T cell epitope polypeptide based on C962R protein and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to T cell epitope polypeptide based on C962R protein and application. The T cell epitope polypeptide comprises one or more of epitope polypeptide C962R-1, epitope polypeptide C962R-2, epitope polypeptide C962R-3 and epitope polypeptide C962R-4, the amino acid sequence is shown as SEQ ID NO.1-4, and the amino acid sequence is shown as SEQ ID NO.2-4. The epitope polypeptide 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] The application belongs to the technical field of biotechnology, and particularly relates to a T cell epitope polypeptide based on C962R protein and application thereof. BACKGROUND

[0002] African swine fever (ASF) is an acute, fever, and highly contagious disease caused by African swine fever virus (ASFV) infection in susceptible hosts such as domestic pigs and European wild boars. The infected pigs show symptoms such as high fever, hemorrhage, anorexia, and dyspnea, and the mortality rate can be as high as 100%. ASFV is the only virus of the African swine fever virus family of the African swine fever virus genus found so far, and is a double-stranded DNA virus. Its genome is about 190 kb in size, has 151-167 open reading frames, encodes more than 150 proteins, about 50 of which are structure-related proteins, and also encodes RAN polymerase, DAN primase, DAN replicase, topoisomerase, and other key enzymes supporting the life cycle of ASFV. It also encodes functional factors containing various host immune response regulators. However, the functions of about half of the ASFV genes encoding proteins have not been determined. Current ASF vaccine development mainly focuses on structural proteins such as P72 protein, P54 protein, and P30 protein, which induce neutralizing antibodies to play a protective role to block virus transmission. However, in recent years, the protection of subunit vaccines developed based on the above-mentioned proteins in the clinic needs to be further improved. Especially under the high variability of ASFV, the ASFV epidemic strain in China's pig population has also changed from type II strain to I / II recombinant strain, which makes the development of ASF vaccine products also need to consider the cross-protection ability.

[0003] The protein encoded by the C962R gene is highly conserved in ASFV strains. As a late transcription gene of ASFV, it can participate in the regulation of processes such as DNA damage repair by its polymerase-helicase dual-function activity. As one of the key components of the virus replication and gene expression machine, the C962R gene encodes its own regulatory protein to precisely regulate the expression of viral genes to cope with the stress pressure in the virus life cycle, affecting the replication efficiency, pathogenicity, immune escape strategy, and remodeling of host cell metabolism of the virus in the host cell. Therefore, focusing on the development of vaccines and other products based on the T cell epitope of C962R protein to induce specific T cell response not only helps to reveal the complex life cycle of ASFV, which is of great significance for the development of safe and effective ASF polypeptide vaccines based on T cell epitopes, but also provides a key target for the development of effective antiviral strategies and drugs. SUMMARY

[0004] The present application aims to identify T cell epitopes of C962R protein to effectively stimulate specific T cell immune response to exert antiviral efficacy.

[0005] To solve the above problems, the present application proposes the following technical solutions: In a first aspect, the present application provides a T cell epitope polypeptide based on C962R protein, wherein the T cell epitope polypeptide comprises one or more of epitope polypeptide C962R-1, epitope polypeptide C962R-2, epitope polypeptide C962R-3 and epitope polypeptide C962R-4, and the amino acid sequences are shown in SEQ ID NO. 1-4.

[0006] Preferably, the T cell epitope polypeptide further comprises an amino acid sequence obtained by substituting, deleting and / or adding one or more amino acids in any of the amino acid sequences shown in SEQ ID NO. 1-4 while maintaining the original functional consistency.

[0007] In a second aspect, the present application provides a nucleotide encoding the T cell epitope polypeptide.

[0008] In a third aspect, the present application provides a recombinant vector comprising the nucleotide.

[0009] In a fourth aspect, the present application provides a recombinant microbial cell comprising the nucleotide.

[0010] In a fifth aspect, the present application provides a vaccine composition comprising the 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 application provides use of the T cell epitope polypeptide, the nucleotide, the recombinant vector or the recombinant microbial cell in the preparation of a medicament for detecting T cell immune response caused by ASFV or treating or preventing ASFV infection.

[0013] The beneficial effects of this invention are as follows: This invention provides a T-cell epitope polypeptide based on the C962R protein. The T-cell epitope polypeptide includes one or more of epitope polypeptides C962R-1, C962R-2, C962R-3, and C962R-4, with amino acid sequences as shown in SEQ ID NO. 1-4. The above-mentioned epitope polypeptides are T-cell epitope polypeptides of the C962R protein obtained through screening, and possess 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 the 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

[0014] Figure 1 To induce specific T cells in vitro using the T cell epitope polypeptide of the C962R protein of this invention, the dot pattern of specific T cells was detected using the ELISPOT assay targeting IFN-γ.

[0015] Figure 2 The T-cell epitope polypeptide of the C962R protein of this invention induces specific T cells in vitro, and the number of specific T cells is detected by the ELISPOT assay targeting IFN-γ, resulting in a statistical graph of ELISPOT spots.

[0016] Figure 3 This is a statistical graph showing the results of specific T cell proliferation stimulated by the C962R-1 polypeptide of this invention, with cell viability detected using CCK-8 assay.

[0017] Figure 4 This is a statistical graph showing the results of C962R-2 polypeptide stimulating polypeptide-specific T cell proliferation, with cell viability detected using CCK-8 assay.

[0018] Figure 5 This is a statistical graph showing the results of C962R-3 peptide stimulating peptide-specific T cell proliferation, with cell viability detected using CCK-8 assay.

[0019] Figure 6 This is a statistical graph showing the results of C962R-4 peptide stimulating peptide-specific T cell proliferation, with cell viability detected using CCK-8 assay. Detailed Implementation

[0020] 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).

[0021] 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.

[0022] 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.

[0023] Example 1: T-cell epitope synthesis and identification of C962R protein 1. T cell epitope peptide synthesis and preparation This invention provides epitope peptides of the C962R protein, namely C962R-1 (number 7033), C962R-2 (number 7034), C962R-3 (number 7035), and C962R-4 (number 7036), whose amino acid sequences are SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, 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.

[0024]

[0025] 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.

[0026] 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).

[0027] 4. Experimental Results The results are as follows Figure 1 , Figure 2 As shown, the T-cell epitope peptide of the C962R protein mentioned in this invention has been verified through in vitro experiments to have the ability to induce T cells to secrete IFN-γ, thereby assisting in the control of ASFV infection and viral clearance.

[0028] Example 2: T cell epitope peptides of C962R protein 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.

[0029] 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.

[0030] 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.

[0031] 2. Preparation of T cells specific to the T cell epitope peptide of C962R protein 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.

[0032] Take the above 1×10 7 One 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.

[0033] 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.

[0034] 3. The T-cell epitope peptide of C962R protein stimulates 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.

[0035] 4. Experimental Results The results are as follows Figures 3-6 As shown, epitope peptide C962R-1, within the experimental concentration range, exhibited approximately 2 times higher cell proliferation than the control group. Epitope peptide C962R-2 showed the most significant proliferation effect at a concentration of 10 μg / mL, approximately 1.5 times higher than the control group. Epitope peptide C962R-3 showed the most significant proliferation effect at a concentration of 5 μg / mL, approximately 1.21 times higher than the control group. Epitope peptide C962R-4 showed the most significant proliferation effects at concentrations of 5 μg / mL and 20 μg / mL, approximately 1.15 times higher than the control group. This demonstrates that the above epitope peptides possess the ability to induce specific T cell proliferation.

[0036] In summary, this invention provides a T-cell epitope polypeptide based on the C962R protein. The T-cell epitope polypeptide includes one or more of epitope polypeptides C962R-1, C962R-2, C962R-3, and C962R-4, with amino acid sequences as shown in SEQ ID NO. 1-4. These epitope polypeptides are T-cell epitope polypeptides of the C962R protein obtained through screening, possessing the properties of inducing ASFV-specific T cells and assisting in the control of ASFV infection and viral clearance. In vitro experiments have verified the ability of the epitope polypeptide 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.

[0037] 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 C962R protein, characterized in that, The T-cell epitope polypeptides include one or more of epitope polypeptides C962R-1, C962R-2, C962R-3, and C962R-4, with amino acid sequences as shown in SEQ ID NO.1~4.

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. 1~4 that has been substituted, deleted, and / or have one or more amino acids added while maintaining the original functional consistency.

3. A nucleotide encoding a T-cell epitope polypeptide as described in claim 1 or 2.

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. The use of the T-cell epitope polypeptide of claim 1 or 2, the nucleotide of claim 3, the recombinant vector of claim 4, or the recombinant microbial cell of claim 5 in the preparation of a drug for detecting T-cell immune responses induced by ASFV or for the preparation of a drug for treating or preventing ASFV infection.

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