African swine fever virus T cell epitope peptide for inducing swine T cell immune response and application of African swine fever virus T cell epitope peptide

Abstract

The invention discloses an African swine fever virus T cell epitope peptide for inducing swine T cell immune response and a composition of the African swine fever virus T cell epitope peptide. The amino acid sequence of the African swine fever virus T cell epitope peptide is shown as any one of SEQ ID NO.1-19. Or an amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO.1-19 and maintains the protein function shown in SEQ ID NO.1-19 through substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1-19; the composition comprises amino acids of which the sequences are SEQ ID NO.1-19. A group of ASFV key T cell antigens and epitope peptides are obtained by combining IFN-gamma ELISpot and flow cytometry screening, the ASFV key T cell antigens and epitope peptides can be used as vaccine components to induce strong T cell immune response, and T cells activated by the antigen peptides have the maximum immune protection potential and are essential key antigens for ASFV vaccine research and development.

Description

Technical Field

[0001] The present invention belongs to the field of biological science and diagnostic reagent technology, relates to the prevention and treatment of animal diseases, and specifically to African swine fever virus T cell epitope peptides that induce pig T cell immune response and applications thereof. Background Art

[0002] African swine fever (ASF) is an acute, hemorrhagic, highly contagious disease caused by the African swine fever virus (ASFV). It can cause 100% mortality in domestic pigs and wild boars and has become the biggest threat to the pig industry. In August 2018, genotype II ASFV was introduced into my country and then spread to Southeast Asia, causing huge economic losses to the pig industry.

[0003] ASFV is the only member of the genus ASFV in the family Asfrigiviridae and the only known insect-borne DNA virus. The viral genome is a 170-194kb linear double-stranded DNA, encoding more than 150 proteins, including 54 structural proteins and more than 100 non-structural proteins. There is still no commercial ASFV vaccine for ASFV. Finding and identifying protective antigens of ASFV is the key to the development of ASFV vaccines. Although the existing attenuated ASFV strains are effective, there are risks such as genetic recombination and virulence reversion. The existing ASFV inactivated vaccines have been shown to be unable to induce immune protection. Subunit vaccines have become an alternative option for ASFV vaccine development due to their reliable safety.

[0004] Existing studies have shown that ASF relies more on T cell immune-mediated immune protection. ASFV inactivated vaccines and subunit vaccines based on structural proteins p72, p54 and p30 cannot provide immune protection against ASFV even if neutralizing antibodies are induced. However, immunization with naturally attenuated OURT88 / 3 and NH / P68 strains on macrophages and bone marrow-derived dendritic cells can induce 100% protection against homologous or heterologous strains, and the lack of CD8 T cells through anti-CD8 antibodies can eliminate the immune protection induced by the vaccine. This shows that the T cell immune response induced by the attenuated strain of ASFV plays a key role in immune protection. Using IFN-γ ELISpot technology, Dixon et al. (2019) identified 38 proteins that can be recognized by T cells from pigs immune recovered from genotype I ASFV. Goatley et al. (2020) further found that the antigen components including p30, p54, p72, pB602L, pE199L, pEP153R, F317L, and MGF505-5R have immune protection. Portugal et al. (2024) proved that vaccination with 6 of the above antigens can induce protective immunity against genotype I ASFV. However, the protective antigens and T cell recognition epitopes of genotype II ASFV are not clear. Although many scientific research institutions at home and abroad have conducted research on protein and peptide vaccines such as p30, p54, and p72, there is no safe and effective ASF vaccine on the market so far. Therefore, using surviving pigs after immunization with genotype II ASFV to identify the ASFV antigens and T cell epitopes specifically recognized by their T cells can provide a theoretical basis for the development of new ASFV vaccines. Summary of the invention

[0005] The present invention aims at the technical problem to be solved, overcomes the shortcomings of the prior art and provides ASFV T cell antigens and epitope polypeptides with strong immunogenicity that can induce pig-specific T cell responses, which are screened by IFN-γ ELISpot technology.

[0006] The present invention also provides a method for identifying T cell subpopulations activated by different T cell epitope polypeptides of ASFV and the functional characteristics of the epitopes using flow cytometry (FCM).

[0007] In order to achieve the above objectives, the technical solution of the present invention is as follows: The present invention provides an African swine fever virus T cell epitope peptide that induces a pig T cell immune response, the amino acid sequence of which is as shown in any one of SEQ ID NO.1-19, or the amino acid sequence of SEQ ID NO.1-19 is obtained by replacing and / or deleting one or several amino acid residues and / or adding an amino acid sequence derived from SEQ ID NO.1-19 and maintaining the protein function shown in SEQ ID NO.1-19.

[0008] The present invention also provides a composition of African swine fever virus T cell epitope peptides that induce pig T cell immune response, wherein the composition comprises amino acids with sequences of SEQ ID NO.1-19.

[0009] The present invention further provides the use of the African swine fever virus T cell epitope peptide or its composition in the preparation of African swine fever virus vaccine, and the epitope peptide or epitope peptide composition is used as a candidate immunogen for the ASFV epitope vector vaccine.

[0010] The technical solution further optimized by the present invention is as follows: Preferably, the amino acid sequence of SEQ ID NO.1 is recognized by CD4 T cells of ASFV recovered pigs and contains SLA-II class restricted T cell epitopes; the amino acid sequence of SEQ ID NO.2-5 is recognized by CD8 T cells of ASFV recovered pigs and contains SLA-I class restricted T cell epitopes; the amino acid sequence of SEQ ID NO.6-19 is recognized by both CD4 and CD8 T cells of ASFV recovered pigs and contains SLA-I and -II class restricted T cell epitopes.

[0011] Preferably, the sequences are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.11 and SEQ ID NO.12, which are recognized by cytotoxic T cells (CTL) of ASFV-recovered pigs and contain SLA-I class-restricted T cell epitopes.

[0012] Preferably, the African swine fever virus T cell epitope peptides are respectively derived from ASFV CP204L , A151R , I215L , E184L , EP153R , E146L The gene or encoded protein, and the African swine fever virus T cell epitope peptide or its derived protein can be used to design the antigen component or combination formulation of the ASFV vaccine.

[0013] Preferably, the African swine fever virus T cell epitope peptide or its source protein is screened and verified from the peripheral blood of surviving recovered pigs immunized with an attenuated ASFV strain and then challenged with a virulent ASFV strain using IFN-γ ELISpot assay, T cell proliferation assay, intracellular cytokine staining and CD107a surface staining.

[0014] The present invention selects the entire amino acid sequence of the potential protective protein antigen of the ASFV epidemic strain in China, utilizes the peptide synthesis technology to synthesize overlapping polypeptides covering the entire sequence of each protein, immunizes piglets with the ASFV attenuated strain, infects the piglets with the ASFV virulent strain 29 days after immunization, collects peripheral blood mononuclear cells (PBMC) of the recovered pigs 5 days, 15 days and 42 days after infection, utilizes immunological test techniques such as IFN-γ ELISpot test, FCM, in vitro culture of T cells, etc., to screen and identify the dominant T cell epitope polypeptides of ASFV, and further analyzes the type of T cell subpopulation (CD4 T or CD8 T cell) activated by the epitope peptide, so as to provide a theoretical basis for the development of a new ASF subunit vaccine.

[0015] Preferably, the African swine fever virus T cell epitope peptide or its derived protein is co-cultured with the peripheral blood of ASFV recovered pigs for 18 hours, and specifically stimulates T cells to secrete IFN-γ (forming spots) in the pig IFN-γ ELISpot test; in the intracellular cytokine staining test, it specifically stimulates CD4 and / or CD8 T cells to secrete IFN-γ, TNF-α or IL-2; in the T cell proliferation test, it is co-cultured with CFSE-labeled peripheral blood of ASFV recovered pigs for 72 hours, and specifically stimulates CD4 and CD8 T cell proliferation (CSFE appears diluted); in the CD107a surface staining method, it specifically stimulates CD8 T cells to express CD107a on the surface.

[0016] Preferably, the coding gene of the African swine fever virus T cell epitope peptide or the protein derived therefrom is used for the design and application of ASFV subunit vaccine, recombinant vector vaccine, mRNA vaccine and DC vaccine, and has immune protection potential; whether the above-mentioned antigen components are used in the vaccine formulation can be determined by detecting whether there is a specific T cell response to each antigen peptide or protein in the body of the corresponding vaccine-immunized pigs through IFN-γ ELISpot test, T cell proliferation test, intracellular cytokine staining and CD107a surface staining method.

[0017] Preferably, at least one African swine fever virus T cell epitope peptide or its derived protein is selected to evaluate the strength of the immune protection and / or T cell immune response induced by any ASFV vaccine using IFN-γ ELISpot assay, T cell proliferation assay, intracellular cytokine staining and CD107a surface staining.

[0018] The present invention screens and identifies the immunodominant T cell antigen epitopes of African swine fever virus. The epitope polypeptide screened by the present invention is an antigen specifically recognized by the T cells of surviving pigs after ASFV immunization and can be used as a candidate immunogen for ASFV epitope vector vaccine. The ELISpot / FCM detection and identification method established by the present invention provides a powerful tool for the evaluation of the protective efficacy of ASFV candidate vaccines and the screening and identification of ASFV dominant epitopes, and also provides a theoretical basis for the research and development of ASFV subunit vaccines.

[0019] The beneficial effects of the present invention are as follows: The present invention combines IFN-γ ELISpot and FCM technology screening to obtain a group of ASFV key T cell antigens and epitope peptides, which can be used as vaccine components to induce strong T cell immune responses. The T cells activated by these antigen peptides have the greatest immune protection potential and are key antigens essential for the development of ASFV vaccines.

[0020] The present invention combines IFN-γ ELISpot test and FCM screening to identify a group of ASFV highly immunogenic T cell epitope peptides, and at the same time analyzes the functional characteristics of T cell subtypes and polypeptides activated by T cell epitope peptides, deepens the understanding of ASFV T cell epitope peptides, and provides an efficient technical solution for high-throughput screening of T cell and B cell epitopes including ASFV, enriches the types of ASFV T cell epitope polypeptides, and can greatly promote the research of ASF subunit vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the ASFV T cell epitope peptide screening assay of the present invention.

[0022] Figure 2 This is a dynamic diagram of the frequency of peptide pool-specific T cells at different time points after the immunization of pigs with the present invention.

[0023] Figure 3 Schematic diagram of the crossover method of the present invention for determining the epitope peptide that induces specific T cells to secrete IFN-γ.

[0024] Figure 4 This is a schematic diagram of the results of the preliminary screening of positive T cell epitope peptides by IFN-γ ELISpot validation of the present invention.

[0025] Figure 5 Schematic diagram of the results of identifying T cell subtypes activated by T cell epitope peptides using the ICS of the present invention.

[0026] Figure 6 This is a schematic diagram of the results of the proliferation assay of the present invention to identify ASFV T cell epitopes that induce CD4 T and CD8 T cell proliferation.

[0027] Figure 7This is a schematic diagram of the results of the FCM test for identifying cytotoxic T cell epitope peptides of the present invention. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail below in conjunction with the embodiments: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection authority of the present invention is not limited to the following embodiments.

[0029] The materials and reagents mentioned in the present invention can be obtained by the public from domestic and foreign commercial channels or free channels. Example 1 Screening of T cell dominant epitope peptides

[0030] 1. ASFV peptide synthesis Based on the amino acid sequences of pA151R, pE184L, pE199L, pI215L, pE146L, pI8L, and pEP153R of the ASFV CADC_HN09 strain prevalent in China (GenBank accession number: MZ614662), overlapping peptides of proteins were synthesized by Nanjing GenScript Biotech Co., Ltd. Each peptide contained 20 amino acids, and adjacent peptides contained 10 amino acids overlapping. The IEDB website was used to predict and synthesize potential epitopes in p72, p54, pB602L, pMGF505-4R, and pM448R proteins that may be recognized by both MHC-I and -II molecules. All peptides were sorted into Figure 3 Arrange as shown, dilute with sterile PBS to prepare a single polypeptide solution with a concentration of 5 mg / mL. Take 20 μL of 10 single polypeptide solutions in each column and mix them into one peptide pool, take 20 μL of 14 single polypeptide solutions in each row and mix them into another peptide pool, and so on, a total of 24 peptide pools are prepared.

[0031] 2. ASFV attenuated strain immunity and challenge protection test Eight healthy piglets aged 6 weeks were randomly divided into two groups, including 5 ASFV attenuated strain immunization groups and 3 control groups. The ASFV CADC_HN09 attenuated strain was used for immunization. The immunization was performed by neck intramuscular injection with an injection volume of 10 6 ASFV / head, the control group was not immunized. 29 days after immunization, the immunized group and the control group were injected intramuscularly with 10 HAD 50 The ASFV CADC_HN09 virulent strain was given a dose of 100 mg / kg. Anticoagulated blood was collected from the immune challenge group 5, 15, 42 days after the challenge and 5 days after the second challenge (the second challenge was 47 days after the first challenge), and PBMC was isolated for IFN-γ ELISpot and FCM identification. The experimental process is as follows: Figure 1After the challenge, the clinical symptoms were continuously observed, and it was found that all the pigs in the control group died about 10 days after the challenge, indicating that the challenge was successful; all the pigs in the immune challenge group survived the entire test and had no obvious clinical symptoms such as fever, indicating that the weak strain induced a good protective effect.

[0032] 3. Isolation of Porcine PBMC At each sampling time point described in step 2, venous blood from the test pigs was collected aseptically using a negative pressure anticoagulation tube, and pig PBMCs were isolated using a porcine peripheral blood mononuclear cell isolation kit (Tianjin Haoyang Biological Company). For specific steps, refer to the manufacturer's instructions. Red blood cells in PBMCs were lysed with red blood cell lysis buffer. After trypan blue staining, live cells were counted using a hemocytometer.

[0033] 4. IFN-γ ELISpot assay to identify T cell epitope peptides Add the PBMC single cell suspension prepared in step 3 above to each well of the ELISpot 96-well plate, and 2×10 5 / well. Add the peptide pool or single polypeptide solution to each well accordingly, and place the culture plate in a cell culture incubator at 37°C and 5% CO2 for 18 hours. Take out the plate, discard the supernatant, and wash with sterile distilled water. Add biotin-labeled anti-swine IFN-γ antibody (P2C11-Biotin Mabtech, Sweden) and incubate for 2 hours. After washing with PBS, add the secondary antibody Streptavidin-ALP (Mabtech, Sweden) and incubate for 1 hour, and repeat the washing steps. Add the substrate BCIP / NBT-plus (Mabtech, Sweden) and let stand until spots appear. Discard the liquid in the well and rinse with ultrapure water. Air dry, and use the ImmunoSpot Analyzer ELISpot (CTL, USA) analyzer to scan and count the number of spots. The results are as follows Figure 2 As shown, peptide pools No. 1, 4, 5, 11, 12, 14, 16, 17, 19, 20, 22, and 24 were recognized by 2 or more pigs and induced the most specific T cells, and were strong positive peptide pools (all ELISpot results were deducted from the natural background). Figure 2 A is the frequency dynamics of peptide-specific T cells 5 days after challenge; B is the frequency dynamics of peptide-specific T cells 15 days after challenge; C is the frequency dynamics of peptide-specific T cells 42 days after challenge; the dotted line is the strong positive dividing line. Figure 3 As shown in Figure 2, 40 strong positive epitope peptides were initially screened using the cross-over method. Figure 4 As shown, different strong positive peptides were used to stimulate the PBMC of recovered pigs to perform IFN-γ ELISpot test to further verify Figure 3 result. Figure 4The upper middle figure shows the identification results of epitope peptides on recovered pig No. 1; the lower figure shows the identification results of epitope peptides on recovered pig No. 2. As a result, 19 strong positive T cell epitope peptides were identified, including pA151R-P6, pA151R-P7, pA151R-P9, pE184L-P1, pE184L-P4, pE184L-P6, pE184L-P7, pE184L-P10, pI215L-P9, pI215L-P11, pI215L-P12, pI215L-P18, pI215L-P20, and pEP153R-P10 (see Table 1).

[0034] Table 1 ASFV T cell epitope peptide sequences and functional characteristics

[0035] Note: +, indicates a positive test; -, indicates a negative test; N, indicates not determined. Example 2 ICS assay to identify T cell subtypes activated by T cell epitope peptides

[0036] 1. Peptide stimulation PBMCs from two recovered pigs were plated in a 96-well U-shaped plate, and 19 single peptide solutions (5 mg / ml) dissolved in PBS were added to each well, with two replicates, and the final peptide concentration was 5 μg / mL. The cells were cultured in a cell culture incubator for 18 h.

[0037] 2. Flow Cytometry (FCM) Surface Staining Add the primary antibody mixture [containing anti-swine CD3ε, CD4, CD8α, TCRγδ molecules flow cytometry antibodies (BD, USA)] to each well, resuspend the cells, and incubate for 25 min. Add PBS solution containing 0.5% BSA, centrifuge and discard the supernatant. Fix the cells with 4% PFA.

[0038] 3.ICS (Intracellular Cytokine Staining) The cells were permeabilized with Perm / Wash permeabilization buffer (BD, USA) for 15 min and incubated with IFN-γ antibody solution (IFN-γ antibody was prepared with Perm / Wash buffer) for 25 min. The antibody solution was washed away with Perm / Wash, and the cells were fixed with 0.5% PFA. The expression of IFN-γ by CD4 T and CD8 T cells under single peptide stimulation was detected by flow cytometry. Figure 5 The left picture in the middle shows an epitope peptide that can stimulate CD4 T cells to secrete IFN-γ; the right picture shows an epitope peptide that can stimulate CD8 T cells to secrete IFN-γ; 1640 in the picture indicates a negative control cultured with 1640 medium; PMA / Iono indicates a positive control. Figure 5As shown, pI215L-P12 can only stimulate CD4 T cells to secrete IFN-γ, and contains SLA-II restricted T cell epitopes. Four peptides, pA151R-P1, pE184L-P2, and pI215L-P13 / P18, can only induce CD8 T cells to secrete IFN-γ, and contain SLA-I restricted T cell epitopes. However, 14 peptides, pA151R-P6 / P7 / P9, pE184L-P2 / P4, pI215L-P9 / P11 / P13 / P18 / P20, pE146L-P8 / 12, and pEP153R-P2 / P3, can simultaneously activate CD4 T and CD8 T cells, and contain SLA-I and -II restricted T cell epitopes. Example 3 Cell proliferation assay to identify epitope peptides that can stimulate CD4 T and CD8 T cell proliferation

[0039] 1. CFSE labeling Take the recovered pig PBMC, resuspend it with an appropriate amount of 37°C CFSE dye solution, and mix gently. Incubate at 37°C in the dark. Then add complete culture medium to stop labeling. Centrifuge and discard the supernatant. Resuspend the cells with an appropriate amount of complete culture medium and spread them on a 96-well U-shaped plate. Add the peptide solution to each well with a final concentration of 5 μg / mL and culture in a cell culture incubator for 3 days.

[0040] 2. FCM Identification Incubate with primary antibody (including flow cytometry antibodies against porcine CD3ε, CD4, CD8α, and TCRγδ molecules): The specific steps are the same as the above flow cytometry surface staining. Figure 6 The left picture in the middle shows that the epitope peptide promotes the proliferation of CD4 T cells; the right picture shows that the epitope peptide promotes the proliferation of CD8 T cells; 1640 represents the negative control; CFSE weak positive represents the proliferating T cells. Figure 6 As shown, pI215L-P12 can only induce CD4 T cells to divide and proliferate, and contains CD4 T cell epitopes; pA151R-P1, pE184L-P2 / P4 and pI215L-P18, a total of 4 polypeptides, can only induce CD8 T cells to divide and proliferate, and contain CD8 T cell epitopes; p30-P14, pA151R-P6 / P7 / P9, pE184L-P1 / P7 and pI215L-P11 can simultaneously induce CD4 T and CD8 T cells to divide and proliferate. This result can confirm the result of Example 2. Example 4 Identification of ASFV Cytotoxic T Cell Epitope Peptides

[0041] In order to further identify the CTL epitope peptides in the CD8 T cell epitope peptides, PBMCs from two recovered pigs were plated in a 96-well U-shaped plate. The peptides recognized by CD8 T cells were added to the wells respectively, and CD107a antibody (Bio-rad, USA) was added at the same time. The cells were cultured in a cell culture incubator for 18 h. Then, flow cytometry surface staining was performed as in Example 2. Figure 7 In the figure, 1# is the result of identification on recovered pig No. 1; 2# is the result of identification on recovered pig No. 2; 1640 is the negative control. Figure 7 As shown, a total of 7 peptides, including pA151R-P1 / P7, pE184L-P1 / P7 and pI215L-P9 / P12 / P13, can induce CD8αβ T cells (CTLs) to express CD107a molecules, which are cytotoxic T cell epitope peptides.

[0042] The above description is only a specific implementation of the present invention, but the protection scope of the present invention is not limited thereto. Any person familiar with the technology can understand and think of any changes or substitutions within the technical scope disclosed by the present invention, which should be included in the scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. An African swine fever virus T cell epitope peptide that induces pig T cell immune response, characterized in that: Its amino acid sequence is as shown in any one of SEQ ID NO.1-19, or the amino acid sequence of SEQ ID NO.1-19 is derived from SEQ ID NO.1-19 by replacing and / or deleting one or several amino acid residues and / or adding an amino acid sequence that retains the protein function shown in SEQ ID NO.1-19.

2. The composition of African swine fever virus T cell epitope peptides that induces pig T cell immune response according to claim 1, characterized in that: The composition comprises amino acids with sequences of SEQ ID NO. 1-19.

3. The African swine fever virus T cell epitope peptide according to claim 1, characterized in that: The amino acid sequence of SEQ ID NO.1 is recognized by the CD4 T cells of ASFV recovered pigs and contains SLA-II class restricted T cell epitopes; the amino acid sequence of SEQ ID NO.2-5 is recognized by the CD8 T cells of ASFV recovered pigs and contains SLA-I class restricted T cell epitopes; the amino acid sequence of SEQ ID NO.6-19 is recognized by both the CD4 and CD8 T cells of ASFV recovered pigs and contains SLA-I and -II class restricted T cell epitopes.

4. The African swine fever virus T cell epitope peptide according to claim 1, characterized in that: The sequences are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.11 and SEQ ID NO.12, which are recognized by cytotoxic T cells of ASFV recovered pigs and contain SLA-I class restricted T cell epitopes.

5. Use of the African swine fever virus T cell epitope peptide according to claim 1 or the combination of the African swine fever virus T cell epitope peptide according to claim 2 in the preparation of an African swine fever virus vaccine, characterized in that: The epitope peptide or epitope peptide composition serves as a candidate immunogen for the ASFV epitope vector vaccine.

6. The African swine fever virus T cell epitope peptide according to claim 1, characterized in that: The African swine fever virus T cell epitope peptides are respectively derived from ASFV CP204L , A151R , I215L , E184L , EP153R , E146L The gene or encoded protein, and the African swine fever virus T cell epitope peptide or its derived protein can be used to design the antigen component or combination formulation of the ASFV vaccine.

7. The African swine fever virus T cell epitope peptide according to claims 1 and 2, characterized in that: The African swine fever virus T cell epitope peptide or its source protein is screened and verified from the peripheral blood of surviving recovered pigs immunized with ASFV attenuated strains and then challenged with ASFV virulent strains using IFN-γ ELISpot test, T cell proliferation test, intracellular cytokine staining and CD107a surface staining.

8. The African swine fever virus T cell epitope peptide according to claim 7, characterized in that: The African swine fever virus T cell epitope peptide or its derived protein was co-cultured with the peripheral blood of ASFV recovered pigs for 18 hours, and specifically stimulated T cells to secrete IFN-γ in the pig IFN-γ ELISpot test; in the intracellular cytokine staining test, it specifically stimulated CD4 and / or CD8 T cells to secrete IFN-γ, TNF-α or IL-2; in the T cell proliferation test, it was co-cultured with CFSE-labeled peripheral blood of ASFV recovered pigs for 72 hours, and specifically stimulated CD4 and CD8 T cell proliferation; in the CD107a surface staining method, it specifically stimulated CD8 T cells to express CD107a on the surface.

9. The African swine fever virus T cell epitope peptide according to claims 1 and 2, characterized in that: The coding gene of the African swine fever virus T cell epitope peptide or the protein derived therefrom is used for the design and application of ASFV subunit vaccine, recombinant vector vaccine, mRNA vaccine and DC vaccine, and has immune protection potential; whether the above-mentioned antigen components are used in the vaccine formula can be determined by detecting whether there is a specific T cell response of each antigen peptide or protein in the body of the corresponding vaccine-immunized pigs through IFN-γ ELISpot test, T cell proliferation test, intracellular cytokine staining and CD107a surface staining method.

10. The African swine fever virus T cell epitope peptide according to claims 1 and 2, characterized in that: At least one African swine fever virus T cell epitope peptide or its source protein was selected and the IFN-γ ELISpot assay, T cell proliferation assay, intracellular cytokine staining and CD107a surface staining were used to evaluate the strength of immune protection and / or T cell immune response induced by any ASFV vaccine.

Images