CN114409743B

CN114409743B - African swine fever virus p54 protein epitope and application thereof

Info

Publication number: CN114409743B
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: 2023-09-12
Anticipated expiration: 2041-12-27
Also published as: CN114409743A

Abstract

The invention relates to an African swine fever virus p54 protein epitope and application thereof, which belong to the field of genetic engineering, wherein a primary structure amino acid sequence of an African swine fever virus p54 (E183L) membrane protein is used as a material, the epitope is predicted by an epitope database, corresponding peptide molecules are synthesized according to the amino acid sequence, GST/epitope recombinant proteins are constructed, and phage (virus-like particles, VLPs) display multiple epitope antigens such as multiple epitopes. Through an african swine fever virus positive serum immunology detection test, 5 epitopes (peptides) are determined to be epitopes of african swine fever virus p54 protein. The acquisition of the antigen epitope lays a foundation for researching and developing african swine fever detection reagents and kits, monoclonal antibody preparation and safety, and strategic prevention and control products which can be used for differential diagnosis of epitope vaccines and the like by taking the antigen epitope as a material. Not only can great economic benefit be generated, but also important social benefit can be achieved.

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 causing swine morbidity by African swine fever virus, and the mortality rate is up to 100%. The world animal health Organization (OIE) lists it as a group of animal infections that must be reported to animals for which our country is a major precaution. Since 2018, 8 is transmitted into China for less than one year, epidemic situation is spread across the country, so that the epidemic situation is not eliminated yet, great economic loss is caused for pig industry in China, the number of live pigs in stock is drastically reduced, and the pork price is drastically increased, so that great economic loss is caused. Various comprehensive measures are adopted by departments of national institutes, national ministry of the sciences and the like to restore the production of live pigs and stabilize the price. However, epidemic situation is spread and spread continuously, extremely serious pollution is caused to the environment, 22 strains including the weak strain with large fragment gene deletion have been separated from the field by 2021, and the environmental pollution is serious, the strain is complex and the prevention and control are difficult. The biggest pig raising country in China is the world, the pork supply exceeds 50%, the African swine fever strikes the pigs, the health sustainable development of the pig raising industry is threatened, a series of social and political problems such as folk life, food safety and biosafety are concerned, and the biggest animal epidemic diseases which are urgently needed to be solved in the country are one of the serious animal epidemic diseases.

African swine fever virus is an important animal virus for centuries, and research on vaccines has never been interrupted, but a safe and effective vaccine which can be used for practice is still lacking. The reason for this is mainly because African swine fever virus has large genome, complex structure, protective antigen, and unclear immune and infection mechanism. The theoretical problem and the technical bottleneck that the African swine fever inactivated vaccine can not provide immune protection can not be explained and solved; attenuated live vaccine causes morbidity and mortality, production capacity is reduced, and biosafety problems such as virulence reversion, genetic recombination and the like can occur; the theoretical problem of poor protective effect of genetic engineering subunit vaccines, DNA vaccines and live vector vaccines. The african swine fever vaccine which is safe, effective and capable of being differentially diagnosed is urgently needed in China, so that the prevention and control and purification capability and the technical level of the african swine fever epidemic situation in China are improved, the healthy and sustainable development of the national pig raising industry which is the first pig raising country in the world is ensured, and the animal-derived food safety and biological safety are ensured.

Based on the above, aiming at African swine fever virus and the complexity thereof, the vaccine research is slow, the safe, effective and identifiable vaccine is lacking, and the state that the prevention and control of products are urgently needed in China. A brand new thought and another development approach are needed to discover more protective antigens so as to solve the dilemma that the genetic engineering vaccine research lacks key materials due to the huge and complex genome of the African swine fever virus and the unclear protective antigens.

Disclosure of Invention

Aiming at the problems, the invention predicts and determines 5 epitopes (peptides) of the African swine fever virus p54 (E183L) membrane protein by taking the primary structure amino acid sequence of the African swine fever virus p54 (E183L) membrane protein as a material, and the obtaining of the epitopes lays a foundation for developing African swine fever detection reagents and kit thereof, monoclonal antibody preparation and safety, differential diagnosis epitope vaccine and other urgent strategy prevention and control products by taking the epitopes as materials.

The invention aims at providing an African swine fever virus p54 protein epitope, wherein the antigen epitope is a single antigen epitope (synthetic peptide), a protein carrier recombinant single antigen epitope or a multi-antigen epitope; the single antigen epitope is a polypeptide with an amino acid sequence shown as SEQ ID NO: 1-5 of any one of the epitope peptides, carrier proteins and epitope recombinant proteins; the multi-antigen epitope is obtained by connecting two or more than two single antigen epitopes in series, adopting a spacer to connect adjacent epitopes, then connecting the adjacent epitopes with phage or self-assembled protein, and expressing VLP to display the multi-epitope.

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

The multi-antigen epitope is preferably p54abc or p54ed, and the p54abc is a polypeptide obtained by combining the amino acid sequence of SEQ ID NO:1, p54a, SEQ ID NO:2 and p54b and SEQ ID NO:3, p54c is obtained by connecting the APs 205 through GS and then GGGGS; said p54ed is the nucleotide sequence of SEQ ID NO:5 and p54e and SEQ ID NO:4, and then SpyCather and AP205 are sequentially connected in series through GS and then through GGGGS.

The second object of the invention is to provide a recombinant protein, which is obtained by connecting the antigen epitope with an expression vector, expressing and purifying.

The third object of the present invention is to provide a polynucleotide encoding the above epitope or recombinant protein.

It is a fourth object of the present invention to provide an expression vector comprising the polynucleotide described above.

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

The invention also provides application of the antigen epitope, the fusion protein, the multi-epitope VLP, the polynucleotide, the expression vector or the host cell in preparation of African swine fever virus detection medicines and African swine fever vaccine.

The beneficial effects are 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 thereof through an epitope database and synthesizes corresponding peptide molecules according to the amino acid sequence. Through african swine fever virus positive serum immunological detection, it is determined that all of the 5 epitopes (peptides) in table 1 are epitopes of african swine fever virus p54 protein, wherein p54d and p54e are dominant epitopes. The acquisition of the antigen epitopes lays a foundation for developing african swine fever detection reagents and kits thereof, monoclonal antibody preparation and safety, and can identify and diagnose strategic prevention and control products such as epitope vaccines, and the like, thereby not only generating great economic benefits, but also having important social benefits.

Drawings

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

FIG. 2 is a graph of the evaluation of immunoreactivity of an epitope recombinant protein;

FIG. 3 is a graph showing the immunoreactivity of an epitope obtained by the present invention with a reference epitope;

annotation: * P <0.0001, P <0.001, P <0.01 indicates that the difference is extremely significant; * Indicating significant differences (P < 0.05); the P >0.05 difference was not significant.

Detailed Description

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

Epitope prediction and screening

The amino acid sequence of p54 protein of African swine fever virus is used as a material, the epitopes of the African swine fever virus are predicted by utilizing IEDB Analysis Resource, ABCpred and Scratch Protein Predictor on-line databases, and 5 epitopes of the p54 protein are determined by using the standard that all score values in the 4 databases are not lower than 0.6 (table 1).

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

2. Synthesis of epitopes (peptides)

The amino acid sequence of the corresponding epitope in Table 1 is entrusted with 'gold Style biotechnology company' to synthesize a corresponding short peptide molecule with purity not lower than 95%.

Establishment of indirect ELISA method

Randomly selecting 3 synthetic short peptides, coating MK100 peptide 96-well ELISA plates at a concentration of 1 mug/ml and 100 mug/well, and standing overnight at 4 ℃; spin-drying the plates, blocking with PBST containing 5% BSA at 37℃for 2 hours, 200. Mu.l/well; spin-drying the plate, and washing with PBST (pH 7.4) for 4-5 times; SPF pig negative serum/ASFV standard positive serum was diluted 1-12 times in columns, starting concentration 1:25,8 well replicates (1A-1H), 100 μl/well, and reacted at 37℃for 1 hour; the liquid was discarded and washed thoroughly with PBST (pH 7.4) 4-5 times; HRP-labeled rabbit anti-IgG was then diluted in row (a-H) fold at an initial concentration of 1:2500, 12 well replicates (A1-a 12), and reacted at 37 ℃ for 1 hour; washing with PBST for 4-5 times, adding chromogenic substrate, and allowing to act for 10-15 min to determine OD _450nm Absorbance values.

Result determination criteria: sample OD _450nm The ratio of the value (S)/the negative control (N) is positive when the ratio is greater than or equal to 2.1 and negative when the ratio is less than 2.1.

Screening and identifying antigen epitope of African swine fever virus positive serum

The antigen epitope of p54 protein was screened and identified by the ELISA described above using african swine fever virus positive serum (purchased from chinese veterinary drug administration) as a standard screening antibody. The results show that the predicted 5 epitopes (peptides) (table 1) can generate specific immune reaction with the african swine fever virus positive serum, and compared with the negative control serum, the statistical difference is obvious (figure 1), which suggests that the predicted 5 epitopes are the epitopes of p54 protein, wherein the immunoreactivity of the two epitopes of p54d and p54e is the dominant epitope of p54 protein.

Further screening and identification of antigenic epitopes by introducing carrier protein GST and epitope tandem

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

In order to further verify the immunoreactivity of the epitope, the GST carrier protein is introduced to make the epitope completely antigenic, the epitope is connected in series to improve the antigen epitope titer, the immunoreactivity of the epitope is improved, and the screening result is more reliable. The present study cloned the above-mentioned screened DNA of the epitopes p54a, p54b, p54c, p54d, p54e into pGEX-4T-1 expression vector to construct recombinant expression plasmids pGEX-4T-p54a, pGEX-4T-p54b, pGEX-4T-p54 c, pGEX-4T-p54 d, pGEX-4T-p54e, and expressed and purified by E.coli BL21 (DE 3) to obtain the target recombinant proteins, which were named GST-p54a, GST-p54b, GST-p54c, GST-p54d, GST-p54e in sequence. The epitope of p54 reported in the literature was expressed by the same method as described above, and was completely antigenized by GST protein and purified by affinity chromatography for immunoreactivity comparison.

The three epitopes of p54a, p54b and p54c of the antigen epitope (table 1) are sequentially connected in series through GS (the p54abc series gene is shown as SEQ ID NO:10, the coding amino acid is shown as SEQ ID NO: 11), and then the antigen epitope is connected with AP205 through GGGGS to form a multi-epitope fusion gene (the p54abc-AP205 gene sequence is shown as SEQ ID NO:12, the coding amino acid is shown as SEQ ID NO: 13); the two dominant epitopes of p54e and p54d are connected in series through GS sequence (the sequence of p54ED series gene is shown as SEQ ID NO:14, the coding amino acid is shown as SEQ ID NO: 15), then GGGGS sequence is utilized to connect SpyCather and AP205, the two dominant epitopes are multi-epitope fusion genes (the sequence of p54ED-AP504 gene is shown as SEQ ID NO:16, the coding amino acid is shown as SEQ ID NO: 17), the fusion genes and phage genes are cloned into pET-28a (+) expression vector respectively, recombinant expression plasmids p54ABC and pET-28/p54ED of phage display series epitopes are constructed, and recombinant proteins (multi-epitope VLPs) of phage display multi-epitopes are expressed through escherichia coli BL21 (DE 3) and named as p54ABC and p54ED respectively.

SDS-PAGE results show that all the recombinant proteins are expressed in a soluble form, and the recombinant proteins for coating the antigen are obtained by adopting an affinity chromatography method.

Introducing carrier protein to make epitope completely antigenic and multi-epitope VLP screen antigen epitope

The immunoreactivity of the single epitope recombinant protein and the multi-epitope VLP was measured by established ELISA, and the epitope was further selected and determined. The result shows that after the carrier protein GST is introduced, the immunoreactivity of the epitope is enhanced (p < 0.05), and the dominant epitopes p54d and p54e are obviously enhanced; in particular, after the epitope is connected in series and expressed in a multi-epitope VLP form, the strong immunoreactivity of the dominant epitope is more prominent, and the immunoreactivity of the single epitope recombinant protein introduced with GST protein is better than that of the single epitope recombinant protein introduced with GST protein, and p54d and p54e are further determined to be the dominant epitope. In a word, the immunogenicity of the multi-epitope VLP is strongest, and the immunogenicity of the single epitope recombinant protein is better than that of the synthetic peptide, so that the antigen epitopes are connected in series to play a remarkable synergistic effect, the immunoreactivity is remarkably enhanced, and the synergistic effect among the dominant antigen epitopes is particularly remarkable; the carrier protein also enhances the immunoreactivity of the epitope, the intensity of which is related to the performance of the epitope (fig. 2). The research method provides a new thought and a new technical means for accurately and efficiently screening dominant epitopes, and provides theoretical basis and technical support for accurately and efficiently screening epitopes, designing strong immunocompetence multi-epitope recombinant antigens, researching specific detection reagents and researching multi-epitope vaccines.

Screening epitope to literature report epitope immunoreactivity comparison

In order to further confirm that the obtained epitope 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 to construct recombinant expression plasmids pGEX-4T-p54G1, pGEX-4T-p54G2, pGEX-4T-p54G3 and pGEX-4T-p54G4, and expresses recombinant proteins, named GST-p54G1, GST-p54G2, GST-p54G3 and GST-p54G4, by BL21 (DE 3).

The immunoreactivity of the epitopes of the screening and literature reporting antigens of the present invention was tested using the ELISA described above. The results show that the immunoreactivity of the dominant epitopes p54d and p54e obtained by the invention and the serum positive to African swine fever is obviously stronger than that of the epitopes reported by the literature (p < 0.0001), and especially after the p54d and the p54e are connected in series and expressed in the form of multi-epitope VLP, the immunoreactivity of the recombinant protein of the tandem epitope is obviously enhanced (figure 3), which fully shows that the immunoreactivity of the epitope obtained by the invention is better than that of the epitope reported by the literature.

Table 2 literature reports epitopes of Georgia 2007 strain p54 protein

Naming the name	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. Sequence analysis of screened epitopes and reported epitopes

The dominant epitope obtained based on the invention has obvious immunological activity difference from the epitope reported in the literature, and the epitope obtained in the invention is different from the epitope reported in the literature for further diagnosis. The research compares and analyzes the amino acid sequences of the epitope obtained by the invention (table 1) and the epitope reported by the literature (table 2), 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 the reported epitope (93 aa-113 aa) in the literature, but the 5 '-end is less than 2 amino Acids (AT) and the 3' -end is less than 3 amino acids (PAT), the immunoreactivity of the epitope is obviously better than that of the epitope reported in the literature, and the amino acids AT the two sides possibly influence the immunoreactivity of the epitope, so that the novel dominant epitope of the p54 protein is obtained; the amino acid sequence of the dominant antigen epitope p54e (115 aa-122 aa) is compared with the amino acid sequence of the antigen epitope p (118 aa-127 aa) reported in the literature, and the results show that the two antigen epitopes are different in length and immunogenicity except PVTD motif, and the immunogenicity of the two antigen epitopes is different.

TABLE 3 screening of epitopes and amino acid sequence differential analysis of the epitopes reported in literature

8. Use of epitopes

The epitope of the p54 protein obtained by the invention, especially 3 new epitopes, namely p54c, p54d and p54e, especially two dominant epitopes of p54d and p54e, has the immunoreactivity remarkably superior to that of the epitope reported in the literature, and has different coding amino acid sequences, thus being the new epitope of the p54 protein. The epitope, especially the dominant epitope with strong immunoreactivity, not only enriches the African swine fever virus epitope database, but also provides a material basis and theoretical basis for researching and developing African swine fever serological detection reagents and kits thereof (ELISA, WB, colloidal gold chromatography test strips and the like), designing epitope antigens, and researching and developing epitope vaccines (including epitope monovalent/multivalent vaccines, synthetic peptide vaccines, serial connection with other pathogenic antigen epitopes or antigens and the like), and has very important significance.

It should be noted that the above-mentioned embodiments are to be understood as illustrative, and not limiting, the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made to the present invention without departing from its spirit or scope.

SEQUENCE LISTING

<110> the animal doctor institute of Lanzhou, china academy of agricultural sciences

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

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<170> PatentIn version 3.3

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ttttttcaac cggtttatcc gcggcattat ggtgagtgtt tgtcaccagg tggttcagct 60

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atgggcagca gcttttttca accggtttat ccgcggcatt atggtgagtg tttgtcacca 60

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atgggcagca gcaaaccagt tacggacaac ccagttggtg gttcaacagc aagtgtaggc 60

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Ser Val Tyr Lys Arg Pro Ala Pro Lys Pro Glu Gly Cys Ala Asp Ala

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Claims

1. The african swine fever virus p54 protein epitope peptide is characterized in that: the antigen epitope peptide is a single antigen epitope peptide, and the single antigen epitope peptide is a polypeptide with an amino acid sequence shown in SEQ ID NO:4 or p54d or SEQ ID NO: p54e shown in fig. 5.

2. A multi-epitope recombinant protein, characterized in that: the polyepitope peptide is p54ed, which p54ed is the polypeptide of SEQ ID NO:5 and p54e and SEQ ID NO:4, sequentially connecting p54d shown in the figure with a linker in series, and sequentially connecting the p54d with SpyCatcher, AP205 multi-epitope recombinant protein p54ED through GGGGS;

the amino acid sequence of the p54ED is shown as SEQ ID NO: shown at 17.

3. A recombinant protein obtained by connecting a gene encoding the epitope peptide according to claim 1 with an expression vector, expressing the gene, and purifying the gene.

4. A polynucleotide encoding the epitope peptide of claim 1 or the recombinant protein of claim 3.

5. An expression vector comprising the polynucleotide of claim 4.

6. A host cell comprising the expression vector of claim 5 or having integrated in the genome the polynucleotide of claim 4.

7. The use of the epitope peptide according to claim 1 in the preparation of African swine fever virus detection medicament.