T cell epitope polypeptide based on ASFV RNA polymerase protein and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a T cell epitope polypeptide based on ASFV RNA polymerase protein, the amino acid sequence of the T cell epitope polypeptide is shown as any one of SEQ ID NO.1-3, the T cell epitope polypeptide is the ASFV RNA polymerase protein and / or subunit-derived T cell epitope polypeptide obtained through screening, and the T cell epitope polypeptide is a T cell epitope polypeptide based on ASFV RNA polymerase protein and / or subunit derived from ASFV RNA polymerase protein and / or subunit derived from ASFV RNA polymerase protein and / or subunit derived from ASFV RNA polymerase protein and / or subunit derived from ASFV RNA polymerase protein. The primer has the characteristics of inducing ASFV specific T cells and assisting in controlling ASFV infection and virus clearance; in-vitro experiments verify that the epitope polypeptide has the capability of inducing ASFV specific T cell response, and a theoretical basis is provided for subsequent development of polypeptide vaccines and diagnostic preparations based on ASFV protein source epitopes.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a T-cell epitope polypeptide based on ASFV RNA polymerase protein and its applications. Background Technology

[0002] African swine fever (ASF) is an acute, febrile, and highly contagious disease caused by the African swine fever virus (ASFV) in susceptible hosts such as domestic pigs and European wild boars. Infected pigs exhibit symptoms including high fever, hemorrhage, anorexia, and respiratory distress, with a mortality rate reaching up to 100%. ASFV is currently the only known virus composed of the genus *African swine fevervirus* within the family *African swine feverviruses*. It is a double-stranded DNA virus. Its genome is approximately 190 kb in size, containing 151-167 open reading frames, encoding over 150 proteins, including about 50 structure-related proteins. It also encodes key enzymes supporting the ASFV life cycle, such as RAN polymerase, DNA replicase, and topoisomerase, as well as various functional factors regulating the host's immune response. However, the functions of about half of the ASFV gene-encoded proteins remain unclear. Current ASF vaccine development mainly focuses on structural proteins, such as P72, P54, and P30 proteins, aiming to induce neutralizing antibodies to provide protection and block viral transmission. However, the protective efficacy of subunit vaccines developed based on the aforementioned proteins in recent years needs further improvement in clinical trials. Especially given the highly variable nature of ASFV, the prevalent ASFV strains in Chinese swine populations have shifted from type II strains to type I / II recombinant strains, necessitating that ASF vaccine development also consider cross-protective capabilities.

[0003] ASFV encodes its own eukaryotic-like RNA polymerase, whose core structure shares conservation with the 12 subunits of eukaryotic RNA polymerase II (PolII) and RNA polymerases from other nucleoplasmic large DNA viruses (NCLDVs). As an indispensable component of the viral replication and gene expression machinery, the ASFV RNA polymerase precisely regulates viral gene expression by encoding its own transcriptional machinery, influencing viral replication efficiency, pathogenicity, immune escape strategies, and metabolic remodeling within host cells. Therefore, focusing on ASFV RNA polymerase T-cell epitopes to develop vaccines and other products, and inducing specific T-cell responses, not only helps to elucidate the complex life cycle of ASFV, which is of great significance for developing safe and effective ASF peptide vaccines based on T-cell antigenic epitopes, but also provides key targets for developing effective antiviral strategies and drugs. Summary of the Invention

[0004] The problem to be solved by this invention is to identify T cell epitopes derived from ASFV RAN polymerase protein and / or subunits in order to effectively stimulate specific T cell immune responses and exert antiviral efficacy.

[0005] To resolve the aforementioned problems, the present invention proposes the following technical solution: In a first aspect, the present invention provides a T-cell epitope polypeptide based on ASFV RNA polymerase protein, wherein the epitope polypeptide includes one or more of epitope polypeptides NP1249L-1, H359L-1, and H359L-2, wherein the amino acid sequence of epitope polypeptide NP1249L-1 is shown in SEQ ID NO.1, the amino acid sequence of epitope polypeptide H359L-1 is shown in SEQ ID NO.2, and the amino acid sequence of epitope polypeptide H359L-2 is shown in SEQ ID NO.3.

[0006] Preferably, the T-cell epitope polypeptide further includes an amino acid sequence as shown in SEQ ID NO. 1~3, in which one or more amino acids are substituted, deleted, and / or added while maintaining the original functional consistency.

[0007] In a second aspect, the present invention provides a nucleotide sequence encoding the aforementioned T-cell epitope polypeptide.

[0008] Thirdly, the present invention provides a recombinant vector comprising the nucleotides described above.

[0009] Fourthly, the present invention provides a recombinant microbial cell, wherein the recombinant microbial cell comprises the aforementioned nucleotides.

[0010] Fifthly, the present invention provides a vaccine composition containing the aforementioned T-cell epitope polypeptide.

[0011] Preferably, the vaccine composition is a monovalent vaccine, a bivalent vaccine, or a multivalent vaccine.

[0012] In a sixth aspect, the present invention provides an antigen-presenting cell sensitized by the aforementioned T-cell epitope polypeptide.

[0013] In a seventh aspect, the present invention provides an African swine fever virus antigen, wherein the African swine fever virus antigen is prepared by coupling the T cell epitope polypeptide with a carrier protein.

[0014] Eighthly, the present invention provides applications of the aforementioned T-cell epitope polypeptide for preparing polypeptide vaccines against African swine fever virus, preparing specific antibodies against African swine fever virus, and preparing reagents or kits for diagnosing or detecting African swine fever virus.

[0015] The beneficial effects of this invention are as follows: This invention provides a T-cell epitope polypeptide based on ASFV RNA polymerase protein. The epitope polypeptide includes one or more of epitope polypeptides NP1249L-1, H359L-1, and H359L-2. The amino acid sequence of epitope polypeptide NP1249L-1 is shown in SEQ ID NO.1, the amino acid sequence of epitope polypeptide H359L-1 is shown in SEQ ID NO.2, and the amino acid sequence of epitope polypeptide H359L-2 is shown in SEQ ID NO.3. The T-cell epitope polypeptide is a T-cell epitope polypeptide derived from ASFV RNA polymerase protein and / or its subunits, obtained through screening. It possesses the characteristics of inducing ASFV-specific T cells and assisting in the control of ASFV infection and viral clearance. In vitro experiments have verified that this epitope polypeptide has the ability to induce ASFV-specific T-cell responses, providing a theoretical basis for the subsequent development of polypeptide vaccines and diagnostic agents based on ASFV protein-derived epitopes. Attached Figure Description

[0016] Figure 1 The present invention uses T cell epitope peptides derived from ASFV RNA polymerase protein and / or subunits to induce specific T cells in vitro, and then uses the ELISPOT assay targeting IFN-γ to detect the dot pattern of specific T cells.

[0017] Figure 2 The present invention uses T cell epitope peptides derived from ASFV RNA polymerase protein and / or subunits to induce specific T cells in vitro, and uses the ELISPOT assay targeting IFN-γ to detect the number of specific T cells, resulting in a statistical graph of ELISPOT spots.

[0018] Figure 3 This is a statistical graph showing the results of specific T cell proliferation stimulated by the NP1249L-1 (number 7030) peptide of this invention, with cell viability detected using CCK-8 assay.

[0019] Figure 4 This is a statistical graph showing the results of peptide-specific T cell proliferation stimulated by the H359L-1 (No. 7031) peptide of this invention, using CCK-8 assay to detect cell viability.

[0020] Figure 5 This is a statistical graph showing the results of peptide-specific T cell proliferation stimulated by the H359L-2 (No. 7032) peptide of this invention, using CCK-8 assay to detect cell viability. Detailed Implementation

[0021] To make the objectives, technical solutions and advantages of the present invention clearer, the following will provide further description in conjunction with the embodiments. Unless otherwise specified, the experimental reagents, experimental equipment and experimental materials involved in the present invention are all commonly used or commercially available products in the field, and the terms and abbreviations involved have the conventional meanings in the field, such as IFN-γ (gamma interferon).

[0022] Experimental methods not specifically described in the following examples are generally performed under standard conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the product manufacturer.

[0023] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0024] Example 1: Synthesis and Identification of T-cell Epitopes of ASFV RNA Polymerase Protein and / or Subunits 1. T cell epitope peptide synthesis and preparation This invention provides epitope peptides derived from any one of the following ASFV RNA polymerase proteins and / or subunits: NP1249L-1 (number 7030), H359L-1 (number 7031), and H359L-2 (number 7032), whose amino acid sequences are SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively, as shown in the table below. The peptides were synthesized by Wuhan Dangang Biotechnology Co., Ltd., and all peptides had a purity higher than 95% and were correctly identified by mass spectrometry. All peptides were dissolved and stored at a final concentration of 1 mg / mL at -80°C for future use.

[0025]

[0026] 2. Separation of PBMCs Three healthy 60-70 day old pigs that tested negative for ASFV were inoculated with the ASFV / GS / 2018 strain. Peripheral anticoagulated blood was collected on the day the pigs showed clinical symptoms (3-5 days) and on the day they were near death (6-8 days). PBMCs were isolated within 4 hours according to the operating instructions of the porcine peripheral blood mononuclear cell isolation kit from Tianjin Haoyang Biological Products Technology Co., Ltd. Red blood cells were removed from the collected PBMCs using erythrocyte lysis buffer (TIANGEN). The cells were resuspended in RPMI 1640 complete medium (containing 10% FBS, 1% penicillin-streptomycin-amphotericidal mixture). 20 μL of this medium was mixed with an equal volume of trypan blue staining solution to label the cells, and the viable cell count was determined using a cell counter.

[0027] 3. ELI-Spot assay to evaluate the ability of T cell epitope peptides to induce IFN-γ release. Pre-coat 96-well plates according to the Porcine IFN-γ (ALP) kit (Mabtech, 3130-2A) instructions. Place the prepared PBMCs (3-5 × 10⁶ per well) into each well. 5 Cells were added to 96-well plates coated with IFN-γ capture antibody, followed by the addition of T epitope peptides (final concentration 10 μg / mL) and controls (complete culture medium), and incubated in a cell culture incubator for 17-24 h. Subsequent ELISpot assays were performed according to the product instructions, and the final results were obtained using an ELISpot analyzer (Mabtech ASTOR 2).

[0028] 4. Experimental Results The results are as follows Figure 1 , Figure 2 As shown, the T-cell epitope peptide of the ASFV RNA polymerase protein and / or subunits mentioned in this invention has been verified through in vitro experiments to have the ability to induce T cells to secrete IFN-γ, thus assisting in the control of ASFV infection and viral clearance. Among them, 7032 showed the best effect for samples with different disease stages, followed by 7031, and 7030 last.

[0029] Example 2: T cell epitope peptides of ASFV RNA polymerase protein and / or subunits induce specific T cell proliferation. 1. Isolation and preparation of porcine BMDCs and spleen lymphocytes Take the femur and tibia from the hind leg of a healthy 60-70 day old pig that is negative for ASFV. Remove the surface tissue from the bones, soak them in 75% absolute ethanol for 5 min, and then rinse three times with sterile PBS. Cut the bones and wash the bone marrow tissue with RPMI 1640 complete medium. Filter the bone marrow wash through a sterile 70 μm filter and collect the wash fluid. Centrifuge at 1500 rpm for 5 min and discard the supernatant. Add 3-5 mL of erythrocyte lysis buffer to remove erythrocytes, centrifuge at 1500 rpm for 5 min to collect the cells, resuspend the cells in 5 mL of RPMI 1640 complete medium, mix 20 μL with an equal volume of trypan blue staining solution to label the cells, and count the viable cells using a cell counter. Plant the cells at a density of 2 × 10⁶ cells per 100 mm cell culture dish. 7 The cells are evenly distributed on the plate.

[0030] BMDCs were induced using RPMI1640 complete medium containing 20 ng / mL porcine GM-CSF and 10 ng / mL porcine IL-4. After 3 days of culture, the medium was replaced with the same medium. On day 5, the differentiation level of BMDCs was observed under a microscope. After induction was completed, the medium was replaced with RPMI1640 complete medium.

[0031] Spleen tissue was taken from the same pig from which BMDCs were isolated. The spleen tissue was washed with sterile PBS under sterile conditions. Three 2cm × 2cm × 2cm pieces of spleen tissue were randomly cut, and the tissue was ground after adding porcine spleen lymphocyte separation medium from Tianjin Haoyang Biological Products Technology Co., Ltd. The mixture was then filtered through a sterile 70μm filter and collected at 800× [a certain temperature range]. g Centrifuge for 25 min, transfer the lymphocyte layer to a new 15 mL centrifuge tube, add 3-5 mL of erythrocyte lysis buffer to remove erythrocytes, centrifuge at 1500 rpm for 5 min to collect cells, resuspend the cells in 5 mL of RPMI 1640 complete culture medium containing 10 ng / mL porcine IL-2, mix 20 μL of the medium with an equal volume of trypan blue staining solution to label the cells, and count the number of viable cells using a cell counter. Culture the cells at a rate of 5 × 10⁶ cells per T25 cell culture flask. 6 After the cells were evenly distributed, they were placed in a cell culture incubator for later use.

[0032] 2. Preparation of T cells specific to T cell epitope peptides of ASFV RNA polymerase protein and / or subunits. The BMDCs prepared above were used in a 6-well plate with 6 × 10⁶ cells per well. 5 After seeding cells, the cells were allowed to rest overnight in a cell culture incubator. After the first replacement with RPMI 1640 medium at a final concentration of 10 μg / mL, the medium was replaced with the same medium every two days. After 3-5 stimulations, antigen-pulse-stimulated BMDCs were obtained.

[0033] Take the above 1×10 7One isolated spleen lymphocyte was placed in a 1 mL centrifuge tube, and 10–25 μL of Pig CD8a-PE (clone: ​​76-2-11) antibody was added. After incubation at 4°C for 30 min, IgG magnetic beads were added according to the recommended protocol in the CST product instructions, and the mixture was incubated at room temperature for 5 min. Subsequently, CD8 cells captured by the antibody were separated using a magnetic rack. + T cells, after discarding the uncaptured cell population, detach from the magnetic rack and collect CD8. + T cell populations were resuspended in RPMI 1640 complete medium containing 10 ng / mL porcine IL-2 and cultured statically in a cell culture incubator for 3 days before use.

[0034] 1×10 6 BMDCs stimulated by antigen pulses and 4 × 10 6 CD8 + T cell suspension was co-seeded into 6-well plates to establish a co-culture system, ensuring the cell culture environment contained 10 ng / mL porcine IL-2. On day 5, each co-culture well was aliquoted into two new wells of the 6-well plate, and an additional 2 mL of fresh culture medium (containing 10 ng / mL IL-2) was added to each well. Cells were cultured for another 3 days. On day 8, cells were collected by gentle aspiration, mixed thoroughly, and 20 μL of the mixture was mixed with an equal volume of trypan blue staining solution to label the cells. The number of viable cells was then counted using a cell counter. CD8 cells were isolated using the magnetic bead method described above. + T cells are antigen-specific T cells.

[0035] 3. T-cell epitope peptides of ASFV RNA polymerase protein and / or subunits stimulate specific CD8. + T cell proliferation Cells were distributed at 5 × 10⁶ cells per well in a 96-well plate. 4 Cells were seeded into plates and allowed to rest overnight. Then, peptides were added to the plates at final concentrations of 5 μg / mL, 10 μg / mL, and 20 μg / mL, respectively. LPS was used as a positive control. After incubation in a cell culture incubator for 48 h, 10 μL of CCK-8 solution was added to each well. After incubation in a cell culture incubator for 4 h, OD450 was measured. The changes in cell viability in each treatment group were calculated, with the normal culture medium group as a control.

[0036] 4. Experimental Results The results are as follows Figure 3 , Figure 4 , Figure 5As shown in the figure, 7030 exhibited a concentration-dependent induction of T cell proliferation, with the best effect observed at a peptide concentration of 20 μg / mL, resulting in cell proliferation 1.6 times higher than the control group. 7031 showed the most significant proliferation effect at a concentration of 5 μg / mL, exceeding the control group by 2.6 times. 7032 also showed the most significant proliferation effect at a concentration of 5 μg / mL, exceeding the control group by 1.9 times. Therefore, the peptides in this invention possess the ability to induce specific T cell proliferation.

[0037] In summary, this invention provides a T-cell epitope polypeptide based on ASFV RNA polymerase protein, wherein the amino acid sequence of the T-cell epitope polypeptide is shown in any one of SEQ ID NO. 1-3. The T-cell epitope polypeptide is a T-cell epitope polypeptide derived from ASFV RNA polymerase protein and / or its subunits, obtained through screening, and possesses the properties of inducing ASFV-specific T cells and assisting in the control of ASFV infection and viral clearance. In vitro experiments have verified that this epitope polypeptide has the ability to induce ASFV-specific T-cell responses, providing a theoretical basis for the subsequent development of polypeptide vaccines and diagnostic agents based on ASFV protein-derived epitopes.

[0038] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A T-cell epitope polypeptide based on ASFV RNA polymerase protein, characterized in that, The epitope polypeptides include one or more of epitope polypeptides NP1249L-1, H359L-1, and H359L-2, wherein the amino acid sequence of epitope polypeptide NP1249L-1 is shown in SEQ ID NO.1, the amino acid sequence of epitope polypeptide H359L-1 is shown in SEQ ID NO.2, and the amino acid sequence of epitope polypeptide H359L-2 is shown in SEQ ID NO.

3.

2. The T-cell epitope polypeptide as described in claim 1, characterized in that, The T-cell epitope polypeptide also includes any amino acid sequence shown in SEQ ID NO. 1~3 that has been substituted, deleted and / or have one or more amino acids added while maintaining the original functional consistency.

3. A nucleotide encoding the T-cell epitope polypeptide as described in claim 1.

4. A recombinant vector, characterized in that, The recombinant vector comprises the nucleotides of claim 3.

5. A recombinant microbial cell, characterized in that, The recombinant microbial cells contain the nucleotides described in claim 3.

6. A vaccine composition, characterized in that, The vaccine composition contains the T-cell epitope polypeptide as described in claim 1 or 2.

7. The vaccine composition according to claim 6, characterized in that, The vaccine composition is a monovalent vaccine, a bivalent vaccine, or a multivalent vaccine.

8. An antigen-presenting cell, characterized in that, The antigen-presenting cells are sensitized with the T-cell epitope polypeptide as described in claim 1.

9. An African swine fever virus antigen, characterized in that, The African swine fever virus antigen is prepared by coupling the T-cell epitope polypeptide of claim 1 or 2 with a carrier protein.

10. The application of the T-cell epitope polypeptide as described in claim 1 or 2, characterized in that, Used for the preparation of peptide vaccines against African swine fever virus, preparation of specific antibodies against African swine fever virus, and preparation of reagents or kits for the diagnosis or detection of African swine fever virus.

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