CN114409743A

CN114409743A - African swine fever virus p54 protein epitope and application thereof

Info

Publication number: CN114409743A
Application number: CN202111610755.XA
Authority: CN
Inventors: 邵军军; 李俊慧; 刘伟; 常惠芸
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2021-12-27
Filing date: 2021-12-27
Publication date: 2022-04-29
Anticipated expiration: 2041-12-27
Also published as: CN114409743B

Abstract

The invention relates to an African swine fever virus p54 protein epitope and application thereof, belonging to the field of genetic engineering, and the invention takes a primary structure amino acid sequence of an African swine fever virus p54(E183L) membrane protein as a material, predicts the epitope through an epitope database, synthesizes corresponding peptide molecules according to the amino acid sequence, constructs GST/epitope recombinant protein, phage (virus-like particles, VLP) display multi-epitope antigen and other multiple epitope antigens. Through African swine fever virus positive serum immunological detection tests, 5 antigen epitopes (peptides) are determined to be the antigen epitopes of the African swine fever virus p54 protein. The obtaining 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 other urgent strategic prevention and control products by taking the antigen epitopes as materials. Not only can generate huge economic benefits, but also has important social benefits.

Description

African swine fever virus p54 protein epitope and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to an African swine fever virus p54 protein epitope and application thereof.

Background

African swine fever is an acute virulent infectious disease caused by swine diseases caused by African swine fever viruses, and the death 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. Since 8 months in 2018 are introduced into China, the epidemic situation is spread across the whole country in less than one year, and the epidemic situation is not eliminated at present, so that the major economic loss is caused to the pig raising industry in China, the number of live pigs in stock is sharply reduced, the pork price is soared, and the huge economic loss is caused. Various comprehensive measures are taken by various departments such as the state institute, the state ministry and the like to recover the production of the live pigs and stabilize the price. However, the continuous spread and epidemic of epidemic situation cause serious pollution to the environment, and 22 strains including large-fragment gene-deleted low virulent strains have been isolated from the field by the early 2021, which indicates that the environmental pollution is serious, the strains are complex and the prevention and control are difficult. As the biggest world pig-raising nation in China, the pork supply exceeds 50 percent, and the attack of African swine fever on pig raising threatens the healthy and sustainable development of the pig raising industry, and also concerns a series of social and political problems of civil life, food safety, biological safety and the like, and becomes one of the major animal epidemic diseases which are urgently needed to be solved by the nation.

The research on the vaccine of the African swine fever virus as an important animal virus with hundred years history is not interrupted, but the safe and effective vaccine which can be used for practice is still lacked to date. The reason is mainly due to the large genome, complex structure, protective antigen and unclear immune and infection mechanisms of the African swine fever virus. The theoretical problem and the technical bottleneck that the African swine fever inactivated vaccine cannot provide immune protection cannot be explained and solved so far; the attenuated live vaccine causes morbidity and mortality, reduces the production capacity, and may cause biological safety problems such as virulence reversion, genetic recombination and the like; the theoretical problem of poor protective effect of genetic engineering subunit vaccines, DNA vaccines and live vector vaccines. The state urgently needs a safe, effective and differential diagnosis African swine fever vaccine to improve the prevention and control of the African swine fever epidemic situation and the purification capability and the technical level of the African swine fever vaccine, ensure the healthy and sustainable development of the first world pig industry of China, and ensure the safety and the biological safety of animal-derived food.

Based on the situation, the vaccine is slow to research, and lacks of safe, effective and identifiable vaccines, and the state of the country urgently needs prevention and control products against the African swine fever virus and the complexity thereof. A brand new thought and a new approach are needed to discover more protective antigens so as to solve the dilemma that the research of the genetic engineering vaccine lacks key materials due to the huge and complex genome of the African swine fever virus and unclear protective antigens.

Disclosure of Invention

Aiming at the problems, the invention takes the primary structure amino acid sequence of the African swine fever virus p54(E183L) membrane protein as a material, predicts and determines 5 antigen epitopes (peptides) of the African swine fever virus p54 protein, and the obtainment of the antigen epitopes lays a foundation for researching and developing African swine fever detection reagents and kits thereof, monoclonal antibody preparation and safe, differential diagnosis epitope vaccines and other urgently needed strategic prevention and control products by taking the antigen epitopes as materials.

The invention aims to provide an African swine fever virus p54 protein epitope, which is a single epitope (synthetic peptide), a protein carrier recombinant single epitope or multiple epitopes; the single epitope is represented by SEQ ID NO: 1-5 any one of the antigen epitope peptide and carrier protein/antigen epitope recombinant protein; the multiple epitope is obtained by connecting two or more than two single epitope in series, connecting adjacent epitopes by adopting a spacer, then connecting the multiple epitope with a bacteriophage or a self-assembly protein, and expressing VLP display multiple epitope.

The single epitope is preferably SEQ ID NO: p54c shown in 3, SEQ ID NO: 4 or p54d shown in SEQ ID NO: p54e shown in fig. 5.

Preferably, the multiple epitope is p54abc or p54ed, wherein p54abc is a residue of SEQ ID NO: 1, p54a, SEQ ID NO: 2 and p54b shown in SEQ ID NO: p54c shown in 3 is obtained by serially connecting GS and then connecting AP205 through GGGGS; p54ed is a peptide of SEQ ID NO: p54e shown in FIG. 5 and SEQ ID NO: p54d shown in FIG. 4 was sequentially connected in series via GS and then sequentially connected to SpyCather and AP205 via GGGGS.

The invention also aims to provide a recombinant protein, which is obtained by connecting the epitope with an expression vector, expressing and purifying.

The third objective of the invention is to provide a polynucleotide, wherein the polynucleotide encodes the epitope or the recombinant protein.

The fourth object of the present invention is to provide an expression vector comprising the polynucleotide.

The fifth object of the present invention is to provide a host cell containing the above expression vector or having the above polynucleotide integrated in its genome.

The sixth purpose of the invention is to provide the application of the antigen epitope, the fusion protein, the multi-epitope VLP, the polynucleotide, the expression vector or the host cell in the preparation of African swine fever virus detection drugs and African swine fever resistant vaccines.

Has the advantages that:

the invention takes the primary structure amino acid sequence of African swine fever virus p54(E183L) membrane protein as a material, predicts the epitope of the African swine fever virus p54(E183L) through an epitope database and synthesizes corresponding peptide molecules according to the amino acid sequence. Through the positive serum immunological detection of the African swine fever virus, 5 epitopes (peptides) in the table 1 are all epitopes of the African swine fever virus p54 protein, wherein p54d and p54e are dominant epitopes. The obtaining 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 and need strategic prevention and control urgently, and not only can generate great economic benefits, but also has important social benefits.

Drawings

FIG. 1 is a graph showing the result of identifying p54 protein epitope by using positive serum of African swine fever virus;

FIG. 2 is a graph showing the evaluation of immunoreactivity of epitope recombinant proteins;

FIG. 3 is a graph comparing the immunoreactivity of the epitopes obtained in the present invention with that of a literature-reported epitope;

note that: p <0.0001, P <0.001, indicating that the difference was extremely significant; indicates significant difference (P < 0.05); p >0.05 differences were not significant.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

Epitope prediction and screening

The amino acid sequence of the p54 Protein of African swine fever virus is taken as a material, the epitope of the Protein is predicted by utilizing an IEDB Analysis Resource, ABCPred and Scatch Protein Predictor online database, and 5 epitopes of the p54 Protein are determined according to the standard that all score values in the 4 databases are not less than 0.6 (Table 1).

TABLE 1 epitope of China/SY18/2018 p54 protein predicted by bioinformatics and immunoinformatics methods

2. Synthesis of antigenic epitopes (peptides)

According to the amino acid sequence of the corresponding epitope in the table 1, the corresponding short peptide molecule is synthesized by the Kinsley biotechnology company, and the purity is not lower than 95%.

Establishment of Indirect ELISA method

Randomly selecting 3 synthetic short peptides, coating an MK100 peptide 96-hole ELISA plate according to the concentration of 1 mu g/ml and 100 mu l/hole, and staying overnight at 4 ℃; spin-dry the plate, block with 5% BSA in PBST for 2 hours at 37 deg.C, 200. mu.l/well; spin-drying the plate, and washing with PBST (pH7.4) for 4-5 times; diluting SPF pig negative serum/ASFV standard positive serum according to the column (1-12) multiple ratio, wherein the initial concentration is 1:25, repeating the steps (1A-1H) in 8 wells, reacting at 100 μ l/well for 1H at 37 ℃; discarding the liquid, washing thoroughly 4-5 times with PBST (pH 7.4); HRP-labeled rabbit anti-IgG was then diluted by line (A-H) at an initial concentration of 1:2500, 12-well repeat (A1-A12), reaction at 37 ℃ for 1 hour; washing with PBST for 4-5 times, adding chromogenic substrate for 10-15 min, and determining OD_450nmAn absorbance value.

And (4) result judgment standard: sample OD_450nmThe value (S)/negative control (N) ratio is positive when the value is 2.1 or more and negative when the value is less than 2.1.

Screening and identification of antigen epitope by positive serum of African swine fever virus

Antibody was screened using african swine fever virus positive serum (purchased from chinese veterinary medicine institute) as a standard, and epitope of p54 protein was screened and identified by ELISA as described above. The results show that the predicted 5 epitopes (peptides) (table 1) can generate specific immune response with the nonserial swine fever virus positive serum, and the statistical difference is significant compared with the negative control serum (figure 1), which indicates that the predicted 5 epitopes are all epitopes of p54 protein, wherein the two epitopes of p54d and p54e have the strongest immune response and are the dominant epitope of p54 protein.

Further screening and identifying antigen epitope by introducing carrier protein GST and epitope tandem

5.1 construction, protein expression and purification of recombinant expression plasmid of single epitope and tandem epitope

In order to further verify the immunoreactivity of the epitope, the invention leads the epitope to be completely antigenic by introducing GST carrier protein, improves the epitope titer by connecting the epitope in series, improves the immunoreactivity of the epitope and leads the screening result to be more reliable. The research clones the DNA of the selected epitope p54a, p54b, p54c, p54d and p54e to pGEX-4T-1 expression vector to construct recombinant expression plasmids pGEX-4T-p54a, pGEX-4T-p54b, pGEX-4T-p54 c, pGEX-4T-p54 d and pGEX-4T-p54e respectively, and obtains the target recombinant protein by expressing and purifying escherichia coli BL21(DE3) and sequentially naming GST-p54a, GST-p54b, GST-p54c, GST-p54d and GST-p54 e. The same method as above is adopted to express the antigenic epitope of p54 reported in the literature, and the antigenic epitope is completely antigenicized by GST protein and purified by affinity chromatography for immunoreactivity comparison.

Three epitopes of p54a, p54b and p54c of the epitope (Table 1) are sequentially connected in series through GS (a p54abc tandem gene is shown as SEQ ID NO: 10, and an encoded amino acid is shown as SEQ ID NO: 11), and then the epitope is connected with AP205 through GGGGS to form a multi-epitope fusion gene (a p54abc-AP205 gene sequence is shown as SEQ ID NO: 12, and an encoded amino acid is shown as SEQ ID NO: 13); two dominant epitopes p54e and p54d are connected in series through a GS sequence (the sequence of a p54ed tandem gene is shown as SEQ ID NO: 14, and the coded amino acid is shown as SEQ ID NO: 15), then a SpyCather and AP205 are connected by a GGGGS sequence to form a multi-epitope fusion gene (the sequence of a p54ed-AP504 gene is shown as SEQ ID NO: 16, and the coded amino acid is shown as SEQ ID NO: 17), the multi-epitope fusion gene and a phage gene are respectively cloned to a pET-28a (+) expression vector to construct recombinant expression plasmids p54ABC and pET-28/p54ed of the phage display tandem epitope, recombinant proteins (multi-epitope VLP) of the phage display multi-epitope are expressed through escherichia coli BL21(DE3), and are respectively named as p54ABC and p54 ED.

SDS-PAGE results show that all the above recombinant proteins are expressed in soluble form, and the recombinant protein for coating antigen is obtained by affinity chromatography.

Introduction of carrier protein to make epitope completely antigenic, multi-epitope VLP screening antigen epitope

The immunoreactivity of the single epitope recombinant protein and the multi-epitope VLP is determined by the established ELISA, and the antigen epitope is further screened and determined. The results show that after introducing the carrier protein GST, the immunoreactivity of the antigen epitope is enhanced (p < 0.05), and the dominant antigen epitopes p54d and p54e are significantly enhanced; particularly, after the epitope is connected in series and expressed in a multi-epitope VLP form, the strong immunoreactivity of the dominant epitope is more outstanding, is better than the immunoreactivity of single-epitope recombinant protein introduced with GST protein, and further determines that p54d and p54e are dominant epitopes. In conclusion, the multi-epitope VLP has the strongest immunogenicity, and the immunogenicity of the single-epitope recombinant protein is superior to that of the synthetic peptide, so that the antigen epitopes play an obvious synergistic effect after being connected in series, the immunoreactivity is obviously enhanced, and the synergistic effect among the dominant antigen epitopes is particularly obvious; the carrier protein also enhances the immunoreactivity of the epitope, and the enhanced strength is related to the property of the epitope (figure 2). The research method provides a new thought and technical means for accurately and efficiently screening dominant antigen epitopes, and provides theoretical basis and technical support for accurately and efficiently screening the antigen epitopes, designing multi-epitope recombinant antigens with strong immunocompetence, researching specific detection reagents and developing multi-epitope vaccines.

The immunoreactivity of the screened epitope is compared with that of the epitope reported in the literature

In order to further determine that the epitope obtained by the present invention is a novel epitope, the present study clones the epitope reported in the literature (table 2) to pGEX-4T-1 expression vector according to the above method, constructs recombinant expression plasmids pGEX-4T-p54G1, pGEX-4T-p54G2, pGEX-4T-p54G3 and pGEX-4T-p54G4, and expresses recombinant proteins via BL21(DE3), named GST-p54G1, GST-p54G2, GST-p54G3 and GST-p54G 4.

The immunoreactivity of the epitope screened and reported in the literature by the invention is detected by adopting the ELISA. The results show that the immunoreactivity of the dominant epitopes p54d and p54e obtained by the invention and the African swine fever positive serum is remarkably stronger than that of the epitope (p < 0.0001) reported in the literature, and particularly, after the p54d and the p54e are connected in series and are expressed in a multi-epitope VLP form, the immunoreactivity of the tandem epitope recombinant protein is remarkably enhanced (figure 3), which fully indicates that the immunoreactivity of the epitope obtained by the invention is better than that of the epitope reported in the literature.

Table 2 reports the epitope of p54 protein of Georgia 2007 strain

Name of	Position (amino acid)	Sequence (5 '-3')	Position number in sequence Listing
				p54G1	65~75	EDIQFINPYQD	SEQ ID NO：6
p54G2	93~113	ATTASVGKPVTGRPATNRPAT	SEQ ID NO：7
				p54G3	118~127	TDNPVTDRLV	SEQ ID NO：8
p54G4	5~9	FFQPV	SEQ ID NO：9

7 epitope screened and epitope sequence analysis reported in literature

The dominant epitope obtained by the invention has obvious immunological activity difference with the epitope reported in the literature, and the epitope obtained by the invention is different from the epitope reported in the literature in order to further confirm diagnosis. The research compares and analyzes the amino acid sequences of the epitope (table 1) obtained by the invention and the epitope (table 2) reported in the literature, and the result shows that (table 3), the epitope p54c (80 aa-95 aa) is found for the first time and is not reported; the dominant epitope p54d (95 aa-110 aa) overlaps with the amino acid sequence of a reported antigen epitope (93 aa-113 aa) in the literature, but 2 amino Acids (AT) are less AT the 5 'end and 3 amino acids (PAT) are less AT the 3' end, the immunoreactivity of the antigen epitope is obviously superior to that reported in the literature, and the immune effect of the antigen epitope is probably influenced by the amino acids AT the two sides, so that the antigen epitope obtained by the invention is a new dominant antigen epitope of the p54 protein; after the comparison of the amino acid sequences of the dominant epitope p54e (115 aa-122 aa) and the amino acid sequences reported in the literature (118 aa-127 aa), the two epitopes are found to have the same PVTD motif, different length, different amino acid sequences at two ends and different immunogenicity.

TABLE 3 analysis of the differences in amino acid sequences between the selected epitope and the literary reporter epitope

8. Use of epitopes

The epitope of the p54 protein obtained by the invention, especially 3 new epitopes, namely two dominant epitopes of p54c, p54d and p54e, especially p54d and p54e, has immunoreactivity which is obviously superior to the epitope reported in the literature, and the encoding amino acid sequences are different and are the new epitope of the p54 protein. The antigen epitopes, especially the dominant antigen epitopes with strong immunoreactivity, enrich the African swine fever virus antigen epitope database, and provide material basis and theoretical basis for developing African swine fever serological detection reagents and kits thereof (ELISA, WB, colloidal gold chromatography test paper strips and the like), designing epitope antigens, developing epitope vaccines (including epitope univalent/multivalent vaccines, synthetic peptide vaccines, and antigen tandem connection with other pathogenic antigen epitopes or antigens and the like), and have very important significance.

It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.

SEQUENCE LISTING

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

<120> African swine fever virus p54 protein epitope and application thereof

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Claims

1. The African swine fever virus p54 protein epitope is characterized in that: the epitope is a single epitope or a recombinant VLP display multi-epitope; the single epitope is represented by SEQ ID NO: 1-5 of any one of the epitope peptides; the multi-epitope is obtained by connecting two or more than two single antigen epitopes in series and displaying the multi-epitope through the recombinant VLP.

2. The epitope according to claim 1, characterized in that: the single epitope is SEQ ID NO: p54c shown in 3, SEQ ID NO: 4 or p54d shown in SEQ ID NO: p54e shown in fig. 5.

3. The epitope according to claim 1, characterized in that: the polyepitope is p54abc or p54ed, and the p54abc is a polypeptide represented by SEQ ID NO: 1, p54a, SEQ ID NO: 2 and p54b shown in SEQ ID NO: p54c shown in 3 is connected in series by a linker, and then is connected with the AP205 through GGGGS; p54ed is a peptide of SEQ ID NO: p54e shown in FIG. 5 and SEQ ID NO: p54d shown in FIG. 4 was sequentially connected in series by a linker, and then sequentially connected to SpyCatcher and AP205 by GGGGS.

4. The epitope according to claim 3, characterized in that: the amino acid sequence of p54abc is shown as SEQ ID NO: 13, the amino acid sequence of the p54ed is shown as SEQ ID NO: shown at 17.

5. A recombinant protein, wherein the recombinant protein is obtained by connecting the epitope as claimed in claim 1 with an expression vector, and then expressing and purifying.

6. A polynucleotide encoding the epitope of claim 1 or the recombinant protein of claim 5.

7. An expression vector comprising the polynucleotide of claim 6.

8. A host cell comprising the expression vector of claim 7 or having the polynucleotide of claim 6 integrated into its genome.

9. The use of the epitope according to claim 1 for the preparation of African swine fever virus detection drugs, anti-African swine fever vaccines.