CN114409743B - African swine fever virus p54 protein antigenic epitope and its application - Google Patents

Abstract

The invention relates to the antigenic epitope of African swine fever virus p54 protein and its application, and belongs to the field of genetic engineering. It uses the primary structural amino acid sequence of the African swine fever virus p54 (E183L) membrane protein as material and predicts its antigen through the antigenic epitope database. Epitopes and synthesize corresponding peptide molecules based on amino acid sequences, construct GST/epitope recombinant proteins, phage (virus-like particles, VLP) display multiple epitopes and other epitope antigens. Through the immunological detection test of African swine fever virus positive serum, 5 antigenic epitopes (peptides) were determined to be the antigenic epitopes of the African swine fever virus p54 protein. The acquisition of these antigenic epitopes has laid the foundation for the development of urgently needed strategic prevention and control products such as African swine fever detection reagents and kits, monoclonal antibody preparation, and safe and differential diagnostic epitope vaccines using antigenic epitopes as materials. Not only will it produce huge economic benefits, but it will also have important social benefits.

Description

African swine fever virus p54 protein antigenic epitope and its application

Technical field

The invention belongs to the technical field of genetic engineering, and specifically relates to the antigenic epitope of African swine fever virus p54 protein and its application.

Background technique

African swine fever is an acute and severe infectious disease in pigs caused by the African swine fever virus, with a mortality rate of up to 100%. The World Organization for Animal Health (OIE) lists it as a notifiable animal disease, and my country lists it as a class of animal infectious disease that is focused on prevention. Since it was introduced into our country in August 2018, the epidemic has spread throughout the country in less than a year. The epidemic has not been eliminated so far, causing major economic losses to our country's pig industry. The number of live pigs has dropped sharply, and the price of pork has soared, causing caused huge economic losses. The State Council, national ministries and commissions and other departments have taken various comprehensive measures to restore pig production and stabilize prices. However, the continuous spread and sporadic epidemic of the epidemic have caused extremely serious pollution to the environment. As of the beginning of 2021, 22 strains have been isolated from the field, including attenuated strains with large gene deletions, indicating that the environment is seriously polluted and the strains are complex. , prevention and control are difficult. As the world's largest pig-raising country, my country supplies more than 50% of its pork supply. The impact of African swine fever on pig-raising not only threatens the healthy and sustainable development of the pig-raising industry, but also affects a series of social and political issues such as people's livelihood, food safety and biosecurity. The problem has become one of the major animal diseases that the country urgently needs to solve.

African swine fever virus is an important animal virus with a century-old history, and research on vaccines has never stopped. However, there is still a lack of safe and effective vaccines that can be used in practice. The reason is mainly due to the large genome and complex structure of African swine fever virus, and the protective antigen, immune and infection mechanisms are unclear. So far, it is impossible to explain and solve the theoretical problems and technical bottlenecks of the inactivated African swine fever vaccine that cannot provide immune protection; the live-attenuated vaccine causes morbidity and death, reduced production capacity, and may cause virulence reversion, genetic recombination and other biological safety issues; gene Theoretical issues surrounding the poor protective efficacy of engineered subunit vaccines, DNA vaccines, and live vector vaccines. The country urgently needs a safe, effective, and differentially diagnostic African swine fever vaccine to improve my country's ability and technical level in the prevention, control and purification of African swine fever epidemics, and to ensure the healthy and sustainable development of my country's pig industry as the world's largest pig-raising country. Food safety and biosecurity of animal origin.

Based on this, the African swine fever virus is extremely complex, vaccine research is slow, there is a lack of safe, effective, and identifiable vaccines, and the country is in urgent need of prevention and control products. It is necessary to use new ideas and find new ways to discover more protective antigens to solve the dilemma of a lack of key materials for genetic engineering vaccine research due to the large and complex genome of African swine fever virus and unclear protective antigens.

Contents of the invention

In response to the above problems, the present invention uses the primary structural amino acid sequence of the African swine fever virus p54 (E183L) membrane protein as material to predict and determine 5 antigenic epitopes (peptides) of the African swine fever virus p54 protein. These antigenic epitopes The acquisition has laid the foundation for the development of urgently needed strategic prevention and control products such as African swine fever detection reagents and kits, monoclonal antibody preparation, and safe and differential diagnostic epitope vaccines using antigenic epitopes as materials.

The first object of the present invention is to provide the African swine fever virus p54 protein antigen epitope, which is a single antigen epitope (synthetic peptide), a protein carrier recombinant single antigen epitope or a multiple antigen epitope; the single antigen epitope The amino acid sequence is an epitope peptide, carrier protein/antigen epitope recombinant protein shown in any one of SEQ ID NO: 1 to 5; the multiple antigen epitopes are two or more single antigen epitopes. The epitopes are connected in tandem and adjacent epitopes are connected using spacers, and then connected to phages or self-assembly proteins to express VLP to display multiple epitopes.

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

The multi-antigen epitope is preferably p54abc or p54ed. The p54abc is p54a shown in SEQ ID NO: 1, p54b shown in SEQ ID NO: 2 and p54c shown in SEQ ID NO: 3 sequentially connected in series through GS, Then, it is obtained by connecting AP205 through GGGGS; the p54ed is obtained by connecting p54e shown in SEQ ID NO: 5 and p54d shown in SEQ ID NO: 4 in series through GS, and then connecting SpyCather and AP205 through GGGGS in sequence.

The second object of the present invention is to provide a recombinant protein, which is obtained by connecting the above-mentioned antigenic epitope to an expression vector and then expressing and purifying the protein.

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

The fourth object of the present invention is to provide an expression vector containing the above-mentioned polynucleotide.

The fifth object of the present invention is to provide a host cell that contains the above-mentioned expression vector or has the above-mentioned polynucleotide integrated into the genome.

The sixth object of the present invention is to provide the application of the above-mentioned antigenic epitopes, fusion proteins, multi-epitope VLPs, polynucleotides, expression vectors or host cells in the preparation of African swine fever virus detection drugs and anti-African swine fever vaccines.

Beneficial effects:

The present invention uses the primary structural amino acid sequence of African swine fever virus p54 (E183L) membrane protein as material, predicts its antigenic epitope through the antigenic epitope database, and synthesizes corresponding peptide molecules based on the amino acid sequence. Through immunological detection of African swine fever virus-positive serum, it was determined that the five antigenic epitopes (peptides) in Table 1 are all antigenic epitopes of the African swine fever virus p54 protein, among which p54d and p54e are the dominant epitopes. The acquisition of these antigenic epitopes has laid the foundation for the development of urgently needed strategic prevention and control products such as African swine fever detection reagents and kits, monoclonal antibody preparation, and safe and differential diagnostic epitope vaccines. It will not only generate huge economic benefits, but also Have important social benefits.

Description of drawings

Figure 1 is a diagram showing the results of identification of p54 protein epitope in African swine fever virus-positive serum;

Figure 2 is a diagram of the immunoreactivity evaluation of epitope recombinant proteins;

Figure 3 is a comparative diagram of the immunoreactivity of the antigen epitopes obtained in the present invention and the antigen epitopes reported in the literature;

Note: ****P<0.0001, ***P<0.001, **P<0.01 means the difference is extremely significant; * means the difference is significant (P<0.05); P>0.05 means the difference is not significant.

Detailed ways

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.

, Antigen epitope prediction and screening

Using the amino acid sequence of the p54 protein of African swine fever virus as material, the IEDB Analysis Resource, ABCpred and Scratch Protein Predictor online databases were used to predict its antigenic epitopes, and p54 was determined based on the standard that all score values in the above four databases were not less than 0.6. 5 antigenic epitopes of the protein (Table 1).

Table 1 Antigenic epitopes 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 antigenic epitope in Table 1, "Genscript Biotechnology Company" was entrusted to synthesize short peptide molecules corresponding to it, with a purity of not less than 95%.

, Establishment of indirect ELISA method

Randomly select 3 synthetic short peptides, at a concentration of 1 μg/ml, 100 μl/well, coat the MK100 peptide 96-well ELISA plate, overnight at 4°C; spin the plate dry, and block with PBST containing 5% BSA at 37°C for 2 hours, 200 μl /well; dry the plate and wash it 4-5 times with PBST (pH7.4); dilute the SPF pig negative serum/ASFV standard positive serum according to the column (1-12) times, the starting concentration is 1:25, 8 Repeat the wells (1A-1H), 100 μl/well, react at 37°C for 1 hour; discard the liquid, wash thoroughly with PBST (pH7.4) 4-5 times; then dilute the HRP-labeled rabbit antibody according to the row (AH) times IgG, the starting concentration is 1:2500, 12 wells are repeated (A1-A12), react at 37°C for 1 hour; wash thoroughly with PBST 4-5 times, add chromogenic substrate and act for 10-15 minutes, measure the OD _450nm absorbance value .

Result judgment standard: The ratio of sample OD _450nm value (S)/negative control (N) is greater than or equal to 2.1 as positive, and less than 2.1 is negative.

, African swine fever virus positive serum screening and identification of antigenic epitopes

African swine fever virus-positive serum (purchased from the China Veterinary Drug Supervision Institute) was used as a standard to screen antibodies, and the antigenic epitopes of p54 protein were screened and identified through the above-mentioned ELISA. The results show that the five predicted antigenic epitopes (peptides) (Table 1) can all produce specific immune reactions with African swine fever virus-positive serum. Compared with the negative control serum, the statistical difference is significant (Figure 1), suggesting that the prediction The five epitopes are all epitopes of p54 protein, among which the two epitopes p54d and p54e have the strongest immunoreactivity and are the dominant epitopes of p54 protein.

, further screen and identify antigenic epitopes by introducing carrier protein GST and epitope tandem

5.1 Construction, protein expression and purification of recombinant expression plasmids for single epitope and tandem epitope

In order to further verify the immunoreactivity of the above-mentioned antigenic epitopes, the present invention introduces GST carrier protein to completely antigenize the antigenic epitopes and connect the epitopes in series to increase the titer of the antigenic epitopes, improve the immunoreactivity of the antigenic epitopes, and make the screening results more accurate. for reliability. In this study, the DNA of the selected antigenic epitopes p54a, p54b, p54c, p54d, and p54e were cloned into the pGEX-4T-1 expression vector to construct recombinant expression plasmids pGEX-4T-p54a, pGEX-4T-p54b, and pGEX-4T. - p54c, pGEX-4T-p54d, pGEX-4T-p54e, the target recombinant proteins were obtained through expression and purification of E. coli BL21 (DE3), and were named GST-p54a, GST-p54b, GST-p54c, GST-p54d, and GST in sequence. -p54e. The same method as above was used to express the epitope of p54 reported in the literature. The epitope was completely antigenized by GST protein and purified by affinity chromatography for immunoreactivity comparison.

The three epitopes p54a, p54b and p54c of the above antigenic epitopes (Table 1) are sequentially concatenated through GS (the p54abc concatenated gene is shown in SEQ ID NO: 10, and the encoded amino acid is shown in SEQ ID NO: 11), and then AP205 is connected through GGGGS, which is a multi-epitope fusion gene (the p54abc-AP205 gene sequence is shown in SEQ ID NO: 12, and the encoded amino acid is shown in SEQ ID NO: 13); the two dominant antigenic epitopes p54e and p54d are connected in series through the GS sequence (The p54ed tandem gene sequence is shown in SEQ ID NO: 14, and the encoded amino acid is shown in SEQ ID NO: 15), and then the GGGGS sequence is used to connect SpyCather and AP205 to form a multi-epitope fusion gene (the p54ed-AP504 gene sequence is shown in SEQ ID NO :16, the encoded amino acid is as shown in SEQ ID NO:17), clone it and the phage gene into the pET-28a (+) expression vector respectively, and construct the recombinant expression plasmids p54abc and pET-28/ in which the phage displays tandem antigen epitopes. p54ed, a recombinant protein (multi-epitope VLP) expressed by phage displaying multiple epitopes through E. coli BL21 (DE3), named p54ABC and p54ED respectively.

SDS-PAGE results showed that all the above-mentioned recombinant proteins were expressed in soluble form, and the recombinant proteins used to coat antigens were obtained by affinity chromatography.

Introduce carrier protein to completely antigenize the epitope, and use multi-epitope VLP to screen antigenic epitopes

The established ELISA was used to measure the immunoreactivity of single-epitope recombinant proteins and multi-epitope VLPs, and further screen and determine antigenic epitopes. The results showed that after the introduction of the carrier protein GST, the immunoreactivity of the antigenic epitopes was enhanced (p<0.05), and the dominant antigenic epitopes p54d and p54e were significantly enhanced; especially after their antigenic epitopes were connected in series and expressed in the form of multi-epitope VLP , the strong immunoreactivity of the dominant antigenic epitopes is more prominent, which is better than the immunoreactivity of the single-epitope recombinant protein introduced into the GST protein, further confirming that p54d and p54e are the dominant antigenic epitopes. In short, multi-epitope VLP has the strongest immunogenicity, followed by single-epitope recombinant protein, which is better than synthetic peptide, indicating that the serial connection of antigen epitopes exerts an obvious synergistic effect and significantly enhances immune reactivity. The synergistic effect between antigenic epitopes is particularly significant; the carrier protein can also increase the immunoreactivity of the antigenic epitope, and the intensity of the improvement is related to the performance of the antigenic epitope (Figure 2). This research method provides new ideas and technical means for accurate and efficient screening of dominant antigen epitopes. Through the research method of the present invention, it is possible to accurately and efficiently screen antigen epitopes, design multi-epitope recombinant antigens with strong immune activity, and study specific detection reagents. , providing theoretical basis and technical support for the development of multi-epitope vaccines.

Comparison of immunoreactivity between screened epitopes and epitopes reported in the literature

In order to further confirm that the antigenic epitope obtained in the present invention is a new antigenic epitope, in this study, the antigenic epitope reported in the literature (Table 2) was cloned into the pGEX-4T-1 expression vector according to the above method, and the recombinant expression plasmid pGEX-4T- was constructed. p54G1, pGEX-4T-p54G2, pGEX-4T-p54G3 and pGEX-4T-p54G4 expressed recombinant proteins through BL21(DE3) and were named GST-p54G1, GST-p54G2, GST-p54G3 and GST-p54G4.

The above-mentioned ELISA was used to detect the immunoreactivity of the antigen epitopes screened in the present invention and reported in the literature. The results show that the present invention obtains dominant antigenic epitopes p54d and p54e, and the immunoreactivity with African swine fever-positive serum is significantly stronger than the antigenic epitopes reported in the literature (p<0.0001), especially when p54d and p54e are connected in series to form a multi-expression After expressed in VLP form, the immunoreactivity of the tandem epitope recombinant protein was significantly enhanced (Figure 3), which fully demonstrates that the immunoreactivity of the antigen epitope obtained by the present invention is better than that reported in the literature.

Table 2 Antigenic epitopes of p54 protein of Georgia 2007 strain reported in the literature

name Position (amino acid) Sequence (5´-3´) Position number in the sequence list 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 Screened antigen epitopes and literature reported antigen epitope sequence analysis

Based on the above-mentioned advantageous antigenic epitopes obtained by the present invention and those reported in the literature, which have obvious differences in immune activity, in order to further confirm the diagnosis, the antigenic epitopes obtained by the present invention are different from those reported in the literature. In this study, a comparative analysis was conducted on the amino acid sequences of the antigenic epitopes obtained in the present invention (Table 1) and the antigenic epitopes reported in the literature (Table 2). The results show (Table 3) that the antigenic epitope p54c (80aa~95aa) is the first of the present invention. Found but not reported; the dominant epitope p54d (95aa~110aa) overlaps with the amino acid sequence of the epitope reported in the literature (93aa~113aa), but there are two amino acids (AT) less at the 5′ end and less at the 3′ end. 3 amino acids (PAT), the immunoreactivity of this antigenic epitope is significantly better than that reported in the literature, indicating that the amino acids on both sides may affect the immune effect of the antigenic epitope. Therefore, the present invention obtains a new protein whose antigenic epitope is p54 protein. Dominant antigenic epitope; After comparing the amino acid sequence of the dominant antigenic epitope p54e (115aa~122aa) with the one reported in the literature (118aa~127aa), it was found that except for the PVTD motif, the two epitopes have the same length and amino acids at both ends. The sequences are all different and the immunogenicity is also different. The immunogenicity of the antigenic epitope obtained by the present invention is better, which fully proves that the antigenic epitope obtained by the present invention is a new antigenic epitope.

Table 3 Analysis of amino acid sequence differences between screened antigen epitopes and literature reported antigen epitopes

8. Use of antigenic epitopes

The antigenic epitopes of the p54 protein obtained by the present invention, especially the three new antigenic epitopes, namely p54c, p54d and p54e, especially the two dominant antigenic epitopes p54d and p54e, have significantly better immune reactivity than the antigens reported in the literature. The epitope, and the encoded amino acid sequence is different, is a new antigenic epitope of p54 protein. The acquisition of these epitopes, especially the dominant epitopes with strong immune reactivity, not only enriches the African swine fever virus epitope database, but also provides a basis for the development of African swine fever serological detection reagents and kits (ELISA, WB, colloid Gold chromatography test strips, etc.), design epitope antigens, and develop epitope vaccines (including epitope monovalent/multivalent vaccines, synthetic peptide vaccines, and other pathogenic antigen epitopes or antigens in series, etc.) provide a material basis and theoretical basis. , is of great significance.

It should be noted that the above-described embodiments should be understood as illustrative rather than limiting the scope of the present invention, which shall be determined by the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present invention still fall within the protection scope of the present invention without departing from the essence and scope of the present invention.

SEQUENCE LISTING

<110> Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences

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

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Claims (7)

1. African swine fever virus p54 protein antigen epitope peptide, characterized in that: the antigen epitope peptide is a single antigen epitope peptide, and the single antigen epitope peptide is p54d with an amino acid sequence as shown in SEQ ID NO: 4 Or p54e shown in SEQ ID NO:5. 2. A multi-epitope recombinant protein, characterized in that: the multi-epitope peptide is p54ed, and the p54ed is p54e shown in SEQ ID NO: 5 and p54d shown in SEQ ID NO: 4 connected in series through a linker, Then it is connected to SpyCatcher and AP205 multi-epitope recombinant protein p54ED through GGGGS sequence; The amino acid sequence of p54ED is shown in SEQ ID NO: 17. 3. A recombinant protein obtained by connecting a gene encoding the epitope peptide as claimed in claim 1 to an expression vector and then expressing and purifying the protein. 4. A polynucleotide encoding the epitope peptide according to claim 1 or the recombinant protein according to claim 3. 5. An expression vector containing the polynucleotide of claim 4. 6. A host cell containing the expression vector of claim 5 or having the polynucleotide of claim 4 integrated into the genome. 7. Application of the antigenic epitope peptide according to claim 1 in the preparation of African swine fever virus detection medicaments.