African swine fever virus p30 antigen epitope protein and preparation method and use thereof

Abstract

The present invention discloses an African swine fever virus p30 antigen epitope protein and a preparation method and use thereof. The p30 antigen epitope protein is selected from any one of p30a, p30b, p30c, p30d, p30e, p30f, p30g, and p30I antigen epitope proteins, or a recombinant multi-epitope protein obtained by connecting any two or more of p30a, p30b, p30c, p30d, p30e, p30f, p30g, and p30I antigen epitope proteins in series. The present invention takes the main structural protein p30 of the ASFVpig / HLJ / 2018 strain isolated in my country as the object, uses bioinformatics technology combined with immunological methods to predict and screen the antigen epitopes of ASFV, draws the antigen epitope maps of these proteins, and finally determines that 8 antigen epitopes (peptides) are the antigen epitopes of the African swine fever virus p30 protein. The acquisition of these candidate antigenic epitopes has greatly saved the cost and time of traditional antigenic epitope screening, and improved the efficiency of immunological methods in verifying antigenic epitopes with good immune protection functions, which is of great significance for the development of new ASF vaccines and detection technologies.

Description

Technical Field

[0001] The present invention relates to an antigen epitope protein and a preparation method and use thereof, and in particular to an African swine fever virus p30 antigen epitope protein and a preparation method and use thereof. The present invention belongs to the field of biomedicine technology. Background Art

[0002] African swine fever (ASF) is an acute and severe infectious disease caused by African swine fever virus (ASFV) infecting pigs, with a mortality rate of up to 100%. The World Organization for Animal Health (OIE) has listed it as a notifiable animal disease, and my country has listed it as a Class I animal infectious disease that requires key prevention.

[0003] As an important animal virus with a history of over a hundred years, research on vaccines for African swine fever virus has never stopped, but there is still a lack of safe, effective, and differentially diagnosed vaccines that can be used in prevention and control practices.

[0004] In view of the complexity of African swine fever virus and the unknown spectrum of protective antigens / antigenic epitopes, it has become a key scientific issue restricting the research of genetic engineering vaccines. The present invention carries out research on the discovery of protective antigens / antigenic epitopes of African swine fever virus to solve the dilemma of the unknown spectrum of protective antigens / antigenic epitopes of African swine fever virus and the lack of key materials restricting the research of genetic engineering vaccines, in order to provide material basis and technical support for the research and development of African swine fever genetic engineering vaccines.

[0005] The present invention uses the primary structure amino acid sequence of p30 (CP204L), one of the membrane proteins of African swine fever virus, as the material, analyzes and predicts its antigenic epitopes through the antigenic epitope database, and synthesizes the corresponding peptides according to the amino acid sequence. The positive serum of pigs infected with African swine fever virus is used to screen specific peptides (antigenic epitopes) that can react specifically with it but not with the positive serum of swine fever, and finally 8 antigenic epitopes (peptides) are determined to be the antigenic epitopes of the p30 protein of African swine fever virus. The acquisition of these antigenic epitopes lays the foundation for the research and development of African swine fever detection reagents and their kits, monoclonal antibody preparation and safe, differential diagnosis epitope vaccines and other urgently needed strategic prevention and control products using antigenic epitopes as materials. It will not only generate huge economic benefits, but also have important social benefits. Summary of the invention

[0006] The purpose of the present invention is to provide an African swine fever virus p30 antigen epitope protein and a preparation method and use thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical means:

[0008] The present invention uses TMHMM software to predict the transmembrane region of ASFVp30 protein. The software is a kind of software based on hidden Markov mathematical model for predicting the transmembrane region of protein, with an accuracy rate of up to 97% to 98% (Krogh et al, 2001). The results show that there is no transmembrane region in p30 protein. Then, IEDBAnalysis Resource and ABCpred software are used to predict the B cell linear epitopes of ASFVp30 protein, and the threshold is set to be not less than 0.5 as the candidate antigenic epitope. IEDBAnalysis Resource predicts antigenic epitopes through neural network (ANN) and support vector machine (SMM) modeling, in which the relevant immune epitopes are selected from scientific publications data available in PubMed, and the parameters such as hydrophilicity, flexibility, accessibility, corner, exposed surface, polarity and antigenic tendency of the polypeptide are analyzed (Jespersen et al, 2017), and 7 antigenic epitopes are identified on the ASFV p30 protein. ABCpred is a predictive B cell antigen epitope database software (Saha et al, 2007) based on an artificial neural network (ANN) model, which was established by training the epitope data of the Bcipep database, and 21 antigen epitopes were identified on the ASFVp30 protein. Next, the present invention determines the antigen epitopes with immunological activity by establishing an indirect ELISA method based on the results of the immune reaction between the antigen epitope (synthetic peptide) and the ASFV infected positive serum. According to the ELISA results, a total of 8 immunologically active p30 protein epitopes were screened from the predicted antigen epitopes of the p30 protein. It is worth mentioning that a new antigen epitope p30b (30-GSLYNW-35) of the ASFVp30 protein was discovered by combining bioinformatics technology with immunological methods. In order to overcome the shortcomings of small molecule characteristics and weak immune response of antigenic epitopes and improve the accuracy of antigenic epitope screening, the present invention uses gene manipulation technology to insert the antigenic epitope DNA sequence into the prokaryotic expression vector pGEX-4T-1 carrying the GST tag, construct a recombinant expression plasmid, and express and purify the GST-epitope recombinant protein. In addition, in order to increase the titer of the antigenic epitope, different antigenic epitopes are connected in series and then displayed on the phage surface through phage display technology. The recombinant protein is purified by affinity chromatography, and the antigenic epitope is identified by the established indirect ELISA method. The results show that after a single peptide segment is introduced into the GST vector, it has a strong affinity with the synthetic peptide OD 450nm In comparison, the immunoreactivity of single epitope recombinant protein was significantly enhanced, and the immunoreactivity enhancement after complete antigenization of the antigen epitope was significantly better than that of a single synthetic peptide. After the multi-epitope protein gene was concatenated with the phage gene, the immunoreactivity of the multi-epitope recombinant protein was significantly better than that of a single antigen epitope after complete antigenization.

[0009] Based on the above research, the present invention proposes an African swine fever virus p30 antigen epitope protein, characterized in that the p30 antigen epitope protein is selected from any one of p30a, p30b, p30c, p30d, p30e, p30f, p30g, and p30I antigen epitope proteins, or a multi-epitope recombinant protein obtained by connecting any two or more of the p30a, p30b, p30c, p30d, p30e, p30f, p30g, and p30I antigen epitope proteins in series, and the amino acid sequences of the p30a, p30b, p30c, p30d, p30e, p30f, p30g, and p30I antigen epitope proteins are shown in SEQ ID NO.1-8, respectively.

[0010] Preferably, one end of the p30 antigen epitope protein is connected to a GST tag.

[0011] Among them, preferably, the p30 antigen epitope protein is composed of p30c, p30d, and p30e antigen epitope proteins connected in series, with a spacer sequence GGGS introduced between adjacent antigen epitopes, and then the encoding gene sequence and the bacteriophage AP205 gene are cloned into the linearized prokaryotic expression vector pET-28a(+) in sequence to construct a phage-displaying multi-epitope recombinant expression plasmid, and the GST-multi-epitope recombinant protein is obtained by expressing it in Escherichia coli.

[0012] Among them, preferably, the p30 antigen epitope protein is composed of p30g and p30I antigen epitope proteins connected in series, with a spacer sequence GGGS introduced between adjacent antigen epitopes, and then the encoding gene sequence and the bacteriophage AP205 gene are cloned into a linearized prokaryotic expression vector pET-28a(+) respectively to construct a phage-displaying multi-epitope recombinant expression plasmid, and the GST-multi-epitope recombinant protein is obtained by expressing it in Escherichia coli.

[0013] Furthermore, the present invention also proposes the use of the African swine fever virus p30 antigen epitope protein in the preparation of an African swine fever virus antibody reagent for detecting African swine fever virus.

[0014] Among them, preferably, the reagent is an ELISA detection reagent.

[0015] Furthermore, the present invention also proposes the use of the African swine fever virus p30 antigen epitope protein or its recombinant protein in the design and preparation of African swine fever vaccine.

[0016] Furthermore, the present invention also proposes an indirect ELISA detection kit for African swine fever virus antibodies, wherein the kit contains the African swine fever virus p30 antigen epitope protein described in any of the above items.

[0017] Preferably, the kit further comprises a diluent, a washing solution, a blocking solution, an HRP-labeled goat anti-pig IgG antibody, a color developing solution and a stop solution.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The results obtained by screening antigenic epitopes using different bioinformatics tools are different, which is caused by the different algorithms used by each software. Although this method has the advantages of fast speed, easy operation and low cost for epitope screening, only one method for screening and identifying antigenic epitopes may have low accuracy and large error in the results, and it is impossible to obtain systematic and comprehensive antigenic epitopes, especially the acquisition of dominant antigenic epitopes in some proteins. Therefore, it is necessary to systematically and comprehensively predict and screen antigenic epitopes through multiple methods. In order to obtain ASFV antigenic epitopes with good immune effects for the study of ASF genetic engineering vaccines, the present invention takes the main structural protein p30 of the ASFVpig / HLJ / 2018 strain isolated in my country as the object, and uses bioinformatics technology combined with immunological methods to predict and screen ASFV antigenic epitopes and draw antigenic epitope maps of these proteins. According to the algorithm results of different software, the data obtained by comprehensive analysis were finally determined to be the antigenic epitopes of the p30 protein of African swine fever virus. The acquisition of these candidate antigen epitopes greatly saves the cost and time of traditional antigen epitope screening, and improves the efficiency of immunological methods in verifying antigen epitopes with good immune protection functions. It will also be of great significance for the development of new ASF vaccines and detection technologies.

[0020] 2. In order to overcome the shortcomings of small molecule characteristics and weak immune response of antigenic epitopes and improve the accuracy of antigenic epitope screening, the present invention uses gene manipulation technology to insert the antigenic epitope DNA sequence into the prokaryotic expression vector pGEX-4T-1 carrying the GST tag, construct a recombinant expression plasmid, and express and purify the GST-epitope recombinant protein. In addition, in order to increase the titer of the antigenic epitope, different antigenic epitopes are connected in series and displayed on the phage surface through phage display technology. The recombinant protein is purified by affinity chromatography, and the antigenic epitope is identified by the established indirect ELISA method. The results show that after a single peptide segment is introduced into the GST vector, it is compatible with the synthetic peptide OD 450nm In comparison, the immunoreactivity of single-epitope recombinant proteins has been significantly enhanced, and the immunoreactivity enhancement after the antigen epitope is fully antigenized is significantly better than that of a single synthetic peptide. After the multi-epitope protein gene is concatenated with the phage gene, the immunoreactivity of the multi-epitope recombinant protein is significantly better than that of a single antigen epitope after complete antigenization. This research strategy provides a theoretical basis and technical support for the efficient screening of antigen epitopes in the future, which is of great significance.

[0021] 3. By combining bioinformatics technology with immunology technology, we can quickly, accurately screen antigen epitopes with high throughput, and draw a map of protective antigen epitopes of African swine fever virus. This provides a material basis for designing recombinant African swine fever epitope antigens and developing vaccines based on identified protective antigen epitopes as elements and self-assembling protein molecules as skeletons, which is of great significance for the development of safe and effective African swine fever vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Predict the antigenic epitopes of p30 protein for IEDB software;

[0023] Figure 2 This is the identification result of the antigenic epitope (synthetic peptide) of the p30 protein of African swine fever virus;

[0024] Note: ****P<0.0001, ***P<0.0001, **P<0.01 indicate extremely significant differences; * indicates significant differences (P<0.05); P>0.05 indicates no significant differences;

[0025] Figure 3 This is the result of identifying the antigenic epitope (recombinant protein) of the African swine fever virus p30 protein;

[0026] Note: ****P<0.0001, ***P<0.0001, **P<0.01 indicate extremely significant differences; * indicates significant differences (P<0.05); P>0.05 indicates insignificant differences. DETAILED DESCRIPTION

[0027] The present invention is described in detail below through specific embodiments so that those skilled in the art can easily implement the present invention according to the disclosure of this specification. The embodiments described below are only preferred examples of the present invention and are not intended to limit the present invention in other forms. 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.

[0028] Example 1 Bioinformatics method predicts the epitope of ASFVp30 protein

[0029] 1 Materials

[0030] Acquisition of ASFV amino acid sequence: The amino acid sequence of p30 (QBH90581.1) in the ASFVpig / HLJ / 2018 strain (NCBI accession number: MK333180.1) was obtained through GenBank in NCBI. The specific amino acid sequence is as follows:

[0031] MDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIK TLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNK VIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLLKKK(SEQ ID NO.9)

[0032] 2 Methods

[0033] 2.1 Bioinformatics prediction of ASFVp30 protein epitopes

[0034] The antigenic epitopes of ASFVp30 protein were predicted using bioinformatics online tools. The bioinformatics prediction software used in the present invention is as follows:

[0035] (1)TMHMM( https: / / services.healthtech.dtu.dk / service.php?TMHMM-2.0 ) is an online software that predicts the transmembrane region of proteins based on the hidden Markov mathematical model. It analyzes the transmembrane region of proteins and identifies the amino acid sequence located in the non-transmembrane region as the candidate antigen epitope region.

[0036] (2)IEDB Analysis Resource http: / / tools.immuneepitope.org / main / ) is to predict antigen epitopes by neural network (ANN) and support vector machine (SMM) modeling, where the relevant immune epitopes are selected from scientific publications available in PubMed, and the parameters such as hydrophilicity, flexibility, accessibility, number of turns, exposed surface, polarity and antigenic tendency of the peptide are analyzed. Judgment criteria: score ≥ 0.5.

[0037] (3)ABCpred( https: / / webs.iiitd.edu.in / raghava / abcpred / ABC_ submission.html ) is a model based on artificial neural network (ANN), which is a database for predicting B cell antigen epitopes established by training epitope data from the Bcipep database. Judgment criteria: score ≥ 0.5.

[0038] 2.2 Determination of candidate antigenic epitopes of ASFVp30 protein

[0039] Based on a comprehensive analysis of the judgment criteria of each software, the common and optimal antigenic epitopes obtained in the three software were determined as the best candidate antigenic epitopes predicted by the present invention.

[0040] 2.3 Comparison and analysis of antigen epitopes reported in literature

[0041] The antigenic epitopes screened by the present invention were compared with the amino acid sequences of antigenic epitopes reported in the literature (Table 1) to determine the similarities and differences between the candidate antigenic epitopes predicted by the present invention using bioinformatics technology and the antigenic epitopes reported in the literature. The reliability, systematicness and integrity of the antigenic epitopes predicted and screened by bioinformatics technology were evaluated. Among them, p30BA1, p30BA2, p30BA3, p30BA4, p30BA5, p30BA6 and p30BA7 are truncated from 120aa-194aa in the ASFV BA71 strain reported in the literature, with each 15 amino acids being a segment.

[0042] Table 1 Amino acid sequences of antigenic epitopes reported in literature

[0043]

[0044]

[0045] 3 Results

[0046] 3.1 Bioinformatics prediction of antigenic epitopes of ASFVp30 protein

[0047] (1) TMHMM online tool predicts the transmembrane region structure of proteins: The transmembrane region of p30 protein was analyzed by TMHMM software. The analysis results showed that p30 protein had no transmembrane region.

[0048] (2) IEDB online tool predicts protein B cell linear antigen epitopes: set the threshold to no less than 0.5 (the yellow area indicates possible antigen epitopes). The results showed that p30 protein has 7 candidate B cell linear antigen epitopes located at 13aa-24aa, 31aa-35aa, 67aa-84aa, 92aa-117aa, 120aa-136aa, 146aa-162aa and 164aa-182aa ( Figure 1 ).

[0049] (3) ABCpred online tool predicts B cell antigen epitopes of proteins: the threshold is set to no less than 0.5, and the epitope length is set to 16 amino acids. 21 antigen epitopes were identified in ASFVp30 (Table 2), and the results are as follows:

[0050] Table 2 Antigenic epitopes of p30 protein predicted by ABCpred software

[0051]

[0052] 3.2 Determination of candidate antigenic epitopes of ASFVp30 protein

[0053] According to the scoring criteria of each prediction software: TMHMM software analyzes the transmembrane region of the protein, IEDB and ABCpred software predict epitopes, the higher the score of the amino acid sequence, the greater the possibility of becoming a candidate antigen epitope. The inventors comprehensively analyzed the results of the three prediction methods and finally determined the most likely antigen epitopes of the p30 protein, that is, 8 possible antigen epitopes of the p30 protein, which were named p30a, p30b, p30c, p30d, p30e, p30f, p30g, and p30I (Table 3).

[0054] Table 3 Candidate antigenic epitopes of ASFVp30 protein

[0055]

[0056]

[0057] 3.3 Comparison analysis results with antigen epitopes reported in literature

[0058] The amino acid sequences of antigenic epitopes screened by the above-mentioned bioinformatics software were compared with those reported in the literature. The results are shown in Table 4. One antigenic epitope was discovered for the first time in the present invention, namely p30b (30-GSLYNW-35). In addition, the study also obtained antigenic epitopes that were related to but different from those reported in the literature, including p30a (15-FKTDLRSSSQ-24), p30c (68-IYAGQGYTEHQAQEEWN-84), p30d (94-ETESSASSENIHEKND-109), p30e (121-TLFEQEPSSEVPKDSK-136), p30f (146-QHIEQYGKAPDFNKVIR-162), p30g (167-IQTIYGTPLKEEEKE-181) and p30I (112-TNECTS-117).

[0059] Table 4 Analysis of amino acid sequence differences between candidate antigenic epitopes of p30 protein and those reported in literature

[0060]

[0061]

[0062] 4 Summary

[0063] The present invention uses TMHMM software to predict the transmembrane region of p30 protein, and the results show that p30 has no transmembrane region. Using IEDB software, 7 candidate antigenic epitopes of the above-mentioned p30 structural protein were determined. The number of candidate antigenic epitopes of the above-mentioned protein determined by ABCpred software is more, that is, 21 p30 proteins. After comprehensive analysis of the above results, the present invention finally determined 8 antigenic epitopes of p30 protein, among which p30b (30-GSLYNW-35) is the antigenic epitope discovered for the first time by the present invention.

[0064] Example 2 Identification of ASFVp30 protein antigenic epitopes

[0065] 1 Materials

[0066] 1.1 Main experimental reagents and instruments

[0067] MK100 was purchased from Takara; African swine fever positive serum was purchased from the China Veterinary Drug Administration; serum diluent was purchased from Bio-Tech Biotechnology Co., Ltd.; TMB colorimetric solution was purchased from SurModics; 10×PBS buffer solution and 10×PBST buffer solution were purchased from Beijing Solebow Technology Co., Ltd.; HRP-labeled goat anti-swine IgG antibody was purchased from Sigma; Carbonate-Bicarbonate Buffer was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; and the microplate reader was purchased from Thermo.

[0068] 1.2 Preparation of main reagents for the experiment

[0069] (1) 5% BSA: 5 g BSA was added to 1× PBST and the volume was adjusted to 100 mL.

[0070] (2) CBS: one capsule dissolved in 100 mL ddH 2 In O.

[0071] (3) 1× PBS buffer solution: 100 mL of 10× PBS buffer solution, add deionized water to make up to 1 L.

[0072] (4) 1× PBST buffer solution: 100 mL 10× PBST buffer solution, add deionized water to make up to 1 L.

[0073] (5) Blocking solution: 5 g skim milk powder, add 1× PBST to make up to 100 mL.

[0074] 2 Methods

[0075] 2.1 Synthesis of candidate antigenic epitopes of ASFVp30 protein

[0076] Based on the results predicted by the bioinformatics software TMHMM, IEDB and ABCpred in Example 1, the 8 epitopes finally identified in the p30 protein were commissioned to a biological reagent company to synthesize peptides with a purity of more than 95%.

[0077] 2.2 Optimization of the indirect ELISA method for screening antigen epitopes

[0078] The optimal ELISA reaction conditions were determined by the chessboard titration method. Three synthetic peptides from the p30 protein epitope peptide were randomly selected as coating antigens. The positive serum infected with African swine fever was used as the primary antibody (the antibody dilution concentrations were 1:50, 1:100 and 1:200, respectively). The HRP-labeled goat anti-swine IgG antibody was used as the secondary antibody. The indirect ELISA method was established and optimized to determine the optimal antibody concentration. The specific operation steps were completed with reference to the Takara Peptide Coating Kit manual, as follows:

[0079] (1) Use the diluent in the kit to dilute the synthesized antigen epitope (peptide) to a concentration of 4 μg / ml, and coat them in the 96-well reaction plate (Reaction plate) provided in the kit, with 50 μL per well.

[0080] (2) Immediately add 10 μL of coupling reagent to each antigen-coated well and incubate at room temperature in the dark for 2 h.

[0081] (3) Discard the liquid, wash five times with deionized water, gently shake each time, and pat dry.

[0082] (4) Blocking: Use the Blocking buffer in the kit, add 200 μL to each well, and block at room temperature in the dark for 1 h.

[0083] (5) Discard the liquid, wash five times with deionized water, shake gently after each wash, and pat dry.

[0084] (6) Incubation with primary antibody: Add serum diluent to dilute ASFV-infected positive serum to 1:50, 1:100, and 1:200 (100 μL / well), incubate at 37°C in the dark for 1 h, and set up a negative control (the serum of the negative control is serum that is not infected with ASFV).

[0085] (7) Discard the solution and wash the tube five times with 1× PBST, gently shaking each time and patting dry.

[0086] (8) Incubation with primary antibody: Add 1:10,000 (recommended concentration of antibody) HRP-labeled goat anti-swine IgG antibody (100 μL / well) to each well, except for the blank control, and incubate at 37°C in the dark for 1 h.

[0087] (9) Discard the solution and wash the tube five times with 1× PBST, gently shaking each time and patting dry.

[0088] (10) Color development: Add TMB substrate solution (100 μL / well) and develop color at 37°C in the dark for 15 min.

[0089] (11) Determination: Add 2M dilute sulfuric acid to terminate the reaction (100 μL / well), mix gently, and measure the OD using an ELISA reader. 450 Absorbance value.

[0090] 2.3 Indirect ELISA screening of antigen epitopes (synthetic peptides)

[0091] Use the diluent in the Takara Peptide Coating Kit to dilute all the synthesized peptides to 4 μg / ml as coating antigens and coat them in 96-well reaction plates. Set up 3 replicates for each sample and set up a negative control. The incubation concentrations of ASFV-infected positive serum in the p30 peptide were 1:100, 1:100, 1:100 and 1:50, respectively. The HRP-labeled goat anti-swine IgG antibody was 1:10000. For other specific operation steps, refer to 2.2.

[0092] 3 Results

[0093] 3.1 Optimization results of the indirect ELISA method

[0094] The optimal antibody concentration for ELISA was determined according to the results of the chessboard titration method (Table 5). The optimal antibody concentration was determined to be the one with the largest positive serum / negative serum ratio. After comprehensive analysis, the optimal antibody concentrations for the p30 protein peptide were determined to be 1:100, 1:100, 1:100 and 1:50, respectively.

[0095] Table 5 Optimization of the optimal concentration of indirect LEISA positive and negative serum

[0096]

[0097] 3.2 Results of indirect ELISA method for screening antigen epitopes

[0098] The positive serum of African swine fever virus was used as the standard to screen antibodies, and the antigenic epitopes of ASFVp30 protein predicted by bioinformatics online tools were screened and identified by ELISA method. Figure 2As shown in the figure, the predicted antigenic epitopes of the p30 protein of the African swine fever virus were identified using the positive serum of the African swine fever virus using the synthetic peptide indirect ELISA established in the laboratory. The results showed that all the predicted antigenic epitopes (Table 3) could react specifically with the positive serum of the African swine fever virus, and the difference was significant compared with the negative control serum (p<0.001). The results fully showed that the antigenic epitopes in Table 3 were all p30 protein epitopes. The first p30 protein epitope discovered using bioinformatics technology combined with immunological methods was p30b (30-GSLYNW-35).

[0099] Example 3 Expression of GST-single epitope and multi-epitope recombinant proteins and identification of antigenic epitopes

[0100] 1 Materials

[0101] 1.1 Main experimental reagents and instruments

[0102] High-affinity GST purification medium and HighAffinity Ni-Charged Resin FF were purchased from Nanjing GenScript Biotechnology Co., Ltd.; reduced glutathione was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; E. coli BL21 (DE3) competent cells, ampicillin, IPTG and kanamycin sulfate were purchased from Shanghai Sangon Biotechnology Co., Ltd.; Rosetta competent cells were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; 12 mL and 30 mL chromatography columns were purchased from Beijing Solebold Technology Co., Ltd.; 96-well plates were purchased from Corning (USA) Incorporated.

[0103] Vertical electric pressure sterilizer (Shanghai Shen'an Medical Equipment Factory), ice machine (Scotsman, USA), electric constant temperature water tank (DK-8D, Shanghai Jinghong Experimental Equipment Co., Ltd.), large centrifuge (Thermo, USA), PCR instrument, electrophoresis instrument, high-resolution gel imager (Bio-Rad, USA), pH meter (Sartorius, Germany), clean bench (Suzhou Antai Air Technology Co., Ltd.), horizontal electrophoresis tank (Beijing Liuyi Biotechnology Co., Ltd.)

[0104] 1.2 Preparation of main reagents for the experiment

[0105] (1) LB liquid medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride (NaCl), add deionized water, adjust to pH 7.0, make up to 1 L, sterilize by high pressure at 121°C, and store at 4°C after cooling.

[0106] (2) 100 mg / mL ampicillin solution: 5 g ampicillin powder was added with autoclaved deionized water to make up to 50 mL, sterilized by filtering with a 0.22 μm filter, divided into aliquots, and stored at -20°C.

[0107] (3) 100 mg / mL kanapencillin solution: 5 g of kanapencillin powder was added with autoclaved deionized water to make up to 50 mL, sterilized by filtering with a 0.22 μm filter, divided into aliquots, and stored at -20°C.

[0108] (4) 8× Binding Buffer: 4 M NaCl, 160 mM Tris-HCl, 40 mM imidazole, add 800 mL of deionized water, adjust to pH 7.9, and make up to 1 L.

[0109] (5) 1× Binding Buffer: 75 mL 8× Binding Buffer, add deionized water to make up to 600 mL.

[0110] (6) 5× SDS-PAGE electrophoresis buffer solution: 15.1 g Tris-HCL, 5.0 g SDS, 94.0 g glycine, add deionized water to make up to 1 L.

[0111] (7) 1× SDS-PAGE electrophoresis buffer solution: 200 mL 5× SDS-PAGE electrophoresis buffer solution, add deionized water to make up to 1 L.

[0112] (8) 10% SDS solution: 10 g SDS, add deionized water to make up to 100 mL.

[0113] (9) 10% ammonium persulfate: 1 g ammonium persulfate was added to deionized water to make up to 10 mL.

[0114] (10) Coomassie Brilliant Blue R-250 staining solution: 1 g Coomassie Brilliant Blue R-250 powder, 250 mL isopropanol, 100 mL glacial acetic acid, add deionized water to make up to 1 L.

[0115] (11) Coomassie Brilliant Blue decolorizing solution: 100 mL of glacial acetic acid, 50 mL of anhydrous ethanol, add deionized water to make up to 1 L.

[0116] (12) 10× Glutathione reconstition buffer: Add 500 mmol Tris-HCl to 800 mL of deionized water, adjust the pH to 8.0, and then add deionized water to make up to 1 L.

[0117] (13) 10×GST Elution Buffer: 1 g of reduced glutathione was dissolved in 32.5 mL of 10×Glutathione Reconstition Buffer.

[0118] (14) 1×GST Elution Buffer: Dilute 10×GST Elution Buffer to 1×GST Elution Buffer and prepare it before use.

[0119] 2 Methods

[0120] 2.1 Construction of pGEX-4T-1 / antigen epitope recombinant expression plasmid, expression and purification of recombinant protein

[0121] 2.1.1 Construction of pGEX-4T-1 / antigen epitope recombinant expression plasmid

[0122] The antigenic epitopes in the candidate ASFVp30 protein predicted by bioinformatics and the 5' and 3' ends of the antigenic epitope base sequences reported in the literature were introduced into the specific restriction sites BamHⅠ / NotⅠ as the target gene, and then inserted into the prokaryotic expression vector pGEX-4T-1 linearized with BamHⅠ / NotⅠ to construct the recombinant plasmid. All recombinant plasmids were commissioned to be synthesized by a biological company. The sequencing results showed a homology of 100%.

[0123] 2.1.2 Expression and purification of antigen epitope recombinant protein

[0124] The specific operation method is as follows:

[0125] (1) The positive recombinant plasmids were transformed into E. coli BL21 (DE3) competent cells by heat shock method, and the positive recombinant bacteria were screened on LB (Amp+) culture plates.

[0126] (2) Select a single positive colony and inoculate it into an appropriate amount of LB (Amp+) liquid culture medium and culture it at 37°C and 220 rpm overnight.

[0127] (3) Inoculate the cells into freshly prepared ampicillin-resistant liquid LB medium at a ratio of 1:100, culture at 37°C, 220 rpm for 4 h, aspirate 1 mL, add IPTG (final concentration 1 mmol / mL), induce at 37°C, 220 rpm for 4 h, harvest the cells by centrifugation at 4°C for 10 min (3000 g), and discard the supernatant.

[0128] (4) Resuspend the pellet with cold 1× PBS (3 mL of cold 1× PBS is required for every 50 mL of culture medium), harvest the pellet by centrifugation at 4°C for 10 min (3000 g), and discard the supernatant.

[0129] (5) Resuspend the pellet in 1× PBS and disrupt the cells by ultrasonication on ice. Centrifuge, collect the supernatant, and resuspend the pellet in the same cold 1× PBS.

[0130] (6) Equal amounts of soluble and insoluble components were taken separately, and the expression level and form of GST-fusion protein were analyzed by SDS-PAGE.

[0131] (7) Add an appropriate amount of high-affinity GST purification medium to the chromatography column and wash with 10 column volumes of cold 1× PBS.

[0132] (8) Add the clarified liquid containing the GST fusion protein to the chromatography column and allow to bind at 4°C for about 1-2 h. After all the liquid has flowed out of the chromatography column, immediately add cold 1× PBS (20 column volumes) to wash the column.

[0133] (9) Elute the fusion protein with freshly prepared 1× GST Elution Buffer (5-10 column volumes) and analyze the eluted protein by SDS-PAGE electrophoresis.

[0134] 2.2 Construction, expression and purification of phage display multi-epitope recombinant expression plasmid

[0135] 2.2.1 Construction of phage display multi-epitope recombinant expression plasmid

[0136] The screened peptides were sequentially connected in series. To ensure the independence of the antigenic epitopes, the spacer sequence GGGS was introduced between adjacent antigenic epitopes. Then, they and the phage AP205 gene were cloned into the NcoⅠ / XhoⅠ linearized prokaryotic expression vector pET-28a(+) to construct a phage-displayed multi-epitope recombinant expression plasmid. The multi-epitope recombinant protein was expressed by Escherichia coli and named as shown in Table 6. All antigenic epitope recombinant expression plasmids were synthesized and identified by biological companies.

[0137] Table 6 Antigen epitope tandem

[0138]

[0139] 2.2.2 Expression and purification of phage-displayed multi-epitope recombinant proteins

[0140] The specific operation method is as follows:

[0141] (1) The positive recombinant plasmids were transformed into E. coli Rosetta competent cells using the heat shock method, and the positive recombinant bacteria were screened on LB (Kan+) culture plates.

[0142] (2) Select a single positive colony and inoculate it into an appropriate amount of LB (Kan+) liquid culture medium and culture it at 37°C, 220 rpm overnight.

[0143] (3) Inoculate the cells into freshly prepared kanamycin-resistant liquid LB medium at a ratio of 1:100, culture at 37°C, 220 rpm for 4 h, aspirate 1 mL, add IPTG (final concentration 1 mmol / mL), induce at 37°C, 220 rpm for 4 h, harvest the cells by centrifugation at 4°C for 10 min (3000 g), and discard the supernatant.

[0144] (4)(5)(6)Same as 2.1.2.

[0145] (7) Add an appropriate amount of high-affinity His purification medium to the chromatography column and wash with 10 times the column volume of 1×Binding.

[0146] (8) Add the clarified liquid containing the His fusion protein to the chromatography column and bind at 4°C for about 1-2 h. After all the liquid flows out of the chromatography column, immediately add 1× Binding to wash, and then wash the column with Binding Buffer containing different concentrations of imidazole, namely 20 mM, 30 mM, 40 mM and 50 mM.

[0147] (9) Finally, the fusion protein was eluted with Binding Buffer containing 500 mM imidazole and analyzed by SDS-PAGE electrophoresis.

[0148] .2.3 Indirect ELISA to identify antigen epitopes (antigen epitope recombinant protein)

[0149] The specific operation method is as follows:

[0150] (1) Coating: Dilute the purified GST-epitope recombinant protein antigen with carbonate buffer (pH 9.6) to a concentration of 1 μg / ml, coat a 96-well reaction plate with 100 μL per well, and replicate 3 wells for each sample at 4°C overnight.

[0151] (2) Washing: Discard the liquid, add 1× PBST to each well for washing, shake gently on a shaker for 5 times, discard the liquid and pat dry.

[0152] (3) Blocking: Add 100 μL of 5% BSA diluted in 1× PBST to each well and incubate at 37°C in the dark for 2 h.

[0153] (4) Washing: Same as (2).

[0154] (5) Incubation of ASFV-positive serum: Add 100 μL (serum diluted to 1 / 100) to each well, set up a negative control (serum not infected with ASFV), and incubate at 37°C for 1 h.

[0155] (6) Washing: Same as (2).

[0156] (7) Incubation with HRP-labeled goat anti-swine IgG antibody: Add serum diluent to each well to dilute to 1 / 10000 (100 μL / well) and incubate at 37°C for 30 min.

[0157] (8) Washing: Same as (2).

[0158] (9) Color development: Add 100 μL of TMB substrate solution to each well and incubate at 37°C in the dark for 15 min.

[0159] (10) Termination: Add 100 μL of 2M sulfuric acid to each well to terminate the reaction. After gently shaking to mix, measure the OD 450 Absorbance value.

[0160] 2.4 Data Analysis

[0161] The ttest test in the statistical software GraphPadPrism 7 was used to analyze the data obtained in the experiment for significant differences (****P<0.0001, ***P<0.001, **P<0.01 indicate extremely significant differences; *P<0.05 indicates significant differences; P>0.05 indicates insignificant differences).

[0162] 3 Results

[0163] 3.1 Expression and purification of GST-antigen epitope recombinant protein

[0164] SDS-PAGE results showed that all antigen epitope proteins of p30 protein were expressed in Escherichia coli, and all proteins were expressed in soluble form. The size was consistent with the expected results, all of which were about 25kDa. Affinity chromatography column purification was used to obtain serial GST-antigen epitope recombinant proteins.

[0165] 3.3 Identification results of antigenic epitopes

[0166] ELISA results showed that Figure 3 As shown, the results showed that the antigen epitope could react immunely with African swine fever virus positive serum, especially after the antigen epitope was concatenated, the immune reactivity was significantly enhanced.

[0167] 4 Summary

[0168] The present invention not only improves the immune activity of antigenic epitopes, but also improves the probability of identifying immune active antigenic epitopes through the strategy of complete epitope antigenization and phage display technology, and is an extremely effective strategy for identifying immune active antigenic epitopes; the immune active antigenic epitopes of ASFVp30 protein are further determined by this method, wherein the p30 protein has 3 minimum core motifs.

[0169] The uses of the obtained p30 protein antigen epitopes include detection reagents and kits for African swine fever virus infection serology (ELISA, WB, colloidal gold chromatography test strips, etc.), the development of African swine fever epitope vaccines and any vaccines related to the epitopes (epitope monovalent / multivalent vaccines, epitope-based synthetic peptide vaccines, and other pathogenic antigen epitopes or antigens in series research diagnostic kit vaccines, etc.), antigen epitope monoclonal antibody preparation, etc. The successful acquisition of these antigen epitopes is of great significance to the serological detection of African swine fever virus, the development of detection kits, the evaluation of immune effects and the development of vaccines. Sequence Listing <110> Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences <120> African swine fever virus p30 antigen epitope protein and preparation method and use thereof <130> klpi220216 <141> 2022-05-04 <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> African swine fever virus <400> 1 Phe Lys Thr Asp Leu Arg Ser Ser Ser Gln 1 5 10 <210> 2 <211> 6 <212> PRT <213> African swine fever virus <400> 2 Gly Ser Leu Tyr Asn Trp 1 5 <210> 3 <211> 17 <212> PRT <213> African swine fever virus <400> 3 Ile Tyr Ala Gly Gln Gly Tyr Thr Glu His Gln Ala Gln Glu Glu Trp 1 5 10 15 Asn <210> 4 <211> 16 <212> PRT <213> African swine fever virus <400> 4 Glu Thr Glu Ser Ser Ala Ser Ser Glu Asn Ile His Glu Lys Asn Asp 1 5 10 15 <210> 5 <211> 16 <212> PRT <213> African swine fever virus <400> 5 Thr Leu Phe Glu Gln Glu Pro Ser Ser Glu Val Pro Lys Asp Ser Lys 1 5 10 15 <210> 6 <211> 17 <212> PRT <213> African swine fever virus <400> 6 Gln His Ile Glu Gln Tyr Gly Lys Ala Pro Asp Phe Asn Lys Val Ile 1 5 10 15 Arg <210> 7 <211> 15 <212> PRT <213> African swine fever virus <400> 7 Ile Gln Thr Ile Tyr Gly Thr Pro Leu Lys Glu Glu Glu Lys Glu 1 5 10 15 <210> 8 <211> 6 <212> PRT <213> African swine fever virus <400> 8 Thr Asn Glu Cys Thr Ser 1 5 <210> 9 <211> 194 <212> PRT <213> African swine fever virus <400> 9 Met Asp Phe Ile Leu Asn Ile Ser Met Lys Met Glu Val Ile Phe Lys 1 5 10 15 Thr Asp Leu Arg Ser Ser Ser Gln Val Val Phe His Ala Gly Ser Leu 20 25 30 Tyr Asn Trp Phe Ser Val Glu Ile Ile Asn Ser Gly Arg Ile Val Thr 35 40 45 Thr Ala Ile Lys Thr Leu Leu Ser Thr Val Lys Tyr Asp Ile Val Lys 50 55 60 Ser Ala Arg Ile Tyr Ala Gly Gln Gly Tyr Thr Glu His Gln Ala Gln 65 70 75 80 Glu Glu Trp Asn Met Ile Leu His Val Leu Phe Glu Glu Glu Thr Glu 85 90 95 Ser Ser Ala Ser Ser Glu Asn Ile His Glu Lys Asn Asp Asn Glu Thr 100 105 110 Asn Glu Cys Thr Ser Ser Phe Glu Thr Leu Phe Glu Gln Glu Pro Ser 115 120 125 Ser Glu Val Pro Lys Asp Ser Lys Leu Tyr Met Leu Ala Gln Lys Thr 130 135 140 Val Gln His Ile Glu Gln Tyr Gly Lys Ala Pro Asp Phe Asn Lys Val 145 150 155 160 Ile Arg Ala His Asn Phe Ile Gln Thr Ile Tyr Gly Thr Pro Leu Lys 165 170 175 Glu Glu Glu Lys Glu Val Val Arg Leu Met Val Ile Lys Leu Leu Lys 180 185 190 Lys Lys

Claims

1. African swine fever virus p30 antigen epitope protein, characterized in that: The p30 antigen epitope protein is p30b, and the amino acid sequence of p30b is shown in SEQ ID NO.

2.

2. The African swine fever virus p30 antigen epitope protein according to claim 1, characterized in that One end of the p30 antigen epitope protein is connected to a GST tag.

3. Use of the African swine fever virus p30 antigen epitope protein according to claim 1 or 2 in the preparation of an antibody reagent for detecting African swine fever virus.

4. The use according to claim 3, characterized in that The reagent is an ELISA detection reagent.

5. An indirect ELISA detection kit for African swine fever virus antibodies, characterized in that: The kit contains the African swine fever virus p30 antigen epitope protein according to claim 1 or 2.

6. The indirect ELISA detection kit for African swine fever virus antibodies according to claim 5, characterized in that: The kit also contains diluent, washing solution, blocking solution, HRP-labeled goat anti-pig IgG antibody, color developing solution and stop solution.

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