CN114940705A - African swine fever virus p30 antigen epitope protein and preparation method and application thereof - Google Patents
African swine fever virus p30 antigen epitope protein and preparation method and application thereof Download PDFInfo
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- CN114940705A CN114940705A CN202210482695.6A CN202210482695A CN114940705A CN 114940705 A CN114940705 A CN 114940705A CN 202210482695 A CN202210482695 A CN 202210482695A CN 114940705 A CN114940705 A CN 114940705A
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Abstract
The invention discloses an African swine fever virus p30 epitope protein and a preparation method and application thereof. The p30 epitope protein is selected from any one of p30a, p30b, p30c, p30d, p30e, p30f, p30g and p30I epitope proteins, or is a recombinant multi-epitope protein obtained by connecting any more than two of p30a, p30b, p30c, p30d, p30e, p30f, p30g and p30I epitope proteins in series. The invention takes main structural protein p30 of ASFV Viig/HLJ/2018 strain separated in China as an object, predicts and screens the epitope of ASFV by using bioinformatics technology and combining an immunological method, draws the epitope map of the proteins and finally determines 8 epitopes (peptides) as the epitope of African swine fever virus p30 protein. The acquisition of the candidate antigen epitopes greatly saves the cost and time of traditional antigen epitope screening, improves the efficiency of verifying the antigen epitopes with good immune protection function by an immunological method, and has very important significance for developing ASF novel vaccines and detection technologies.
Description
Technical Field
The invention relates to an epitope protein, a preparation method and application thereof, in particular to an African swine fever virus p30 epitope protein, a preparation method and application thereof. The invention belongs to the technical field of biological medicines.
Background
African Swine Fever (ASF) is an acute virulent infectious disease caused by African Swine Fever Virus (ASFV) infected pigs, and the mortality rate is up to 100%. The world animal health Organization (OIE) classifies the animal infectious diseases as animal infectious diseases which need to be notified, and China classifies the animal infectious diseases as important precaution.
The research on the vaccine of the African swine fever virus as an important animal virus with hundred years of history is not interrupted, but the vaccine which can be used for prevention and control practice, is safe, effective and can be identified and diagnosed is still lacked up to now.
Aiming at the African swine fever virus and the complex and unknown protective antigen/epitope spectrum, which becomes a key scientific problem restricting the research of genetic engineering vaccines, the invention develops the research and research of protective antigen/epitope of the African swine fever virus, so as to solve the dilemma that the map of the protective antigen/epitope of the African swine fever virus is unknown and the research of the genetic engineering vaccines lacks key materials, and provide material basis and technical support for researching and developing the African swine fever genetic engineering vaccines.
The invention takes the primary structure amino acid sequence of p30(CP204L) which is one of African swine fever virus membrane proteins as a material, analyzes and predicts the epitope of the antigen through an epitope database, and synthesizes the corresponding peptide according to the amino acid sequence. The African swine fever virus infected swine positive serum is used for screening specific peptides (antigen epitopes) which can generate specific immune reaction with the African swine fever virus infected swine positive serum but not generate immune reaction with the swine fever positive serum, and finally 8 antigen epitopes (peptides) are determined to be the antigen epitopes of the African swine fever virus p30 protein. The acquisition of the antigen epitopes lays a foundation for researching and developing African swine fever detection reagents and kits thereof, monoclonal antibody preparation and products which are safe and can identify and diagnose epitope vaccines and the like which need strategic prevention and control urgently by taking the antigen epitopes as materials. Not only will produce huge economic benefits, but also have important social benefits.
Disclosure of Invention
The invention aims to provide an African swine fever virus p30 antigen epitope protein, and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical means:
the invention utilizes TMHMM software to predict the transmembrane domain of the ASFVp30 protein, the software is a transmembrane domain for predicting the protein established based on a hidden Markov mathematical model, and the accuracy rate is up to 97-98% (Krogh et al, 2001). The results show that there is no transmembrane domain in the p30 protein. Then, the IEDBanalysis Resource and ABCPred software is used for predicting the B cell linear epitope of the ASFVp30 protein, and the candidate epitope is set to be the threshold value not less than 0.5. IEDBAnalysis Resource predicts epitopes by neural network (ANN) and support vector machine (SMM) modeling, where the relevant immune epitopes are selected from scientific published data available in PubMed, and the parameters of hydrophilicity, flexibility, accessibility, turning angle, exposed surface, polarity and antigenic propensity of the polypeptide are analyzed (Jespersen et al,2017), and 7 epitopes are identified on the ASFV p30 protein. ABCpred is a model based on an Artificial Neural Network (ANN), and 21 epitopes are determined on ASFVp30 protein by creating predicted B-cell epitope database software (Saha et al,2007) by training epitope data of a Bcipep database. Then, the invention determines the antigen epitope with immunological activity by establishing an indirect ELISA method according to the result of immunoreaction between the antigen epitope (synthetic peptide) and ASFV infection positive serum. According to ELISA results, 8 immunologically active p30 protein epitopes are obtained by co-screening from the predicted epitopes of the p30 protein. It is worth mentioning that a new epitope p30b (30-GSLYNW-35) of ASFVp30 protein is discovered by bioinformatics technology and immunological method. In order to overcome the defects of small molecular characteristics and weak immunoreaction of the epitope and improve the accuracy of epitope screening, the invention utilizes a gene operation technology to insert an epitope DNA sequence into a prokaryotic expression vector pGEX-4T-1 carrying a GST label, construct a recombinant expression plasmid, express and purify GST-epitopeA recombinant protein. In addition, in order to improve the titer of the antigen epitope, different antigen epitopes are displayed on the surface of the phage by the phage display technology after being connected in series. The recombinant protein is purified by affinity chromatography, and the epitope is identified by the established indirect ELISA method. The results show that the single peptide fragment is introduced into GST carrier and then is combined with synthetic peptide OD 450nm Compared with the single synthetic peptide, the immunoreactivity of the single epitope recombinant protein is obviously enhanced, and the immunoreactivity enhancement after complete antigenication of the epitope is obviously superior to that of the single synthetic peptide. After the multi-epitope protein gene and the phage gene are connected in series, the immunoreactivity of the multi-epitope recombinant protein is obviously superior to that of single-antigen epitope complete antigenication.
On the basis of the research, the invention provides an African swine fever virus p30 epitope protein, which is characterized in that the p30 epitope protein is selected from any one of p30a, p30b, p30c, p30d, p30e, p30f, p30g and p30I epitope proteins or a multi-epitope recombinant protein obtained by connecting any two or more of p30a, p30b, p30c, p30d, p30e, p30f, p30g and p30I epitope proteins in series, and the amino acid sequences of the p30a, p30b, p30c, p30d, p30e, p30f, p30g and p30I epitope proteins are respectively shown as SEQ ID NO. 1-8.
Preferably, one end of the p30 epitope protein is linked to the GST tag.
Preferably, the p30 epitope protein is a GST-multi-epitope recombinant protein obtained by connecting p30c, p30d and p30e epitope proteins in series, introducing a spacer sequence GGGS between adjacent antigen epitopes, sequentially cloning a coding gene sequence and a phage AP205 gene to a linearized prokaryotic expression vector pET-28a (+) respectively, constructing a phage display multi-epitope recombinant expression plasmid, and expressing the recombinant plasmid through escherichia coli.
Preferably, the p30 epitope protein is a GST-multi-epitope recombinant protein obtained by connecting p30g and p30I epitope proteins in series, introducing a spacer sequence GGGS between adjacent antigen epitopes, sequentially cloning a coding gene sequence and a phage AP205 gene to a linearized prokaryotic expression vector pET-28a (+) respectively, constructing a phage display multi-epitope recombinant expression plasmid, and expressing the recombinant expression plasmid through escherichia coli.
Furthermore, the invention also provides application of the African swine fever virus p30 epitope protein in preparation of an antibody reagent for detecting the African swine fever virus.
Preferably, the reagent is an ELISA detection reagent.
Furthermore, the invention also provides application of the African swine fever virus p30 epitope protein or the recombinant protein thereof in designing and preparing an African swine fever vaccine.
Furthermore, the invention also provides an indirect ELISA detection kit for the African swine fever virus antibody, and the kit contains the African swine fever virus p30 epitope protein.
Preferably, the kit further comprises a diluent, a washing solution, a blocking solution, an HRP-labeled goat anti-pig IgG antibody, a developing solution and a stopping solution.
Compared with the prior art, the invention has the beneficial effects that:
1. the results obtained from screening epitopes with different bioinformatic tools are different due to the different algorithms used for each software. Although the method has the advantages of high speed, easy operation, low cost and the like for screening the epitope, the method for screening and identifying the epitope by only adopting one method has the defects of low accuracy, large result error and incapability of obtaining systematic and comprehensive epitope, particularly the dominant epitope in some proteins, so that the epitope needs to be systematically and comprehensively predicted and screened by various methods. In order to obtain ASFV epitope with good immune effect for research of ASF genetic engineering vaccine, the invention takes main structural protein p30 of ASFVPig/HLJ/2018 strain separated in China as an object, predicts and screens ASFV epitope by using bioinformatics technology and immunological method, and draws the epitope map of the proteins. According to the algorithm results of different software, data are obtained by comprehensive analysis, and finally 8 antigen epitopes (peptides) are determined to be the antigen epitopes of the African swine fever virus p30 protein. The acquisition of the candidate epitope greatly saves the cost and time of traditional epitope screening and improves the efficiency of the immunological method for verifying the epitope with good immune protection function. Also has important significance for developing novel ASF vaccines and detection technologies.
2. In order to overcome the defects of small molecular characteristics and weak immunoreaction of the epitope and improve the accuracy of epitope screening, the invention utilizes a gene operation technology to insert an epitope DNA sequence into a prokaryotic expression vector pGEX-4T-1 carrying a GST label, construct a recombinant expression plasmid, and express and purify GST-epitope recombinant protein. In addition, in order to improve the titer of the antigen epitope, different antigen epitopes are displayed on the surface of the phage by the phage display technology after being connected in series. The recombinant protein is purified by affinity chromatography, and the epitope is identified by the established indirect ELISA method. The results show that the single peptide fragment is introduced into GST carrier and then is combined with synthetic peptide OD 450nm Compared with the single synthetic peptide, the immunoreactivity of the single epitope recombinant protein is obviously enhanced, and the immunoreactivity enhancement after complete antigenication of the epitope is obviously superior to that of the single synthetic peptide. After the multi-epitope protein gene and the phage gene are connected in series, the immunoreactivity of the multi-epitope recombinant protein is obviously superior to that of single-antigen epitope complete antigenication. The research strategy provides theoretical basis and technical support for efficient screening of the epitope in the future, and has very important significance.
3. By combining the bioinformatics technology with the immunology technology, the epitope can be rapidly and accurately screened in high flux, the African swine fever virus protective epitope map is drawn, a material basis is provided for designing and recombining the African swine fever epitope antigen and researching and developing the vaccine by taking the identified protective epitope as an element and the self-assembled protein molecule as a framework, and the method has very important significance for developing safe and effective African swine fever vaccines.
Drawings
FIG. 1 is IEDB software predicting the epitope of p30 protein;
FIG. 2 shows the result of identifying the epitope (synthetic peptide) of P30 protein of African swine fever virus;
note that: p <0.0001, indicating that the difference was extremely significant; indicates significant difference (P < 0.05); p >0.05 was not significantly different;
FIG. 3 shows the result of the epitope (recombinant protein) identification of P30 protein of African swine fever virus;
note that: p <0.0001, P <0.0001 indicates that the difference was very significant; indicates significant difference (P < 0.05); p >0.05 differences were not significant.
Detailed Description
The present invention is described in detail below with reference to specific examples so that those skilled in the art can easily practice the present invention in light of the disclosure of the present specification. The embodiments described below are merely preferred examples of the present invention, and are not intended to limit the present invention in any way. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 bioinformatics approach to predicting epitopes of ASFVp30 protein
1 Material
Obtaining of an ASFV amino acid sequence: the amino acid sequence of p30(QBH90581.1) in the ASFVpig/HLJ/2018 strain (NCBI accession number: MK333180.1) is obtained through GenBank in NCBI, and the specific amino acid sequence is as follows:
MDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIK TLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHE KNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNK VIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLLKKK(SEQ ID NO.9)
2 method
2.1 bioinformatics prediction of epitope of ASFVp30 protein
The method utilizes a bioinformatics online tool to predict the epitope of the ASFVp30 protein, and the bioinformatics prediction software adopted by the method is as follows:
(1)TMHMM(https://services.healthtech.dtu.dk/service.phpTMHMM-2.0) Is an on-line software for predicting the transmembrane region of protein based on hidden Markov mathematical model, and analyzes the transmembrane region of proteinThe amino acid sequence of the transmembrane region is identified as a candidate epitope region.
(2)IEDB Analysis Resource(http://tools.immuneepitope.org/main/) Antigenic epitopes are predicted by neural network (ANN) and support vector machine (SMM) modeling, wherein relevant immune epitopes are selected from scientific publication data available in PubMed, and parameters of hydrophilicity, flexibility, accessibility, number of turns, exposed surface, polarity, and antigenic propensity of the polypeptide are analyzed. And (4) judging the standard: the score is more than or equal to 0.5.
(3)ABCpred(https://webs.iiitd.edu.in/raghava/abcpred/ABC_ submission.html) The prediction B cell epitope database is established by training epitope data of a Bcipep database based on an Artificial Neural Network (ANN) model. And (4) judging the standard: the score is more than or equal to 0.5.
2.2 determination of candidate epitopes of ASFVp30 protein
And comprehensively analyzing according to the judgment standard of each software, and determining the common and optimal epitope obtained from the three software as the optimal candidate epitope predicted by the invention.
2.3 epitope comparison with literature reports
The epitope screened by the invention is compared and analyzed with the epitope amino acid sequences (table 1) reported in the literature to determine the similarity and difference between the candidate epitope predicted by the bioinformatics technology and the epitope reported in the literature. And evaluating the reliability, systematicness and integrity of the epitope predicted and screened by the bioinformatics technology. Wherein p30BA1, p30BA2, p30BA3, p30BA4, p30BA5, p30BA6 and p30BA7 are truncated by 120aa-194aa in ASFV BA71 strain reported in literature, and 15 amino acids are one fragment.
Table 1 reports the epitope amino acid sequences
3 results
3.1 bioinformatics prediction of epitope of ASFVp30 protein
(1) The TMHMM online tool predicts the transmembrane domain structure of proteins: the transmembrane region of the p30 protein is analyzed by TMHMM software, and the analysis result shows that the p30 protein has no transmembrane region.
(2) IEDB online tool predicts B-cell linear epitopes of proteins: the threshold was set to not less than 0.5 (yellow areas indicate possible epitopes). The results showed that the p30 protein has 7 candidate B cell linear epitopes located at 13aa-24aa, 31aa-35aa, 67aa-84aa, 92aa-117aa, 120aa-136aa, 146aa-162aa and 164aa-182aa, respectively (FIG. 1).
(3) The ABCpred online tool predicts B cell epitopes of proteins: the threshold was set to not less than 0.5 and the epitope length was set to 16 amino acids. 21 epitopes were identified in ASFVp30 (table 2), respectively, with the following results:
TABLE 2 ABCPred software predicts the epitope of p30 protein
3.2 determination of candidate epitopes of ASFVp30 protein
According to each predictive software scoring criterion: TMHMM software analyzes the transmembrane region of the protein, IEDB and ABCPred software predicts epitopes, and the higher the amino acid sequence score, the higher the possibility of becoming candidate epitopes, and the standard is taken as the standard. The inventor comprehensively analyzes the results of the 3 prediction methods, and finally determines the most possible epitope of the p30 protein, namely 8 possible epitopes of the p30 protein, which are respectively named as p30a, p30b, p30c, p30d, p30e, p30f, p30g and p30I in turn (Table 3).
TABLE 3 candidate epitopes of ASFVp30 protein
3.3 comparison and analysis of epitopes with the reported antigens
The results of comparison analysis of the bioinformatics software screening and the epitope amino acid sequences reported in the literature are shown in Table 4, and 1 epitope is found for the first time in the invention and is p30b (30-GSLYNW-35); in addition, the research also obtains the epitope which is related to but distinguished from the literature reports and comprises 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).
TABLE 4 amino acid sequence difference analysis between candidate epitope and literature reported epitope of p30 protein
4 small knot
The TMHMM software is used for predicting the transmembrane region of the p30 protein, and the result shows that the p30 has no transmembrane region. The IEDB software is used for determining 7 candidate epitopes of the p30 structural protein. The ABCPred software determines that the number of candidate epitopes of the protein is more, namely 21 p30 proteins. After the comprehensive analysis of the results, 8 epitopes of the p30 protein are finally determined, wherein p30b (30-GSLYNW-35) is the epitope firstly discovered by the invention.
Example 2 identification of epitopes of ASFVp30 protein
1 Material
1.1 Main Experimental reagents and instruments
MK100 is purchased from Takara; the African swine fever positive serum is purchased from Chinese veterinary medicine inspection institute; serum dilutions were purchased from Baiditai Bio Inc; TMB color developing solutions were purchased from surfmodics; 10 XPBS buffer and 10 XPBST buffer were purchased from Beijing Solaibao Tech Co., Ltd; the HRP-labeled goat anti-pig IgG antibody was purchased from Sigma; Carbonate-Bicarbonate Buffer is available from Sigma Aldrich trade, Inc.; enzyme-labeling instruments were purchased from Thermo corporation.
1.2 preparation of main reagents for experiment
(1) 5% BSA: 5g BSA, 1 XPBST was added to bring the volume to 100 mL.
(2) And (3) CBS: one capsule dissolved in 100mL ddH 2 And (4) in O.
(3)1 × PBS buffer solution: 100mL of 10 XPBS buffer was added to 1L of deionized water.
(4)1 × PBST buffer solution: 100mL of 10 XPBST buffer was added to 1L of deionized water.
(5) Sealing liquid: 5g of skimmed milk powder, and 1 XPBST is added to make the volume of the skimmed milk powder to 100 mL.
2 method
2.1 Synthesis of candidate epitope of ASFVp30 protein
By comprehensive analysis of the results of the bioinformatics software TMHMM, IEDB and ABCPred in example 1, 8 epitopes finally determined in the p30 protein were assigned to the biological reagent company to synthesize peptide fragments, and the purity of the peptide fragments was more than 95%.
2.2 optimization of epitope Indirect ELISA method for screening
The optimal ELISA reaction condition is determined by a chessboard titration method, 3 synthetic peptides in p30 protein epitope peptides are randomly selected as coating antigens, African swine fever infection positive serum is used as a primary antibody (the dilution concentration of the antibodies is 1:50, 1:100 and 1; 200), an HRP-labeled goat anti-pig IgG antibody is used as a secondary antibody, and an indirect ELISA method is established and optimized to determine the optimal antibody concentration. The specific operation steps are completed by referring to the specification of Takara peptide Coating Kit, and are as follows:
(1) the epitope (peptide) was synthesized by diluting the dilution solution in the kit to a concentration of 4. mu.g/ml, and the resulting solution was coated in a 96-well reaction plate (reaction plate) provided in the kit, 50. mu.L per well.
(2) Immediately, 10. mu.L of each Coupling reagent was added to the antigen-coated wells, and incubated for 2h at room temperature in the dark.
(3) The solution was discarded, washed 5 times with deionized water, shaken slightly for each wash, and patted dry.
(4) And (3) sealing: using Blockingbuffer in the kit, 200. mu.L of the buffer solution was added to each well, and the wells were sealed in the dark at room temperature for 1 hour.
(5) Discarding the solution, washing with deionized water for 5 times, shaking slightly for each washing, and patting dry.
(6) Incubation of primary antibody: adding serum diluent to dilute to 1:50, 1:100 and 1; 200 ASFV infection positive serum (100. mu.L/well), incubated at 37 ℃ for 1h in the dark, and a negative control (the serum of the negative control is the serum without ASFV infection) was set.
(7) Discard solution, wash 5 times with 1 × PBST, shake gently for each wash, and pat dry.
(8) Incubation of primary antibody: add 1:10000 (recommended concentration of antibody) of HRP-labeled goat anti-pig IgG antibody (100. mu.L/well), except blank control, and incubation at 37 ℃ for 1h in the dark.
(9) Discard solution, wash 5 times with 1 × PBST, shake gently for each wash, and pat dry.
(10) Color development: TMB substrate solution (100. mu.L/well) was added and developed in the dark at 37 ℃ for 15 min.
(11) And (3) determination: the reaction was stopped by adding 2M dilute sulfuric acid (100. mu.L/well), gently mixed, and OD measured using a microplate reader 450 And (4) an absorbance value.
2.3 Indirect ELISA screening of epitopes (synthetic peptides)
All synthesized Peptide fragments are diluted to 4 mu g/ml by using a diluent in a Takara Peptide Coating Kit to serve as Coating antigens, the Coating antigens are coated on 96-well reactionplates respectively, 3 times of each sample are set, negative controls are set, the incubation concentrations of ASFV infection positive serum in p30 Peptide fragments are 1:100, 1:100 and 1:50 respectively, HRP-labeled goat anti-pig IgG antibody is 1:10000, and other specific operation steps refer to 2.2.
3 results
3.1 optimization results of Indirect ELISA method
According to the chessboard titration results (Table 5), the optimal antibody concentration of ELISA is determined, the ratio of positive serum/negative serum is determined to be the maximum, the optimal antibody concentration is determined, and the optimal reactive antibody concentrations of the p30 protein peptide fragment are determined to be 1:100, 1:100 and 1:50 respectively through comprehensive analysis.
TABLE 5 Indirect optimization of negative and positive serum concentrations of LEISA
3.2 results of epitope screening by Indirect ELISA method
The antibody is screened by taking African swine fever virus positive serum as a standard, and the epitope of the ASFVp30 protein predicted by the bioinformatics online tool is screened and identified by adopting an ELISA method. As shown in FIG. 2, the epitope of the predicted African swine fever virus p30 protein was identified using a laboratory-established synthetic peptide indirect ELISA with African swine fever virus positive serum. The results show that all the predicted epitopes (table 3) can generate specific immune response with the nonserial swine fever virus positive serum, the differences are obvious (p is less than 0.001) compared with the negative control serum, and the results fully show that the epitopes in the table 3 are p30 protein epitopes. The epitope of the p30 protein is found to be p30b (30-GSLYNW-35) for the first time by using a bioinformatics technology and an immunological method.
Example 3 GST-Single epitope, Multi-epitope recombinant protein expression and epitope identification
1 Material
1.1 Main Experimental reagents and instruments
High affinity GST purification media and HighAffinityNi-Charged Resin FF were purchased from Ossie Biotech, Inc. of Nanjing; the reduced glutathione is purchased from Shanghai Michelin Biotechnology Ltd; coli BL21(DE3) competent cells, ampicillin, IPTG and kanamycin sulfate were purchased from shanghai bio-engineering gmbh; rosetta competent cells were purchased from Beijing Tiangen Biochemical technology, Inc.; 12mL and 30mL chromatography columns were purchased from Beijing Solaibao Tech Co., Ltd; 96-well plates were purchased from corning (usa).
Vertical electric pressure sterilizer (Shanghai Shenan medical equipment factory), ice maker (Scotsman, USA), electric heating constant temperature water tank (DK-8D type, Shanghai Jing Macro laboratory instruments Co., Ltd.), large centrifuge (Thermo, USA), PCR instrument, electrophoresis instrument, high resolution gel imager (Bio-Rad, USA), PH meter (Sartorius, Germany), ultra-clean bench (Suzhou Antai air technology Co., Ltd.), horizontal electrophoresis tank (Beijing six biosciences Co., Ltd.)
1.2 preparation of main reagents for experiment
(1) LB liquid medium: 10g Tryptone (Tryptone), 5g Yeast extract (Yeast extract), 10g sodium chloride (NaCl), adding deionized water, adjusting pH to 7.0, diluting to 1L, autoclaving at 121 deg.C, cooling, and storing at 4 deg.C.
(2)100mg/mL ampicillin solution: 5g of ampicillin powder, adding high-pressure deionized water to the solution until the volume is 50mL, filtering and sterilizing the solution by a 0.22 mu m filter, subpackaging the solution and storing the solution at the temperature of minus 20 ℃.
(3)100mg/mL kanamycin solution: 5g kanamycin powder, adding high-pressure deionized water to the solution until the volume is 50mL, filtering and sterilizing the solution by a 0.22 mu m filter, subpackaging the solution and storing the solution at the temperature of minus 20 ℃.
(4)8 × Binding Buffer solution: 4M NaCl, 160mM Tris-Hcl and 40mM imidazole, adding 800mL deionized water, adjusting the pH value to 7.9, and fixing the volume to 1L.
(5)1 × Binding Buffer solution: 75mL of 8 × Binding Buffer solution was added to 600mL of deionized water.
(6)5 × SDS-PAGE electrophoresis buffer solution: 15.1g Tris-HCL, 5.0g SDS, 94.0g glycine, adding deionized water to make the volume constant to 1L.
(7)1 × SDS-PAGE electrophoresis buffer solution: 200mL of 5 XSDS-PAGE electrophoresis buffer was added with deionized water to a volume of 1L.
(8) 10% SDS solution: 10g SDS, deionized water was added to make 100 mL.
(9) 10% ammonium persulfate: 1g of ammonium persulfate, deionized water is added to the solution to reach a volume of 10 mL.
(10) Coomassie brilliant blue R-250 staining solution: 1g of Coomassie brilliant blue R-250 powder, 250mL of isopropanol and 100mL of glacial acetic acid, and deionized water is added to the mixture to make the volume of the mixture constant to 1L.
(11) Coomassie brilliant blue destaining solution: 100mL of glacial acetic acid and 50mL of absolute ethyl alcohol are added with deionized water to reach the volume of 1L.
(12)10 × Glutathione reception Buffer: adding 500mmol Tris-HCl into 800mL deionized water, adjusting pH to 8.0, and adding deionized water to a constant volume of 1L.
(13)10 × GST Elution Buffer: 1g reduced Glutathione was dissolved in 32.5mL 10 × Glutathione reception Buffer.
(14)1 × GST Elution Buffer: 10 × GST Elution Buffer is diluted to 1 × GST Elution Buffer, and is ready for use.
2 method
2.1 construction of pGEX-4T-1/epitope recombinant expression plasmid, expression and purification of recombinant protein
2.1.1 construction of pGEX-4T-1/epitope recombinant expression plasmid
Respectively introducing specific enzyme cutting sites BamHI/Not I into an epitope in candidate ASFVp30 protein predicted by bioinformatics and a5 'end and a 3' end of an epitope base sequence reported in a literature as target genes, and then inserting the target genes into a BamHI/Not I linearized prokaryotic expression vector pGEX-4T-1 to construct recombinant plasmids. All recombinant plasmids were completely consigned to the organism company for synthesis. The homology was 100% as a result of sequencing.
2.1.2 expression and purification of epitope recombinant proteins
The specific operation method comprises the following steps:
(1) positive recombinant plasmids are respectively transformed into E.coli BL21(DE3) competent cells by a heat shock method, and positive recombinant bacteria are screened by an LB (Amp +) culture plate.
(2) Positive single colonies were selected and inoculated in an appropriate amount of LB (Amp +) liquid medium and cultured overnight at 37 ℃ at 220 rpm.
(3) According to the following steps: 100 was inoculated into a freshly prepared liquid LB medium for ampicillin resistance, cultured at 37 ℃ and 220rpm for 4 hours, 1mL was aspirated, IPTG (final concentration of 1mmol/mL) was added, induction was carried out at 37 ℃ and 220rpm for 4 hours, the cells were centrifuged at 4 ℃ for 10min (3000g) to harvest the cells, and the supernatant was discarded.
(4) The pellet was resuspended in cold 1 XPBS (3 mL of cold 1 XPBS per 50mL of medium), centrifuged at 4 ℃ for 10min (3000g) to harvest the pellet, and the supernatant discarded.
(5) The pellet was resuspended in 1 XPBS and the cells were disrupted on ice by ultrasonication. The supernatant was collected by centrifugation and the pellet resuspended with the same cold 1 × PBS.
(6) Equal amounts of soluble and insoluble fractions were aspirated separately, and the expression content and form of GST-fusion protein were analyzed by SDS-PAGE.
(7) An appropriate amount of high affinity GST purification medium was applied to a chromatography column and washed with 10 column volumes of cold 1 × PBS.
(8) The clarified liquid containing the GST fusion protein is added to a chromatography column, bound at 4 ℃ for about 1-2h, and after all the liquid has flowed out of the column, cold 1 XPBS (20 column volumes) is added immediately to wash the column.
(9) The fusion protein was eluted using the freshly prepared 1 × GST Elution Buffer (5-10 column volumes) and the eluted protein was analyzed by SDS-PAGE electrophoresis.
2.2 construction, expression and purification of phage display Multi-epitope recombinant expression plasmid
2.2.1 construction of phage display Multi-epitope recombinant expression plasmids
The screened peptide fragments are connected in series in sequence, in order to ensure the independence of the epitope, a spacer sequence GGGS is introduced between adjacent epitopes, then the epitope and a phage AP205 gene are respectively cloned to a prokaryotic expression vector pET-28a (+) with Nco I/Xho I linearization, a phage display multi-epitope recombinant expression plasmid is constructed, the multi-epitope recombinant protein is expressed by escherichia coli, and the nomenclature is shown in Table 6. All epitope recombinant expression plasmids were synthesized by the organism company and the identification was completed.
TABLE 6 epitope tandem mode
2.2.2 expression and purification of phage-displayed Multi-epitope recombinant proteins
The specific operation method comprises the following steps:
(1) positive recombinant plasmids are respectively transformed into E.coli Rosetta competent cells by a heat shock method, and positive recombinant bacteria are screened by an LB (Kan +) culture plate.
(2) Positive single colonies were selected and inoculated in an appropriate amount of LB (Kan +) liquid medium and cultured overnight at 37 ℃ and 220 rpm.
(3) According to the following steps: 100 was inoculated into a freshly prepared kanamycin-resistant liquid LB medium, cultured at 37 ℃ for 4 hours at 220rpm, aspirated to 1mL, IPTG (final concentration of 1mmol/mL) was added, induced at 37 ℃ for 4 hours at 220rpm, centrifuged at 4 ℃ for 10min (3000g) to harvest the cells, and the supernatant was discarded.
(4) (5) and (6) the same as 2.1.2.
(7) An appropriate amount of high affinity His purification media was loaded onto a chromatography column and washed with 1 × Binding 10 column volumes.
(8) Adding the clear liquid containing the His fusion protein into a chromatographic column, Binding for about 1-2h at 4 ℃, immediately adding 1 × Binding for washing after all the liquid flows out of the chromatographic column, and then washing the column by Binding buffers containing imidazole with different concentrations, wherein the concentrations are 20mM, 30mM, 40mM and 50mM in sequence.
(9) Finally, the fusion protein is eluted by Binding Buffer containing 500mM imidazole, and the eluted protein is analyzed by SDS-PAGE electrophoresis.
2.3 identification of epitopes by Indirect ELISA (epitope recombinant protein)
The specific operation method comprises the following steps:
(1) coating: the purified GST-epitope recombinant protein antigen was diluted with carbonate buffer (pH9.6) at a concentration of 1. mu.g/ml, and the 96-well reaction plate was coated with 100. mu.L of the antigen per well, and the reaction was repeated at 3 wells per sample overnight at 4 ℃.
(2) Washing: discard solution, add 1 × PBST to each well separately for washing, shake gently using shaker for 5 times total, discard solution and pat dry.
(3) And (3) sealing: mu.L of 5% BSA diluted in 1 XPBST was added to each well and incubated at 37 ℃ for 2h in the absence of light.
(4) Washing: the same as (2).
(5) Incubation of ASFV positive sera: mu.L of each well (serum dilution to 1/100) was added, and a negative control (serum without ASFV infection) was set up and incubated at 37 ℃ for 1 h.
(6) Washing: the same as (2).
(7) Incubation of HRP-labeled goat anti-pig IgG antibody: serum dilutions were added to each well to 1/10000 (100. mu.L/well) and incubated for 30min at 37 ℃.
(8) Washing: the same as (2).
(9) Color development: 100 mu LTMB substrate solution is added to each well, and the mixture is developed for 15min at 37 ℃ in the dark.
(10) And (4) terminating: add 100. mu.L of 2M sulfuric acid to each well to terminate the reaction, mix the mixture by gentle shaking, and measure the OD 450 An absorbance value.
2.4 data analysis
The data obtained from the experiments were analyzed for differential significance using the ttest test in the statistical GraphPadPrism 7 software (P <0.0001, P <0.001, P <0.01 indicates very significant differences;. P <0.05 indicates significant differences; P >0.05 does not significantly differ).
3 results
3.1 expression and purification of GST-epitope recombinant protein
SDS-PAGE result shows that all the epitope proteins of the p30 protein are expressed in colon bacillus, all the proteins are expressed in a soluble form, the size of the proteins is consistent with the expected result, the expression is about 25kDa, and affinity chromatography column purification is adopted to obtain the series GST-epitope recombinant protein.
3.3 identification of epitopes
The ELISA result shows that, as shown in FIG. 3, the result shows that the epitope can generate immunoreaction with African swine fever virus positive serum, and particularly, after the epitope is connected in series, the immunoreaction is obviously enhanced.
4 small knot
According to the invention, through an epitope complete antigenicity strategy and a phage display technology, the immunocompetence of the epitope is improved, the probability of identifying the immunocompetent epitope is improved, and the strategy is an effective strategy for identifying the immunocompetent epitope; the method is used for further determining the immune active epitope of the ASFVp30 protein, wherein the p30 protein has 3 minimum core motifs.
The applications of the obtained p30 protein epitope comprise researches on detection reagents and kits thereof (ELISA, WB, colloidal gold chromatography test paper strips and the like) for African swine fever virus infection serology, development of African swine fever epitope vaccines and any vaccines related to the epitope (epitope univalent/multivalent vaccines, epitope-based synthetic peptide vaccines, and other pathogen epitope or antigen tandem research diagnosis kit vaccines and the like), preparation of epitope monoclonal antibodies and the like. The successful acquisition of the epitope has very important significance on serological detection of African swine fever virus, detection kit development, immune effect evaluation, vaccine development and the like.
Sequence listing
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> African swine fever virus p30 antigen epitope protein, preparation method and application 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
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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 (8)
1. The African swine fever virus p30 epitope protein is characterized in that the p30 epitope protein is selected from any one of p30a, p30b, p30c, p30d, p30e, p30f, p30g and p30I epitope proteins or a multi-epitope recombinant protein obtained by connecting any two or more of p30a, p30b, p30c, p30d, p30e, p30f, p30g and p30I epitope proteins in series, and the amino acid sequences of the p30a, p30b, p30c, p30d, p30e, p30f, p30g and p30I epitope proteins are respectively shown as SEQ ID NO. 1-8.
2. The African swine fever virus p30 epitope protein of claim 1, wherein one end of the p30 epitope protein is linked to a GST tag.
3. The African swine fever virus p30 epitope protein of claim 1, wherein the p30 epitope protein is GST-multi epitope recombinant protein obtained by connecting p30c, p30d and p30e epitope proteins in series, introducing a spacer sequence GGGS between adjacent antigen epitopes, then cloning the coding gene sequence and the phage AP205 gene to a linearized prokaryotic expression vector pET-28a (+) in sequence respectively, constructing a phage display multi-epitope recombinant expression plasmid, and expressing the recombinant expression plasmid by Escherichia coli.
4. The African swine fever virus p30 epitope protein of claim 1, wherein the p30 epitope protein is GST-multi epitope recombinant protein obtained by connecting p30g and p30I epitope proteins in series, introducing a spacer sequence GGGS between adjacent antigen epitopes, cloning the coding gene sequence and the phage AP205 gene to a linearized prokaryotic expression vector pET-28a (+) in sequence, respectively, constructing phage display multi-epitope recombinant expression plasmid, and expressing the recombinant expression plasmid by Escherichia coli.
5. Use of the African swine fever virus p30 epitope protein of any one of claims 1-4 in preparation of an antibody reagent for detecting African swine fever virus.
6. The use of claim 5, wherein the reagent is an ELISA detection reagent.
7. An indirect ELISA detection kit for African swine fever virus antibody, characterized in that the kit contains the African swine fever virus p30 epitope protein of any one of claims 1-4.
8. The indirect ELISA detection kit of claim 7 wherein the kit further comprises a diluent, a washing solution, a blocking solution, an HRP-labeled goat anti-pig IgG antibody, a chromogenic solution and a stop solution.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113512095A (en) * | 2021-08-10 | 2021-10-19 | 郑州大学 | anti-African swine fever virus p30 protein monoclonal antibody, preparation method and B cell epitope screening and identification |
| CN113607952A (en) * | 2021-08-18 | 2021-11-05 | 杭州恒奥科技有限公司 | African swine fever virus blocking ELISA antibody detection kit and preparation method and application thereof |
| US20220047694A1 (en) * | 2018-09-18 | 2022-02-17 | Stichting Wageningen Research | African swine fever virus vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20220047694A1 (en) * | 2018-09-18 | 2022-02-17 | Stichting Wageningen Research | African swine fever virus vaccine |
| CN113512095A (en) * | 2021-08-10 | 2021-10-19 | 郑州大学 | anti-African swine fever virus p30 protein monoclonal antibody, preparation method and B cell epitope screening and identification |
| CN113607952A (en) * | 2021-08-18 | 2021-11-05 | 杭州恒奥科技有限公司 | African swine fever virus blocking ELISA antibody detection kit and preparation method and application thereof |
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| CN116987155A (en) * | 2022-09-15 | 2023-11-03 | 河南农业大学 | African swine fever virus epitope peptide and application thereof |
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