination is composed of a p30 protein antigen peptide, a p54 protein antigen peptide and a p72 protein antigen peptide; results show that a plurality of antigen peptides of p30, p54 and p72 proteins and ASFV antiserum show positive reaction, the antigen peptides p30-10, p54-12 and p72-3 show the strongest reaction to the ASFV antiserum, are selected as coating antigens and have good specificity, and the sensitivity of the antigen peptide ELISA kit is 1: 1600; compared detection of an ASFV antigen peptide ELISA kit and a commercial kit is carried out on 89 clinical serum samples, the positive coincidence rate is 92%, the negative coincidence rate is 82.8%, the total coincidence rate is 87.4%, and the ASFV antigen peptide ELISA kit has a wide clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to an African swine fever virus antigen peptide combination, an ELISA kit and application. BACKGROUND

[0002] African swine fever (ASF) is an acute, highly virulent and highly contagious disease caused by African swine fever virus (ASFV). ASFV can infect pigs of all ages, and the mortality rate of acute infection in pigs is as high as 100%. ASFV is a linear double-stranded DNA virus with a capsid, with a genome of about 170-190 kbp, encoding more than 160 proteins. The p30 protein with a molecular weight of 26 kDa is encoded by the CP204L gene and is an important antigen protein, which plays a role in resisting ASFV invasion and internalization. The p30 protein can be expressed about 2-4 hours after ASFV infection, and therefore, it is used as a marker of early viral infection and is an important target for early diagnosis of ASF. The p54 protein with a molecular weight of 25 kDa is encoded by the E183L gene and is the main antigen protein of ASFV, which plays an important role in inducing specific antibodies after pigs are infected with ASFV. The p72 protein with a molecular weight of 73 kDa is encoded by the B646L gene and is the main component of the icosahedral capsid of the virus, which is also a key structural protein, accounting for about 32% of the mass of the virus particle, and has strong antigenicity and immunogenicity. Notably, p72 has antigen stability and high conservation in different ASFV isolates. The combined use of p30, p54 and p72 proteins in serological detection can improve the diagnostic sensitivity.

[0003] Currently, there is no effective commercially available vaccine or effective treatment method, and the prevention and control of ASF relies on the early detection of viral infection and the timely implementation of biosafety measures. Therefore, it is particularly crucial to establish a rapid and accurate early diagnosis technology. Serum adsorption test, virus isolation, fluorescent antibody test, antigen detection, ELISA, conventional PCR and real-time PCR are the most widely used ASFV detection methods. Among them, the ELISA technology has the advantages of rapidity, high sensitivity, no need for special facilities and equipment, etc. In this study, we developed an indirect ELISA kit based on the antigen peptides screened from p30, p54 and p72 proteins, which has higher sensitivity and specificity than the commercial ELISA kit. The present application provides a new and potentially effective method for serological detection of ASF. SUMMARY

[0004] The primary objective of this invention is to provide an African swine fever virus antigenic peptide combination, which comprises a p30 protein antigenic peptide, a p54 protein antigenic peptide, and a p72 protein antigenic peptide. The amino acid sequence of the p30 protein antigenic peptide is shown in SEQ ID No. 1, the amino acid sequence of the p54 protein antigenic peptide is shown in SEQ ID No. 2, and the amino acid sequence of the p72 protein antigenic peptide is shown in SEQ ID No. 3.

[0005] A second objective of this invention is to provide the application of the aforementioned antigen peptide combination in the preparation of diagnostic reagents for African swine fever virus.

[0006] A third objective of this invention is to provide an ELISA detection kit for African swine fever virus, wherein the detection kit contains the aforementioned African swine fever virus antigen peptide combination.

[0007] Preferably, the detection kit is prepared by the following method: (1) The p30 protein antigen peptide, p54 protein antigen peptide and p72 protein antigen peptide were diluted with CBS at a ratio of 1:1:1 and mixed overnight to coat the ELISA plate. (2) 7.5% skim milk was blocked at room temperature; serum was tested by 1:200 dilution and reacted at room temperature; (3) The enzyme-labeled secondary antibody was diluted at 1:50000 and reacted at 37℃; TMB was incubated at 37℃.

[0008] The beneficial effects of this invention are: (1) The present invention uses indirect ELISA method and ASFV positive pig serum to accurately detect the antigenic peptides of African swine fever virus p30, p54 and p72 proteins, and screens out peptides that are suitable for ELISA detection and react strongly with ASFV positive pig serum. These antigenic peptides have the advantages of strong reactivity, high sensitivity and strong specificity.

[0009] (2) The present invention uses ASFV p30, p54 and p72 protein antigen peptides as coating antigens to make a kit that can effectively detect whether the tested animals are infected with ASFV. Experiments have shown that the kit has high specificity, high sensitivity and good repeatability. Attached Figure Description

[0010] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1 Antigen peptide screening results Figure 2 ELISA method condition optimization results Figure 3 ELISA method critical value determination results Figure 4 ELISA method specificity detection results Figure 5 ELISA method sensitivity test results Figure 6 Clinical application results of ELISA method Figure 7 Comparison results of ELISA method with other patents Detailed Implementation

[0012] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0013] It should be noted that, unless otherwise specified, the methods described in the following embodiments are all conventional methods, and the reagents described are all commercially available.

[0014] Invention patent CN117229367A discloses an African swine fever virus antigenic epitope peptide and an ELISA antibody detection kit. The antigenic epitope peptide is composed of African swine fever virus p72 protein antigenic epitope peptide and pp62 protein antigenic epitope peptide. The sequence of the p72 protein antigenic epitope peptide is NKSYGKPDPEPTLSQI, located at positions 37-52 aa; the sequence of the pp62 protein antigenic epitope peptide is DFLYTAIPEEKVGGNE, located at positions 450-465 aa. In this invention, the inventors used the aforementioned patent as a control group to evaluate the efficacy of the antigenic peptide of this invention.

[0015] Example 1: Screening of Antigenic Peptides 1. Method 1.1 Serum Samples Positive sera for ASFV (hyperimmune serum), porcine reproductive and respiratory syndrome virus (PRRSV), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), and classical swine fever virus (CSFV) were all preserved in the laboratory. Serum from SPF pigs tested negative for ASFV antibodies and was used as the negative serum for this study, preserved in our laboratory. Clinical serum samples were obtained from pig farms across my country. SPF pigs were immunized with the ASFV-ΔH240R live attenuated vaccine and challenged with the ASFV HLJ / 18 strain. Serum samples were collected from the experimental pigs at different time points and preserved in our laboratory.

[0016] 1.2 Antigen peptide design and synthesis We designed antigenic peptides containing the full-length amino acid sequences of ASFV p30, p54, and p72, each peptide being 20 amino acids in length. However, some antigenic peptides could not be synthesized due to hydrophobicity or other reasons. These antigenic peptides were synthesized by GenScript (Nanjing, China).

[0017] 1.3 Indirect ELISA method for screening immunodominant antigen peptides The synthesized antigenic peptide was coated at a concentration of 2 μg / ml onto a 96-well ELISA plate and incubated overnight at 4°C in CBS (0.05 mol / L, pH 9.6). The plate was washed three times with PBST (0.05% Tween 20, PBS) and blocked with 200 μL of 5% skim milk at 37°C for 1 hour. After washing, porcine ASFV-positive serum diluted 1:200 was used as the primary antibody and incubated at 37°C for 1 hour. HRP-conjugated goat anti-porcine IgG (1:10000 dilution) was used as the secondary antibody and incubated at 37°C for 0.5 hours. The reaction was terminated with 2 mol / L H₂SO₄ after reacting with TMB at room temperature for 10 minutes. OD was then detected. 450nm Absorbance value.

[0018] 2. Results To screen for immunogenic peptides, this study designed and synthesized a series of antigenic peptides covering the full-length sequences of ASFV p30, p54, and p72 proteins, each consisting of 20 amino acids (aa). The synthesized antigenic peptides were dissolved in DMSO at a concentration of 2 mg / mL.

[0019] The reactivity of these antigenic peptides with ASFV-positive serum was evaluated using ELISA. The results showed that multiple antigenic peptides of p30, p54, and p72 proteins exhibited positive reactions with ASFV antiserum. Among them, antigenic peptide p30-10 (with the sequence "1") showed a particularly strong reaction. 11 ETNECTSSFETLFEQEPSSE 130 p54-12 (sequence "1")41 SAPAHPAEPYTTVTTQNTAS 160 p72-3 (sequence "4") and p72-3 (sequence "4") 1 GKPDPEPTLSQIEETHLVH 60 The antigen showed the strongest response to ASFV antiserum and was therefore selected as the coating antigen. Figure 1 ).

[0020] Example 2: Establishment of an indirect ELISA method based on p30, p54, and p72 antigenic peptides (1) The antigenic peptides of p30, p54 and p72 were mixed in equal proportions (100ng / ml to 3.125ng / ml) and coated onto ELISA plates respectively. ASFV positive and negative sera were diluted at a ratio of 1:10 to 1:600 ​​and added to ELISA plates for incubation. Finally, the optimal coating concentration of 100ng / ml for each antigenic peptide and the optimal serum dilution of 1:200 were selected.

[0021] (2) The antigen peptides were coated in carbonate buffer (CBS, 0.05 mol / L, pH 9.6) and phosphate buffer (PBS, pH 7.4), respectively. CBS was finally selected as the best antigen peptide coating solution. The antigen peptides were coated on ELISA plates at 37°C for 1 hour, 37°C for 2 hours, 4°C for 6 hours and 4°C overnight. After screening, 4°C overnight was finally selected as the best antigen peptide coating condition.

[0022] (3) The optimal blocking solution was selected from 2.5%, 5%, and 7.5% skim milk and 2.5%, 5%, and 7.5% bovine serum albumin (BSA), respectively. The optimal blocking conditions were screened from room temperature (25℃±2℃) for 0.5 hours, 1 hour, 1.5 hours, and 2 hours and 37℃ for 0.5 hours, 1 hour, 1.5 hours, and 2 hours. Finally, 7.5% skim milk was determined to be the optimal blocking condition for room temperature blocking for 1 hour.

[0023] (4) The serum was incubated at room temperature and 37°C for 30, 45, 60, 75 and 90 minutes respectively, so that the optimal serum reaction conditions were selected as room temperature reaction for 45 minutes.

[0024] (5) Dilute the enzyme-labeled secondary antibody at ratios of 1:15000, 1:20000, 1:25000, 1:30000, 1:35000, 1:40000, 1:45000, and 1:50000, and incubate at room temperature (25℃±2℃) and 37℃ for 15, 30, 45, and 60 minutes, respectively. The optimal reaction conditions for screening the secondary antibody are a 1:50000 dilution and a reaction at 37℃ for 30 minutes.

[0025] (6) TMB was incubated at 37°C for 5, 10, 15, 20 and 25 minutes respectively. The optimal TMB reaction condition was screened as 37°C for 10 minutes for color development.

[0026] Therefore, the optimal reaction conditions for the ELISA method are as follows: antigen peptides p30-10, p54-12, and p72-3 are diluted with CBS (0.05 mol / L, pH 9.6) and mixed at a concentration of 100 ng / mL to coat the ELISA plate overnight at 4°C; 7.5% skim milk is used for blocking at room temperature for 1 hour; the detection serum is diluted 1:200 and reacted at room temperature for 45 minutes; the enzyme-labeled secondary antibody is diluted 1:50000 and reacted at 37°C for 30 minutes; and TMB is developed at 37°C for 10 minutes.

[0027] Example 3: Characteristic Analysis of the ELISA Method 1. Determination of critical values The cutoff value for the antigen peptide ELISA kit was determined using 30 clinically negative serum samples. The mean OD was calculated through statistical analysis. 450nm Value and standard deviation (SD), the critical value is determined as OD 450nm Samples with a value greater than or equal to the critical value are considered positive, while samples with a value less than the critical value are considered negative.

[0028] Thirty clinically negative serum samples from different pig farms were tested, with a mean value of 0.180 and a SD of 0.050. The final cutoff value was 0.330. Figure 2 A serum OD value greater than or equal to 0.330 is considered positive; otherwise, it is considered negative.

[0029] 2. Sensitivity and specificity assays The specificity of the antigen peptide ELISA kit was determined by detecting PRRSV, TGEV, PEDV, PRV, PCV2, and CSFV positive sera under optimized conditions. ASFV positive sera were diluted at ratios of 1:200–1:12800 and detected under optimized conditions to determine the sensitivity of the kit.

[0030] The antigen peptide ELISA kit was used to detect PRRSV, TGEV, PEDV, PRV, PCV2, CSFV, and ASFV-positive and ASFV-negative sera, respectively, with good specificity. Figure 3 The sensitivity of the antigen peptide ELISA kit is 1:1600. Figure 4 ).

[0031] 3. Reproducibility of the antigen peptide ELISA kit To assess the reproducibility of the antigen peptide ELISA kit, serum samples with four known backgrounds were tested five times each using the same and different batches of the antigen peptide ELISA kit to detect intra-batch and inter-batch reproducibility.

[0032] The intra-assay repeatability of the four ASFV serum samples ranged from 2.43% to 9.22%, and the inter-assay repeatability ranged from 2.82% to 9.28%, indicating good repeatability (see Table 1).

[0033] Table 1. Reproducibility of antigen peptide ELISA kits prepared in the same and different batches.

[0034] 4. Clinical serum sample testing All clinical serum samples were tested and compared using a commercially available ASFV indirect ELISA antibody detection kit (based on ASFV p30, Beijing Jinno Biotech Co., Ltd., Beijing, China) and the antigen peptide ELISA kit used in this study.

[0035] Eighty-nine clinical serum samples were compared between the ASFV antigen peptide ELISA kit and a commercial kit. The antigen peptide ELISA kit detected 34 positive serum samples and 55 negative serum samples, while the commercial kit detected 25 positive serum samples and 64 negative serum samples. The positive concordance rate was 92%, the negative concordance rate was 82.8%, and the overall concordance rate was 87.4% (Table 2).

[0036] Table 2. Conformity rate of antigen peptide ELISA kits

[0037] Example 4: Animal Experiment 1. Method Three healthy 6-week-old SPF pigs were immunized with ASFV-ΔH240R live attenuated vaccine at a dose of 10. 3 HAD 50 / head; 28 days after immunization, challenge with ASFV HLJ / 18 wild-type strain, challenge dose was 10. 2.5 HAD 50 / head. Serum samples were collected from experimental pigs at different time points after immunization and challenge, and detected using an antigen peptide ELISA kit.

[0038] 2. Results Serum from experimental pigs was collected at 1, 4, 7, 11, 16, 21, and 28 days post-vaccination and post-challenge. The antibodies were detected using a commercially available ASFV indirect ELISA antibody detection kit (based on ASFV p30, Beijing Jinno Biotech Co., Ltd., Beijing, China) and the antigen peptide ELISA kit used in this study, respectively. Results showed that the antigen peptide ELISA kit detected antibody seroconversion as early as day 4 post-immunization; and that serum antibody titers in two pigs began to show a gradual upward trend starting on day 7 post-immunization. Figure 5 ); however, using commercially available kits, antibody seroconversion was only detected in two pigs up to 11 days post-immunization, and the upward trend in antibody titers was not obvious. Figure 6 This indicates that the ASFV antigen peptide ELISA kit developed in this study has higher sensitivity when detecting serum in the early stages of infection.

[0039] Comparative Example 1 method 1.1 Comparative Sources This invention conducted a comparative detection of the "p72 protein antigenic epitope polypeptide sequence NKSYGKPDPEPTLSQI, position 37-52aa" shown in the invention patent "An African Swine Fever Virus Antigenic Epitope Polypeptide and ELISA Antibody Detection Kit (Patent No. CN 117229367A)". The coating antigenic polypeptide is the antigenic peptide at position 31-50aa of the p72 protein.

[0040] 1.2 Serum Samples The five serum samples used in the comparative experiment of this invention were all clinically submitted serum samples, which tested positive using a commercially available ASFV indirect ELISA antibody detection kit and were stored in our laboratory.

[0041] 1.3 Comparison and Testing (1) Five clinical serum samples were tested using the methods in Examples 2 and 3 of the invention patent “CN 117229367 A”.

[0042] (2) Five clinical serum samples were tested using the test steps determined in Example 2 of the present invention.

[0043] (3) Compare the two test results.

[0044] result Of the five serum samples that tested positive using a commercially available ASFV indirect ELISA antibody detection kit, all five serum samples tested using this invention were also positive. However, when tested using the peptide assay described in patent "CN 117229367 A", serum sample number 1 was negative, while the other four were positive, but their positive values ​​were all lower than those detected by this invention. This indicates that the results from this invention have a higher concordance rate with those from the commercially available kit.

[0045] In summary, this invention provides a combination of African swine fever virus (ASFV) antigenic peptides, comprising p30, p54, and p72 protein antigenic peptides. The amino acid sequence of the p30 protein antigenic peptide is shown in SEQ ID No. 1, the amino acid sequence of the p54 protein antigenic peptide is shown in SEQ ID No. 2, and the amino acid sequence of the p72 protein antigenic peptide is shown in SEQ ID No. 3. The reactivity of these antigenic peptides with ASFV-positive serum was evaluated using an ELISA method. The results showed that multiple antigenic peptides of p30, p54, and p72 proteins showed a positive reaction with ASFV antiserum, among which, antigenic peptide p30-10 (sequence "1") showed a positive reaction. 11 ETNECTSSFETLFEQEPSSE 130 p54-12 (sequence "1") 41 SAPAHPAEPYTTVTTQNTAS 160 p72-3 (sequence "4") and p72-3 (sequence "4") 1 GKPDPEPTLSQIEETHLVH 60 The antigen p30-10 showed the strongest response to ASFV antiserum and was therefore selected as the coating antigen. The optimal reaction conditions for the ELISA method were: antigen peptides p30-10, p54-12, and p72-3 were reacted with CBS (0.05 mol / L, pH 10). 9.6) Dilute and mix at a concentration of 100 ng / mL, then coat the ELISA plate overnight at 4°C; block with 7.5% skim milk at room temperature for 1 hour; dilute the test serum 1:200 and react at room temperature for 45 minutes; dilute the enzyme-labeled secondary antibody 1:50000 and react at 37°C for 30 minutes; develop TMB at 37°C for 10 minutes. The antigen peptide ELISA kit was used to detect PRRSV, TGEV, PEDV, PRV, PCV2, CSFV, and ASFV positive and ASFV negative sera, showing good specificity. The sensitivity of the antigen peptide ELISA kit was 1:1600; a comparison was made between the ASFV antigen peptide ELISA kit and a commercial kit for 89 clinical serum samples. The antigen peptide ELISA kit detected 34 positive sera and 55 negative sera, while the commercial kit detected 25 positive sera and 64 negative sera, with a positive concordance rate of 92%, a negative concordance rate of 82.8%, and an overall concordance rate of 87.4%.

[0046] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A combination of African swine fever virus antigen peptides, characterized in that, The antigenic peptide combination consists of p30 protein antigenic peptide, p54 protein antigenic peptide and p72 protein antigenic peptide. The amino acid sequence of the p30 protein antigenic peptide is shown in SEQ ID No. 1, the amino acid sequence of the p54 protein antigenic peptide is shown in SEQ ID No. 2, and the amino acid sequence of the p72 protein antigenic peptide is shown in SEQ ID No.

3.

2. The application of the antigen peptide combination as described in claim 1 in the preparation of diagnostic reagents for African swine fever virus.

3. An ELISA detection kit for African swine fever virus, characterized in that, The test kit contains the African swine fever virus antigen peptide combination as described in claim 1.

4. The ELISA detection kit as described in claim 3, characterized in that, The detection kit is prepared by the following method: (1) The p30 protein antigen peptide, p54 protein antigen peptide and p72 protein antigen peptide were diluted with CBS at a ratio of 1:1:1 and mixed overnight to coat ELISA plates; (2) 7.5% skim milk was blocked at room temperature; serum was tested by dilution at 1:200 and reacted at room temperature; (3) The enzyme-labeled secondary antibody was diluted at 1:50000 and reacted at 37℃; TMB was incubated at 37℃.

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