ALV-J MHC-B21 restricted epitope peptide and its application

Abstract

The invention discloses an ALV-J MHC-B21 restricted epitope peptide and its application, belonging to the field of bioengineering. The invention utilizes the identified restriction motif of B21 haplotype SPF chicken MHC class I molecule binding polypeptide to systematically screen the potential epitopes of MHC B21 restricted ALV-J, and finally determines the immunogenic polypeptide epitope through functional experimental verification, the polypeptide sequence of which is PRLMGPFYEQL, which provides a material and theoretical basis for the research and development of ALV-J epitope vaccine.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to an ALV-J MHC-B21 restricted epitope peptide and application thereof. Background Art

[0002] Avian Leukosis Virus is a single-stranded, positive-strand RNA virus belonging to the genus Alpharetrovirus of the family Retroviridae. It can be divided into 11 subtypes based on host range and antigenic differences of viral envelope proteins. Among them, infection with subtype J avian leukosis virus can induce tumors such as myelocytoma and hemangioma in chickens. Since its discovery, it has caused serious damage to the global poultry industry. Like other subtypes of avian leukosis virus, the J subtype avian leukosis virus has three main coding genes in its genome, namely capsid protein gene (gag), polymerase gene (pol) and envelope glycoprotein gene (env), expressing four proteins: Gag, Pol, Gp85 and Gp37.

[0003] Type I major histocompatibility complex is expressed on the surface of all nucleated cells and is mainly involved in endogenous antigen presentation. It processes intracellular antigens into peptides and presents them to downstream CD8+T cells for recognition, thereby activating the body's cellular immunity to eliminate abnormal cells. The presented antigen peptides are called cytotoxic T cell (CTL) epitopes. Vaccines developed based on CTL epitopes can enable the body to produce high levels of cellular immunity, which is an important direction for the development of vaccines for various livestock and poultry diseases. Reports have shown that CD8+T cells play a key role in resisting ALV infection, which shows that the development of epitope vaccines that can stimulate the body to produce cellular immunity is of great significance for the prevention and control of ALV. However, there are currently few reports on ALV epitope vaccines.

[0004] Immunogenic epitopes are a prerequisite for inducing T cells to produce immune effects, so the preparation of epitope vaccines must first screen for immunogenic peptide epitopes, and antigenic epitopes are recognized by downstream TCRs by binding to MHC class I molecules. Therefore, understanding the restriction motifs of MHC class I molecules binding epitopes is of great significance for the development of epitope vaccines. At present, chickens can be divided into 29 haplotypes from B1 to B29 based on the gene sequence of the MHC B gene region; MHC class I molecules are polymorphic, and the restriction motifs of MHC class I molecules binding epitopes of different ploidy types are not the same. The motifs of the haplotypes B4, B12, B15, B19, and B21 have been reported for chicken MHC class I molecules, and the motif of the B21 haplotype was determined by Michael Koch et al. At the same time, according to reports, we know that B21 haplotype SPF chickens are resistant to Marek's disease virus infection, and avian leukosis virus and Marek's disease virus are both tumor diseases, so this ploidy type is also an excellent material for ALV experimental research. Summary of the invention

[0005] The purpose of the present invention is to provide an ALV-J MHC-B21 restricted epitope peptide and its application, so as to solve the problems existing in the above-mentioned prior art, detect the immunogenicity of candidate polypeptide epitopes, screen out advantageous epitope peptides against J subtype avian leukosis virus, and provide material and theoretical basis for the research and development of J subtype avian leukosis virus epitope vaccine.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an ALV-J MHC-B21 restricted epitope peptide, the amino acid sequence of the epitope peptide is PRLMGPFYEQL (SEQ ID NO: 1).

[0008] The present invention also provides a gene encoding the ALV-J MHC-B21 restricted epitope peptide.

[0009] The present invention also provides the use of the ALV-J MHC-B21 restricted epitope peptide or the gene in preparing an anti-J subtype avian leukosis virus epitope vaccine.

[0010] The present invention also provides the use of the ALV-J MHC-B21 restricted epitope peptide or the gene in preparing a drug against J subtype avian leukosis virus.

[0011] The present invention discloses the following technical effects:

[0012] The present invention firstly infects B21 (BW / G6) haplotype SPF chickens with ALV-J SCAU-HN06 strain, and successfully establishes an infection response model to serve as subsequent test materials by detecting the cloacal excretion of the chickens, serum antibody levels, viremia, changes in T cell subtypes in PBMCs, and changes in cell immunity-related genes in PBMCs. Then, according to the motif (XH / K / RXXXXX-(X)-E / DXA / V / L / I / F / M) of the B21 haplotype SPF chicken MHC class I molecule binding polypeptide reported in the literature, candidate polypeptide epitopes for ALV-J are screened, and finally the immunogenicity of the polypeptide is verified by ELISpot test, and an effective T cell epitope peptide (PRLMGPFYEQL) is determined, so as to provide a basis for the research and development of ALV epitope vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 The changes of viremia in chickens after virus attack;

[0015] Figure 2 The cloaca excretion of toxins in chickens after the challenge;

[0016] Figure 3 is the serum antibody level of chickens after challenge;

[0017] Figure 4 This is a schematic diagram of the flow gate strategy;

[0018] Figure 5 CD8 after attack + Changes in T cell proportions;

[0019] Figure 6 CD4 after virus attack + Changes in T cell proportions;

[0020] Figure 7 CD4 after virus attack + CD8α + Changes in T cell proportions;

[0021] Figure 8 The expression of innate immunity-related genes in PBMC of B21 haplotype chickens after challenge;

[0022] Fig. 9 The expression of CTLs-related genes in PBMC of B21 haplotype chickens after virus attack;

[0023] Fig.10 The expression of Th2-related genes in PBMC of B21 haplotype chickens after challenge;

[0024] Fig.11 The expression of chemokines and inflammatory cytokines in PBMC of B21 haplotype chickens after challenge;

[0025] Fig.12 This is a partial spot pattern of the #1 chicken ELISpot test;

[0026] Fig.13 This is a partial spot pattern of the #2 chicken ELISpot test;

[0027] Fig.14Partial spot pattern for #3 chicken ELISpot test;

[0028] Fig.15 The expression level of IFN-γ after peptide stimulation of lymphocytes; statistical analysis was performed using unpaired t-test. Except for the positive control, each peptide was repeated three times. *: P<0.05, significant difference; ***: P<0.001, extremely significant difference. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Explanation of relevant terms in the present invention:

[0035] ALV-J: avian leukosis virus subtype J;

[0036] MHC-I: major histocompatibility complex class I;

[0037] ELISpot: enzyme-linked immunospot assay;

[0038] DPI: days post-challenge;

[0039] PBMC: peripheral blood mononuclear cells;

[0040] SPF chicken: Specific pathogen free chicken

[0041] CTL: Cytotoxic T cell

[0042] IFN-γ: interferon gamma

[0043] Example 1

[0044] 1. Test materials

[0045] 1.1 Experimental animals and viruses

[0046] The chickens used in this experiment were 4-week-old B21 haplotype (BW / G6) SPF chickens purchased from the National Poultry Laboratory Animal Resource Bank; the ALV-J SCAU-HN06 strain was from the National and Local Joint Engineering Laboratory for Zoonotic Disease Prevention and Control Preparations of South China Agricultural University.

[0047] 1.2 Main reagents for the test

[0048] RPMI-1640 culture medium and FBS Australian fetal bovine serum were purchased from GIBCO, USA; dimethyl sulfoxide (DMSO), saponin, and brefeldin A (BFA) were purchased from Sigma, USA; phorbol ester and ionomycin (PMA+Ionomycin), and TMB ELISpot special colorimetric solution were purchased from Dakoway, China; Chicken IFN-γ ELISpot BASIC kit was purchased from Mabtech,

[0049] Anti-chicken CD3 antibody, Anti-chicken CD4 antibody, Anti-chicken CD8αantibody, Anti-chicken IFN-γ-FITC antibody were purchased from Southern Biotech. Chicken peripheral blood lymphocyte separation kit, chicken organ tissue mononuclear cell separation kit, and red blood cell lysis solution were purchased from Tianjin Haoyang Biological Company; ChamQ SYRB qPCR Master Mix was purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0050] 1.3 Preparation of main solution

[0051] Flow cytometry buffer: 2% FBS + 98% PBS. After preparation, place at 4°C for later use.

[0052] Cell freezing solution: 10% DMSO + 90% FBS. After preparation, store at 4°C for future use.

[0053] 2. Test methods

[0054] 2.1 Establishment of an animal model of ALV-J infected B21 haplotype SPF chickens

[0055] Twelve 4-week-old B21 haplotype SPF chickens were randomly divided into a challenge group and a control group, with 6 chickens in each group. The virus titer of the challenge group was 10 4.7 TCID 50 1 mL of ALV-J virus solution / 100 μl was injected into the peritoneal cavity of the control group, and 1 mL of PBS was injected into the peritoneal cavity of the control group. After the challenge, the corresponding samples were collected from each chicken for the following tests:

[0056] 2.1.1 Detection of cloaca toxin excretion in B21 haplotype SPF chickens after infection

[0057] The cloacal swab was collected and added to 1 mL of PBS and stored at -80°C, and returned to room temperature before testing. The ELISA test was performed using the avian leukosis p27 antigen detection kit from IDEXX to detect the detoxification status.

[0058] First, select the positive and negative control wells and sample wells on the coated plate, add 100 μL of the corresponding standard to each positive and negative control well according to the instructions, repeat 2 times for each positive and negative control, and add 100 μL of cloaca swab fluid to each sample well. After incubation at 18-26°C for 60 minutes, add 350 μL of distilled water to each well for washing, repeat 5 times, and then add enzyme-labeled antibody, 100 μL per well. After reacting at 18-26°C for 60 minutes, wash 5 times as above, and then add TMB substrate solution, 100 μL per well; after reacting at 18-26°C for 15 minutes, add stop solution to stop the reaction, 100 μL per well, and finally use an enzyme reader to measure and record the absorbance value A of the positive and negative control wells and each sample at a wavelength of 650 nm, and calculate the S / P value according to the instructions.

[0059] 2.1.2 Detection of changes in viremia in B21 haplotype SPF chickens after infection

[0060] DF-1 cells were plated in a 24-well adherent culture plate. When the cells grew to 90%, 1 mL of chicken anticoagulated whole blood was collected and centrifuged at 2000 g for 8 min at 4 °C to separate the plasma. 100 μL of plasma was inoculated into DF-1 cells and placed in a 37 °C incubator for 24 h. After washing with PBS, the cells were added with a cell maintenance solution consisting of 1640 medium containing 2% fetal bovine serum and cultured for 5 more days. On the 6th to 7th day, the cell culture supernatant was taken for ELISA test to detect viral antigens. The specific steps were the same as in 2.1.1.

[0061] 2.1.3 Detection of serum antibody levels

[0062] The non-anticoagulated blood was collected and placed at room temperature until the serum precipitated, and the serum was collected in a 1.5 mL centrifuge tube. The red blood cells were then removed by centrifugation at 4°C and 2000 rpm for 10 min, and the remaining serum was used to detect the serum antibody level.

[0063] The antibody level was detected by ELISA test using the avian leukosis J subgroup antibody detection kit from IDEXX.

[0064] 2.1.4 Detection of changes in T cell subtypes in PBMC of B21 haplotype SPF chickens after infection

[0065] B21 haplotype SPF chicken peripheral blood lymphocytes were isolated by conventional method, and 1×10 6 Each cell was placed in a flow tube, and a channel control group and a blank control group were set up at the same time. Then 1 mL of PBS was added to each tube for washing, centrifuged at 400 g for 5 min, and the supernatant was discarded. Then anti-chicken CD3, CD4 and CD8α flow antibodies were used for staining. Each antibody was diluted with flow buffer according to the proportion in the instructions. 100 μL of diluted antibody was added to each sample to resuspend the cells and incubated at 4 ° C for 30 min. After incubation, PBS was added to the final volume of 1 mL to terminate the staining reaction, and centrifuged at 400 g for 5 min. After discarding PBS, 200 μL of flow buffer was added to each tube to resuspend the cells and detect on the machine.

[0066] 2.1.5 Detection of changes in cellular immunity-related genes in PBMC of B21 haplotype SPF chickens after infection

[0067] The RNA of peripheral blood lymphocytes isolated in 2.1.4 was extracted by TRIZOL method: 1 mL TRIZOL was added to the cells, and the cells were lysed by shaking. Then 200 μL chloroform was added, and the mixture was allowed to stand at room temperature for 10 min after thorough shaking. The mixture was centrifuged at 12000 g and 4°C for 15 min, and the upper transparent liquid was transferred to a new enzyme-free centrifuge tube. An equal volume of isopropanol was added, and the mixture was mixed upside down. The mixture was placed at -20°C for 30 min, and the sample was taken out. The mixture was centrifuged at 12000 g and 4°C for 10 min, and the RNA was precipitated at the bottom of the tube. After discarding the supernatant, 1 mL 75% ethanol was added, and the mixture was shaken and centrifuged at 12000 g and 4°C for 10 min to wash the RNA. After repeating twice, the supernatant was discarded, the tube was opened to allow the ethanol to evaporate naturally, and finally 30 μL 0.1% DEPC water preheated to 65°C was added to the precipitate to dissolve the RNA. The RNA concentration was measured by spectrophotometer.

[0068] RNA reverse transcription was performed using the PrimeScriptTM RT Master Mix (Perfect Real Time) kit from TAKARA, and the system is shown in Table 1. Reaction procedure: 37°C for 15 min, inactivation at 85°C for 5 s.

[0069] Table 1 Reverse transcription system

[0070]

[0071] Fluorescence quantitative PCR amplified the reverse transcribed cDNA to detect changes in cellular immunity-related genes in PBMC. The target genes and primers for detection are shown in Table 2, and the system is shown in Table 3. Reaction procedure: pre-denaturation at 95°C, 30s; cycle reaction at 95°C, 10s, 60°C, 30s, for a total of 40 cycles; melting curve analysis at 95°C, 15s, 60°C, 60s, 95°C, 15s.

[0072] Table 2 qPCR primers for immune-related genes

[0073]

[0074]

[0075]

[0076] Table 3 Fluorescence quantification system

[0077]

[0078] 2.2 Identification of the Motifs of the B21 Haplotype SPF Chicken MHC I Molecules

[0079] 2.2.1 Screening and synthesis of epitopes based on motifs

[0080] According to the motif of the B21 haplotype SPF chicken MHC class I molecule binding polypeptide published in the article (XH / K / RXXXXX-(X)-E / DXA / V / L / I / F / M), the candidate epitopes in the protein sequences of the ALV-JHN06 strain were screened by stepping one amino acid at a time, as shown in Table 4. The polypeptides were synthesized by GenScript Biotech Co., Ltd. using a chemical synthesis method, and three reverse HPLC (RP-HPLC) and mass spectrometry (MS) analyses were performed to ensure the quality of each polypeptide, with a purity of more than 95%.

[0081] Table 4 ALV-J epitopes based on motif screening

[0082]

[0083] 2.3ELISpot assay to detect immunogenicity of candidate peptides

[0084] Take the B21 haplotype SPF chicken infected 28 days after 2.1, isolate the spleen lymphocytes of the chicken, and the isolation procedure is carried out according to the instructions of the chicken organ tissue mononuclear cell isolation fluid kit, and then perform an ELISpot test to detect the immunogenicity of the candidate polypeptide epitope. The specific steps are as follows:

[0085] (1) Activate PVDF 96-well plate: add 15 μL 35% ethanol to each well, wash with sterile water 5 times after 30 seconds to 1 minute;

[0086] (2) Coating antibody: add 100 μL of anti-chicken IFN-γ diluted to 15 μg / mL to each well and incubate at 4°C overnight;

[0087] (3) Blocking: Discard the coating solution, wash with DPBS 5 times, and then pat the plate dry on sterile absorbent paper. Add RMPI 1640 medium containing 10% FBS to the PVDF 96-well plate, 200 μL per well, and perform blocking reaction at room temperature for 30 minutes;

[0088] (4) Stimulation: Discard the blocking solution and add 100 μL of pre-prepared lymphocyte suspension to each well. The concentration of the cell suspension is 5×10 6 cells / mL. At the same time, 50 μL of diluted antigen peptide was added to the experimental group to make the final concentration 10 μg / mL, PMA was added to the positive control group at a final concentration of 10 μg / mL, and no stimulant was added to the negative control group. After all samples were added, the PVDF 96-well plate was placed in a 37°C CO2 incubator for 24 to 48 hours;

[0089] (5) Secondary antibody incubation: After the incubation, the cells were shaken off, washed 5 times with DPBS, and a biotin-labeled secondary antibody at a concentration of 1 μg / mL was added, 100 μL per well, and incubated at room temperature for 2 h;

[0090] (6) HRP incubation: discard the secondary antibody solution, wash with DPBS 5 times, then add streptavidin-labeled HRP, 100 μL per well, and incubate at room temperature for 1 h;

[0091] (7) Color development: discard the HRP incubation solution, wash five times with DPBS, then add TMB color development substrate, 100 μL per well, and incubate at room temperature or 37°C for 15-30 min. When obvious spots appear in the positive control group, discard the color development solution, terminate the color development with pure water, and read the plate after air drying.

[0092] 2.4 Data Analysis

[0093] All experimental data were statistically analyzed using GraphPad Prism 8 software, where ns means P>0.05, no significant difference; * means P<0.05, significant difference; ** means P<0.01, extremely significant difference; *** means P<0.001, extremely significant difference; **** means P<0.0001, extremely significant difference.

[0094] 3. Results

[0095] 3.1 Viremia, cloacal excretion and serum antibody levels in B21 haplotype SPF chickens after ALV-J infection

[0096] The changes of viremia in chickens after infection are as follows Figure 1 As shown in the figure, the virus content in the blood was the highest at 7 DPI, indicating that all chickens in the challenge group were successfully infected with ALV-J. The virus content in the blood had significantly decreased at 14 DPI (P<0.001), and the virus was no longer detectable at 21 DPI. Figure 2 As shown, no detoxification was observed. Figure 3 These are the antibody ELISA test results of B21 haplotype SPF chickens. The antibody level of B21 haplotype SPF chickens was negative at 7DPI, and 50% of the chickens tested positive for antibodies at 14DPI and 21DPI, indicating that the infection group of B21 haplotype SPF chickens was successfully infected (Note: unpaired t-test was used for statistical analysis).

[0097] 3.2 Changes in T cell subtypes in PBMC of B21 haplotype SPF chickens after ALV-J infection

[0098] In order to detect the T cell immune response of B21 haplotype SPF chickens after challenge, peripheral blood was collected from the jugular vein according to the experimental arrangement. PBMCs were isolated from peripheral blood of B21 haplotype SPF chickens on days 7, 14, and 21 after challenge, and the proportions of CD8α+T cells, CD4+T cells, and CD4+CD8α+T cells were detected by flow cytometry. The gating strategy for CD8α+T cells, CD4+T cells, and CD4+CD8α+T cells is as follows: Figure 4 The changes in the proportion of CD8α+T cells are shown in Figure 5 As shown in the figure, the proportion of CD8α+T cells in the challenge group increased at 7 DPI (P<0.05), and a significant increase in the proportion of CD8α+T cells in the challenge group was detected at 14 DPI and 21 DPI (P<0.001), indicating that viral infection may induce CD8α+T cell immune response in chickens. Figure 6 , Figure 7 As shown, during the entire detection process, there was no statistical difference in the CD4+T cell ratio and CD4+CD8α+T cell ratio between the challenge group and the control group (Note: unpaired t-test was used for statistical analysis).

[0099] 3.3 Changes in the expression of cellular immunity-related genes in PBMCs of B21 haplotype SPF chickens after infection

[0100] To further verify the role of cellular immune response in ALV-J infection of B21 haplotype SPF chickens, fluorescence quantitative PCR was used to detect the changes in mRNA expression of important immune genes in PBMCs 14 and 21 days after infection. The detection mainly included four parts: CTLs genes, Th2 genes, innate immunity-related genes and inflammation-related genes.

[0101] Innate immunity related genes Figure 8 ), compared with the control group, the expression of antiviral gene ISG12-2 (Interferon-stimulated gene 12-2) was significantly upregulated at 14DPI and 21DPI (P<0.05 or P<0.01); the expression of IFIT5 (Interferon-induced proteins with tetratricopeptide repeats 5) was significantly upregulated at 14DPI (P<0.05); the expression of MX1 (Myxovirus resistance 1) was significantly upregulated at 21DPI (P<0.05). CTLs gene part ( Fig. 9), NK-lysin, HMG-2 (high mobility group box 2) and PARP (Poly (ADP-ribose) polymerase) expression levels were significantly upregulated at 14 DPI (P<0.05); Granzyme A expression levels were significantly upregulated at 14 DPI and 21 DPI (P<0.01); Granzyme K and MHC-I expression levels were extremely significantly upregulated at 21 DPI (P<0.01). Detected Th2 genes ( Fig.10 ), the expression of MHC-II was significantly upregulated at 14DPI and 21DPI (P<0.05 or P<0.01); the expression of IL-13 was significantly upregulated at 21DPI (P<0.05). Fig.11 ), compared with the control group, there was no significant difference in the expression of each gene. Figure 5 The increase in the proportion of CD8α+T cells in the B21 haplotype SPF chickens further indicated that ALV-J infection successfully activated the T cell immune response of B21 haplotype SPF chickens. Based on the above results, the infection model of ALV-J SCAU-HN06 strain infecting B21 haplotype SPF chickens and effectively inducing cellular immune response was successfully established (Note: unpaired t-test was used for statistical analysis).

[0102] 3.4ELISpot assay to detect immunogenicity of candidate peptides

[0103] The synthetic peptides were used to stimulate the spleen lymphocytes of B21 haplotype SPF chickens infected with ALV-J for 28 days. Fig.12 , Fig.13 , Fig.14 As shown in the figure, under the premise of establishing a positive control group, statistical analysis found that compared with the negative control group, the experimental group using ALV-J-M70 as a stimulus could significantly produce more spots (P<0.05), indicating that this polypeptide can stimulate cells to secrete IFN-γ ( Fig.15 ), which is an immunogenic T cell epitope, provides a material and theoretical basis for the development of ALV-J epitope vaccine. Fig.12 , Fig.13 , Fig.14 M76 and L53 are two other representative test group peptides shown.

[0104] The specific epitope information of M70 is shown in Table 5.

[0105] Table 5 ALV-J T cell epitope information for B21 haplotype chicken

[0106]

[0107] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An ALV-J MHC-B21 restricted epitope peptide, characterized in that: The amino acid sequence of the epitope peptide is PRLMGPFYEQL.

2. A gene encoding the ALV-J MHC-B21 restricted epitope peptide according to claim 1.

3. Use of the ALV-J MHC-B21 restricted epitope peptide according to claim 1 or the gene according to claim 2 in preparing an anti-J subtype avian leukosis virus epitope vaccine.

4. Use of the ALV-J MHC-B21 restricted epitope peptide according to claim 1 or the gene according to claim 2 in the preparation of an anti-J subtype avian leukosis virus drug.

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