H9N2 subtype avian influenza virus TCRγδ+CD8α+ T cell recognition epitope peptide and its application

Abstract

The application belongs to the technical field of biology, and discloses an avian influenza virus TCR gamma delta + CD8 alpha + T cell recognizes an epitope polypeptide, and an amino acid sequence of the epitope polypeptide is shown in any one of SEQ ID NO. 1 to SEQ ID NO. 3. The application takes H9N2 subtype AIV infection-induced SPF chicken TCR gamma delta + CD8 alpha + T cell as a research object, and screens a virus target protein recognized by the cell subpopulation; on the basis, a long peptide of the target protein is designed according to structure prediction and antigenicity score, and a dominant long peptide region recognized by the TCR gamma delta + CD8 alpha + T cell is further screened; finally, the TCR gamma delta + CD8 alpha + T cell is precisely positioned and obtained by step moving and truncation screening, and recognizes a core epitope sequence. Meanwhile, the application also provides an antigen and a use of the antigen.

Description

Technical Field

[0001] This invention relates to the field of biology, specifically to an H9N2 subtype avian influenza virus TCRγδ. + CD8α + T-cell recognition epitope peptides. Background Technology

[0002] The H9N2 subtype of avian influenza virus (H9N2 AIV) is one of the important low-pathogenic avian influenza viruses that are currently widespread in poultry farming. Infection with this virus can cause respiratory symptoms, decreased production performance, immunosuppression, and an increased risk of secondary infections in chickens.

[0003] Currently, the prevention and control of H9N2 AIV mainly relies on traditional vaccine strategies such as inactivated vaccines. Although inactivated vaccines have some effect in reducing clinical symptoms, due to continuous viral mutation, insufficient antigen matching, and limited ability to induce mucosal and cellular immunity, there may still be problems such as incomplete immune protection, difficulty in effectively blocking viral shedding, and insufficient cross-protection. Therefore, relying solely on humoral immune indicators to evaluate vaccine efficacy has certain limitations. There is an urgent need to deeply analyze the protective immune mechanism after H9N2 AIV infection or immunization from the perspective of cellular immunity, so as to provide a basis for the design of novel vaccines and immune enhancement strategies.

[0004] T cell-mediated cellular immunity plays a crucial role in the recognition and clearance of infected cells and in long-term immune protection. Unlike antibodies, which primarily recognize viral surface antigens, T cells can recognize antigenic peptide epitopes derived from viral structural or non-structural proteins. Therefore, they have significant applications in addressing viral antigenic drift, enhancing cross-protective potential, and constructing epitope vaccines. Screening for antigenic proteins and dominant epitopes that can induce effective T cell responses against avian influenza viruses is a fundamental step in developing broad-spectrum, durable cellular immune vaccines.

[0005] The following technical solutions exist for screening existing T-cell antigenic peptide epitopes: The patent application, with publication number CN117964718A, is for a T-cell epitope polypeptide based on the HA1 protein of H9 subtype avian influenza virus and its application. It predicts 55 polypeptides of the HA1 protein and verifies 2 of them.

[0006] No existing protocols have been reported for the development of epitope peptides that contribute to the immune response of γδT cells.

[0007] γδ T cells are an important population of T cells in the avian immune system, with a high proportion in chickens, especially in peripheral blood, spleen, and mucosal-associated tissues, where they possess strong immune response potential. In particular, TCRγδ... +CD8α + T cell subsets may be closely related to effector function, cytotoxic activity, and immune protection after viral infection. Previous studies have shown that TCRγδ... + CD8α + T cells play a crucial role in the early stages of H9N2 AIV infection. This suggests that this cell subset may be one of the important effector cells in chicken cellular immunity against H9N2 AIV. However, previous studies on γδ T cell antigen recognition have largely focused on recognition mechanisms such as non-classical antigens, stress-related molecules, lipid antigens, or conformation-dependent ligands. Whether viral epitopes can be recognized by chicken TCR γδ T cells remains a mystery. + CD8α + T cell recognition currently lacks clear reports. Existing H9N2 AIV-related epitopes mainly originate from traditional TCRαβ. + CD8α + T cell epitope selection is difficult to directly reflect TCRγδ + CD8α + T cell antigen recognition characteristics and effector response patterns. Therefore, which viral proteins in H9N2 AIV can be recognized by chicken TCRγδ? + CD8α + T cell recognition, which viral peptides can induce a response in this cell subpopulation, and what its core recognition sequence is, remain unsolved problems in existing technologies.

[0008] Therefore, the analysis of H9N2 AIV can be performed on chicken TCRγδ + CD8α + Identifying viral-derived peptide epitopes recognized by T cells, defining their target proteins, dominant long peptide regions, and core epitope sequences, is crucial for demonstrating the effectiveness of chicken TCRγδ. + CD8α + T cells possess the ability to recognize viral-derived peptide epitopes, revealing the mediated anti-H9N2 AIV cellular immune mechanism, enriching avian influenza virus T cell epitope resources, and contributing to subsequent targeting of TCRγδ. + CD8α + Providing candidate targets for T-cell epitope vaccine design and immune control strategy formulation is of great significance.

[0009] Based on this premise, this application is hereby submitted. Summary of the Invention

[0010] The main objective of this invention is to provide an H9N2 subtype avian influenza virus TCRγδ + CD8α + T cell recognition epitope polypeptide, the present invention uses SPF chicken TCRγδ induced by H9N2 subtype AIV infection. + CD8α +Using T cells as the research subject, the RCSABP reverse genetics system was used to rescue recombinant viruses expressing 10 viral proteins of H9N2 AIV, and these proteins were then stimulated in vitro to activate TCRγδ. + CD8α + T cells were used to assess their immune response, and viral target proteins recognized by this cell subset were screened. Based on this, long peptides of the target proteins were designed according to structural prediction and antigenicity scores, and TCRγδ was further screened. + CD8α + The dominant long peptide regions recognized by T cells were finally precisely located and obtained through walking and truncation screening. + CD8α + The core epitope sequence recognized by T cells. Through the above experiments, this invention demonstrates that chicken TCRγδ + CD8α + T cells can respond to H9N2 AIV-derived peptide epitopes and provide candidate epitopes for subsequent research on γδT cell antigen recognition mechanisms, cellular immunity evaluation, and epitope vaccine design.

[0011] In addition, the present invention also provides an antigen and the use of the antigen.

[0012] To achieve the above objectives, the present invention provides the following technical solution: A type of avian influenza virus TCRγδ + CD8α + T cell recognition epitope polypeptide, wherein the amino acid sequence of the epitope polypeptide is shown in any one of SEQ ID NO.1 to SEQ ID NO.3.

[0013] Meanwhile, the present invention also discloses an avian influenza antigen having an amino acid fragment as shown in any of SEQ ID NO.1 to SEQ ID NO.3, or an overlap of multiple amino acid fragments.

[0014] In the antigens described above, polypeptides with a fragment length of 0 to 15 amino acids are attached to both ends of the antigen.

[0015] In the above-mentioned antigens, the polypeptide is: (1) The polypeptide located at one or both ends of the epitope in the avian influenza virus; or, (2) Peptides used to stimulate cells to produce an immune response.

[0016] Among the antigens mentioned above, the avian influenza virus is one of H5N1, H5N6, H5N8, H7N9, and H9N2.

[0017] Finally, this invention discloses the preparation of a method for stimulating TCRγδ using the epitope peptides described above or any of the antigens described above. + CD8α + Uses of immune enhancers that promote T-cell immune responses.

[0018] One of the above-described technical solutions of the present invention has at least one of the following advantages or beneficial effects: 1. This invention identifies three TCRγδ pathways targeting H9N2 AIV. + CD8α + Dominant antigenic epitopes recognized by T cells.

[0019] 2. Two of the three dominant antigenic epitopes of this invention are highly conserved in various subtypes of avian influenza viruses.

[0020] 3. This invention proves that chicken TCRγδ + CD8α + T cells can respond to H9N2 AIV-derived peptide epitopes and provide candidate epitopes for subsequent research on γδT cell antigen recognition mechanisms, cellular immunity evaluation, and epitope vaccine design. Attached Figure Description

[0021] Figure 1A The plasmid map of the vector RCSABP; Figure 1B The plasmid map of plasmid RCASBP-HA; Figure 1C The results show the PCR identification of each recombinant virus; lane M in the figure is the marker, and lane R is the empty RCASBP vector. Figure 2 A curve showing the S / P ratio of p27 antigen in cell culture supernatant detected by ELISA; Figure 3 The results of Western Blot identification of each recombinant virus are shown in the figure; lane M is the marker, lane R is the protein lane of DF-1 cells infected with RCASBP empty vector, and the rest are the protein lanes of DF-1 cells infected with each recombinant virus. Figure 4A For Flag + DF-1 cell swarm gate strategy diagram; Figure 4B For Flag + DF-1 cell flow cytometry single-gated plot; Figure 4C Flags for F1-F3 generations after three independent replicate experiments transfecting recombinant plasmids. + DF-1 cell percentage curve; Figure 5AFor IFN-γ + TCRγδ + CD8α + Flow cytometry gating strategy diagram for T cells; Figure 5B For IFN-γ + TCRγδ + CD8α + T cell flow cytometry single-gated loop diagram; Figure 5C For IFN-γ + TCRγδ + CD8α + A bar chart showing the percentage of T cells; Figure 6A This is a bar chart showing the IFN-γ response levels as detected by IFN-γ ELISpot after stimulation of HA candidate epitopes. Figure 6B This is a bar chart showing the IFN-γ response levels as detected by IFN-γ ELISpot after stimulation of PA candidate epitopes. Figure 6C This is a bar chart showing the IFN-γ response levels as detected by IFN-γ ELISpot after stimulation of PB2 candidate epitopes. Figure 6D IFN-γ levels as detected by ICS after stimulation of HA candidate epitopes + TCRγδ + CD8α + T cell proportion bar chart; Figure 6E IFN-γ as detected by ICS after stimulation of PA candidate epitopes + TCRγδ + CD8α + T cell proportion bar chart; Figure 6F IFN-γ as detected by ICS after stimulation of PB2 candidate epitopes + TCRγδ + CD8α + T cell proportion bar chart; Figure 7A For HA 330-338 Figure showing the results of conservation analysis of positive epitope peptide sequences; Figure 7B PA 414-427 Figure showing the results of conservation analysis of positive epitope peptide sequences; Figure 7C For PB2 698-706 Figure showing the results of sequence conservation analysis of positive epitope peptides. Detailed Implementation

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] I. Terminology Explanation Terminology Explanation: AIV: Avian influenza virus; APC: Antigen presenting cells; SPF chickens: Specific pathogen-free chickens; CTL: Cytotoxic T cells; IFN-γ: Interferon gamma; DPI: Days after virus infection; EID50: Median infection dose in chicken embryos; ELISpot: Enzyme-linked immunospot assay; FBS: Fetal bovine serum; ICS: Intracellular factor staining.

[0024] II. Reagents and Materials 1.1 Background of the strain A / Chicken / HuNan / HN / 2015 (H9N2 subtype AIV) was isolated and preserved by the National-Local Joint Engineering Laboratory for Zoonotic Disease Prevention and Control.

[0025] 1.2 Chicken embryos and laboratory animals The 4-week-old SPF chickens and 9-11-day-old SPF chicken embryos were provided by Guangdong Xinxing Dahua Agricultural Poultry and Egg Co., Ltd.

[0026] 1.3 Main Reagents RPMI-1640 medium, FBS (Australian fetal bovine serum), PBS (pH 7.4 basic 1×), 10000 UI of penicillin-dextrin antibiotics, L-glutamine (200 mM), HEPES (100×), sodium pyruvate (100 mM), non-essential amino acids (100×), and 0.25% Trypsin-EDTA trypsin were purchased from GIBCO, USA. Chicken peripheral blood lymphocyte separation kit, chicken organ tissue mononuclear lymphocyte separation kit, and erythrocyte lysis buffer were purchased from Tianjin Haoyang Biotechnology Co., Ltd.; 2-mercaptoethanol (55 mM), concanavalin A (ConA), and TPCK-treated trypsin were purchased from Sigma-Aldrich (USA); ChamQ SYRB... RqPCR Master Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd.; TMB ELISpot chromogenic reagent was purchased from China Daktronics Co., Ltd.; Chicken IFN-γ ELISpot BASIC The kit was purchased from Mabtech; Viability Dye eFluor TM 780 was purchased from Thermo Fisher Scientific; flow cytometry antibodies Anti-chicken CD3 antibody, Anti-chicken TCRγδ antibody, and Anti-chicken CD8α antibody were purchased from Southern Biotech. All candidate peptides and epitopes were synthesized by Shanghai Taopu Technology Co., Ltd., with the purity of the candidate peptides preferably not less than 95%. The synthesized candidate peptides were dissolved in dimethyl sulfoxide and prepared into stock solutions, preferably at a concentration of 10 mmol / L, aliquoted, and stored at −80℃ for later use. The RCASBP(A) expression system and recombinant viral plasmid were stored in this laboratory for future use.

[0027] 1.4 Preparation of main reagents (1) 10% complete culture medium (RP-10): 10% FBS + 90% 1640, after preparation, store at 4℃.

[0028] (2) T cell culture medium: 10% FBS + 1% non-essential amino acids, 1% glutamine, 1% sodium pyruvate, 1% penicillin and antibiotics, β-mercaptoethanol + 90% RMPI 1640. After thorough mixing, the culture medium is divided into portions and stored at 4°C for use.

[0029] (3) Flow cytometry buffer: 2% FBS + 98% PBS, after preparation, store at 4℃ for later use.

[0030] (4) Cell cryopreservation solution: 10% DMSO + 90% FBS. After preparation, store at 4℃ for later use.

[0031] III. Brief Description of the Plan This invention first induced TCRγδ in SPF chickens by infecting them with H9N2 subtype AIV. + CD8α + Using T cells as the research subject, the RCSABP reverse genetics system was used to rescue recombinant viruses expressing different viral proteins of H9N2 AIV. These viruses were then stimulated in vitro to target H9N2 AIV-specific TCRγδ. + CD8α + T cells were used to assess their immune response and screen for cells that could be converted to TCRγδ. + CD8α+ T cells recognize viral-derived target proteins. Then, based on the amino acid sequences of the screened target proteins, structural prediction and antigenicity scoring are performed to design long peptides of the target proteins, which are then used to stimulate the corresponding dominant protein-specific TCRγδ. + CD8α + T cells, by detecting cell activation or effector response, are screened for TCRγδ. + CD8α + The dominant long peptide regions recognized by T cells were then identified. Finally, based on the identification of these dominant long peptides, a walking and truncation screening strategy was employed to finely locate candidate epitope regions and determine the TCRγδ region. + CD8α + The core epitope sequence recognized by T cells.

[0032] Using the above method, dominant antigenic epitopes targeting H9N2 AIV in chicken TCRγδ+CD8α+ T cells were obtained.

[0033] Example 1 1.1 Expression and identification of recombinant plasmid RCASBP(A) 1.1.1 Transfection of DF-1 cells with recombinant plasmid RCASBP(A) The day before transfection, prepare a 6-well cell culture plate containing DF-1 cells, ensuring a density of 80%-100% at transfection. Dilute 3 μg of donor plasmid DNA in 250 μL OPTI-MEM I medium according to the manufacturer's instructions, and vortex to mix. Dilute 6 μL Nulen PlusTrans in 250 μL OPTI-MEM I medium. TM Transfection reagent: After dilution, incubate at room temperature for 5 minutes. Mix diluted donor plasmid DNA and diluted Nulen Plus Transfection reagent. TM Mix the transfection reagent (total volume 500 μL) gently and incubate at room temperature for 20 min. Add 500 μL of DNA-Nulen PlusTrans... TM The complex was added dropwise to 6-well cell culture plates (with 1.5 mL of culture medium in the plates), the plates were gently shaken to mix, and then incubated at 37°C in a 5% CO2 incubator. Five hours after transfection, the medium was replaced with DF-1 cell maintenance medium.

[0034] The recombinant plasmids RCASBP(A) mentioned above are: RCASBP-HA, RCASBP-NA, RCASBP-PA, RCASBP-NP, RCASBP-PB1, RCASBP-PB2, RCASBP-M1, RCASBP-M2, RCASBP-NS1, and RCASBP-NS2; the plasmid map of the vector plasmid RCASBP is shown below. Figure 1A As shown, the plasmid map of recombinant plasmid RCASBP-HA is as follows. Figure 1B As shown in the diagram; the insertion sites for the remaining recombinant plasmids are referenced in RCASBP-HA; the sequences of the target genes inserted into the vector are as follows: HA gene as shown in SEQ ID NO.4; NA gene as shown in SEQ ID NO.5; PA gene as shown in SEQ ID NO.6; NP gene as shown in SEQ ID NO.7; PB1 gene as shown in SEQ ID NO.8; PB2 gene as shown in SEQ ID NO.9; M1 gene as shown in SEQ ID NO.10; M2 gene as shown in SEQ ID NO.11; NS1 gene as shown in SEQ ID NO.12; NS2 gene as shown in SEQ ID NO.13.

[0035] Four days after transfecting DF-1 cells with each recombinant plasmid, viral RNA was extracted from the cell culture supernatant and reverse transcribed into cDNA. The recombinant virus was then identified by PCR using primers RCASBP-F and RCASBP-R targeting the RCASBP(A) insertion site. The results are as follows: Figure 1C The specific bands at approximately 2067 bp, 1785 bp, 2535 bp, 1881 bp, 2658 bp, 2664 bp, 1143 bp, 678 bp, 1038 bp, and 750 bp show RCASBP-HA, RCASBP-NA, RCASBP-PA, RCASBP-NP, RCASBP-PB1, RCASBP-PB2, RCASBP-M1, RCASBP-M2, RCASBP-NS1, and RCASBP-NS2. Sequencing confirmed that the sequences were correct and represented the target protein genes of H9N2 AIV. This indicates that the recombinant plasmid can successfully and correctly express the H9N2 AIV protein genes after transfection into DF-1 cells.

[0036] 1.1.2 Identification of expression of recombinant plasmid RCASBP(A) After 4 days of culture, the supernatant of F1 generation cells was collected, and the cells in the 6-well plates were digested with 500 μL of trypsin. The reaction was terminated by adding 100 μL of FBS. The cells were centrifuged at 440 × g for 5 min, the supernatant was discarded, and the cells were resuspended in DF-1 cell growth medium. The cells were then seeded back into the 6-well plates and cultured for 4 days, labeled as F2 generation cells. The culture supernatant was collected, and the above operation was repeated to obtain F3 generation cells and culture supernatant.

[0037] (1) PCR identification Viral RNA was extracted from the supernatant of the three-generation cell culture using the Vazyme FastPure Viral DNA / RNA Mini Kit Pro. The concentration of the extracted RNA was measured using a spectrophotometer and stored at -80°C. Viral cDNA was obtained by reverse transcription of the extracted RNA. All operations were performed on ice. The reaction program was: 37°C for 15 min, 85°C for 5 s inactivation, and storage at 4°C.

[0038] Specific primers targeting the RCASBP(A) insertion site were used to perform PCR amplification with recombinant viral cDNA as a template. The PCR products were analyzed by DNA gel electrophoresis on a 1% agarose gel and sequenced after agarose gel recovery to determine whether each recombinant virus correctly expressed the target protein genes of H9N2 AIV.

[0039] (2) ELISA detection of p27 antigen in cell supernatant The recombinant virus solution from the supernatant of the three generations of cell culture was used to detect the p27 antigen in the cell supernatant according to the instructions of the ALV p27 ELISA kit from Harbin Guosheng Biotechnology.

[0040] To detect whether each recombinant plasmid could successfully construct and express recombinant viruses after transfection, each recombinant plasmid was transfected into DF-1 cells for 4 days and then passaged to F3. Cell culture supernatants from F1 to F3 were collected, and the p27 antigen of the recombinant virus was detected by ELISA and its S / P value was calculated according to the kit instructions. like Figure 2 As shown, a positive result was obtained in the culture supernatant of F1 to F3 cells with an S / P value >0.2 for the p27 antigen of the recombinant virus. The results of three independent replicate experiments showed that the S / P value of the p27 antigen of each recombinant virus in F1 to F3 was >1, indicating that the constructed recombinant virus had a high titer and good reproducibility.

[0041] (3) Western Blot identification Six-well cell culture plates containing DF-1 cells transfected with recombinant plasmid RCASBP(A) from F1, F2, and F3 generations were cultured at 37°C and 5% CO2 for 4 days. Western blotting was then performed as follows: Cell supernatant was discarded, cells were washed with pre-chilled PBS, and 200 μL of RIPA lysis buffer containing a protease inhibitor was added to each well. Lysis was performed on ice or at 4°C for 15 min. The liquid in the wells was then collected, thoroughly mixed, and centrifuged at 12000 × g at 4°C for 5 min. The supernatant was collected, and 5× SDS-PAGE loading buffer was added proportionally. After mixing, the mixture was boiled for 10 min before use. Add appropriate amounts of protein marker and sample sequentially to the sample wells. Stop electrophoresis after the protein band reaches the appropriate position. Cut off the PAGE gel containing the target protein and prepare a PVDF membrane of appropriate size (the PVDF membrane needs to be soaked in methanol for 30 s beforehand). Clamp the gel and PVDF membrane together using the "sandwich" method and place them in a transfer apparatus. Transfer the membrane at 400 mA for 20 min using rapid transfer buffer. Transfer the PVDF membrane to rapid blocking buffer and block at room temperature for 1 h. Wash 4 times with TBST for 5 min each time, then add rabbit-derived Flag monoclonal antibody and incubate overnight at 4°C. Wash 4 times with TBST for 5 min each time, then add HRP-conjugated goat anti-rabbit IgG antibody and incubate at room temperature in the dark for 1 h. Wash 4 times with TBST for 5 min each time, add chromogenic solution, and observe the results using a two-color infrared laser imaging analysis system.

[0042] The expression of each recombinant virus was detected by Western blotting, such as... Figure 3 As shown, no specific bands were detected in the DF-1 cell pellet infected with the empty RCASBP vector, while clear protein bands were visible in the DF-1 cell pellet infected with the recombinant virus. The protein sizes of NP-Flag, NA-Flag, PB1-Flag, PB2-Flag, HA-Flag, PA-Flag, NS1-Flag, NS2-Flag, M1-Flag, and M2-Flag were approximately 58 kDa, 57 kDa, 84 kDa, 84 kDa, 63 kDa, 82 kDa, 20 kDa, 16 kDa, 21 kDa, and 15 kDa, respectively, indicating that each recombinant virus could successfully express its respective target protein.

[0043] (4) Flow cytometry identification DF-1 cells transfected with recombinant plasmid RCASBP(A) in F1, F2, and F3 generations were cultured in 6-well cell culture plates at 37°C and 5% CO2 for 4 days. Flow cytometry analysis was then performed as follows: cells were washed three times with PBS, and trypsin-digested for 1 min to detach the cells from the culture dish. 1 mL of DF-1 cell growth medium was gently pipetted to form a single-cell suspension. The suspension was diluted 1:1 with 0.08% trypan blue and counted. Ten cells were collected. 6 Centrifuge at 440 × g for 5 min, discard the culture medium, wash once with flow cytometry buffer, and centrifuge at 440 × g for 5 min. Discard the liquid and use Viability DyeeFluoride. TM Flow cytometry staining was performed at 780°C, followed by antibody incubation at 4°C in the dark for 30 min. After incubation, 1 mL of flow cytometry buffer was added for washing, followed by centrifugation at 440 g for 5 min. After centrifugation and discarding the buffer, fixation / permeabilization was performed using a Fixation / Permeabilization Kit at 4°C for 20 min, followed by washing with wash buffer. Then, rabbit-derived Flag monoclonal antibody was used for incubation at 4°C for 30 min, followed by washing with wash buffer. Finally, the cells were labeled with goat anti-rabbit IgG-FITC secondary antibody, incubated at 4°C in the dark for 30 min, centrifuged, and resuspended in wash buffer. Flag was detected by flow cytometry. + Changes in cell proportions.

[0044] To detect the protein expression efficiency of DF-1 cells after transfection with various recombinant plasmids, DF-1 cells from F1 to F3 after transfection were collected, stained with Flag antibody, and analyzed by flow cytometry. + DF-1 cell swarm gating strategy, such as Figure 4A As shown, Flag + DF-1 cells single phylum such as Figure 4B Statistical analysis, such as Figure 4C After transfection with recombinant plasmids, three independent replicate experiments showed that DF-1 cells from F1 to F3 generations could express all single antigens of H9N2 AIV. The expression efficiency showed an increasing trend in F2 and F3 generations. F2 generation cells were sufficient for subsequent use as APCs to screen and identify H9N2 AIV antigen-specific TCRγδ. + CD8α + T cell target proteins.

[0045] Example 2 TCRγδ + CD8α + T cell in vitro expansion 2.1.1 In vitro stimulation of chicken TCRγδ by H9N2 subtype AIV +CD8α + T cell expansion Chicken spleens were aseptically collected, chopped, and added to RP-10 medium. Cell suspension was prepared by gentle pipetting and filtration through a 40 μm cell filter. The suspension was then subjected to density gradient centrifugation using chicken organ tissue mononuclear lymphocyte separation medium. Intermediate layer cells were collected, washed twice, resuspended in RP-10, and counted using 0.08% trypan blue.

[0046] Subsequently, H9N2 AIV was inoculated at an MOI of 2 × 10⁻⁶ cells. 6 Splenic mononuclear lymphocytes were incubated for 1 h, then replaced with RPMI 1640 medium containing 0.25 μg / mL TPCK and 10% FBS, and cultured at 39°C for another 5 h to obtain H9N2 AIV-infected splenic mononuclear lymphocytes as antigen-presenting cells (APCs).

[0047] Freshly isolated spleen mononuclear lymphocytes were also collected and divided into an H9N2 group, a positive control group, and a negative control group, and were administered at a dose of 3×10⁻⁶. 6 Cells were seeded at a density of 100 cells / mL in 48-well plates. After 6 h of culture, infected APCs were added to the H9N2 group at a ratio of 5:1 (cells to be stimulated:APCs), 2.5 μg / mL ConA was added to the positive control group, and no treatment was given to the negative control group. Cells were cultured at 39°C, and cell morphology was observed daily. Cell counts were also recorded using trypan blue staining.

[0048] 2.2.2 In vitro stimulation of chicken TCRγδ by dominant protein + CD8α + T cell expansion (1) H9N2 subtype AIV in vitro stimulation of chicken TCRγδ + CD8α + T cell expansion The H9N2 AIV-specific TCRγδ was analyzed according to the method in 2.1.1. + CD8α + T cells were expanded in vitro.

[0049] (2) RCASBP(A) recombinant virus infects antigen-presenting cells Take another 6×10 per hole 5 APC was performed on cells in a biochemical reaction tube. After centrifugation at 440×g for 5 min, the cells were resuspended in 1640 medium and cultured at 1000 mg / mL. 3 TCID 50Recombinant RCASBP(A) virus was inoculated at a ratio of 150 μL per well, with the virus solution to cell suspension at 1:1. After thorough mixing, the mixture was incubated at 39°C for 5 hours. Splenic mononuclear cells infected with recombinant RCASBP(A) virus were used as antigen-presenting cells in the experiment.

[0050] (3) APC stimulation of H9N2 AIV-specific TCRγδ + CD8α + T cells Stimulate according to the method in 2.1.1.

[0051] 2.3 H9N2 AIV-specific TCRγδ + CD8α + Screening and identification of T cell target proteins To further clarify TCRγδ + CD8α + T cells and their subpopulations, such as TCRγδ + CD8α + T cell-recognized target proteins of the H9N2AIV dominant immune response were analyzed using ICS (Intracytoplasmic Stromaling). DF-1 cells cultured for 4 days after transfection with ten recombinant plasmids RCASBP(A) that were successfully identified and expressed were collected as APCs. Cell counts were performed using 0.08% trypan blue staining. H9N2AIV antigen-specific TCRγδ proteins were added at a 5:1 ratio. + CD8α + T cells were added to APCs. APCs and T cells were co-cultured for 6 h, with BFA added simultaneously. A negative control consisted of DF-1 cells transfected with the empty vector, while the experimental group consisted of DF-1 cells transfected with each recombinant plasmid. A positive control consisted of DF-1 cells infected with H9N2 AIV. After co-culturing at 39℃ for 6 h, flow cytometry staining was performed, and the results were analyzed.

[0052] The H9N2 AIV antigen-specific TCRγδ was validated using the ICS method. + CD8α + The dominant target protein of T cells was amplified in vitro using the method described in section 2.2 for H9N2-specific TCRγδ. + CD8α + After T cells are incubated, APCs are incubated according to method 2.3. The incubated APCs and BFA are then added to the cultured specific TCRγδ. + CD8α + Intracellular factor staining was performed on T cells. IFN-γ + TCRγδ + CD8α + T-cell flow cytometry gate strategies, such as Figure 5A As shown, IFN-γ + TCRγδ + CD8α + T cell single-ring gate, such as Figure 5B As shown, the statistical analysis results are as follows: Figure 5C As shown. For TCRγδ + CD8α + Compared with the RCASBP empty vector group, T cells stimulated by the RCASBP-HA group, RCASBP-PA group and RCASBP-PB2 group produced significantly higher levels of IFN-γ (P < 0.05 or P < 0.01).

[0053] The results above show that for H9N2 AIV-specific TCRγδ + CD8α + The dominant target proteins of T cells are HA, PA, and PB2 proteins.

[0054] 2.4 Peptide Synthesis The amino acid sequence of the target viral protein was submitted to a protein structure feature prediction platform for analysis of its structural features at the amino acid residue level, including information on surface accessibility, secondary structure, and disordered regions. Based on the prediction results, protein regions with high predicted surface exposure and easy recognition by immune cells were preferentially selected as preliminary candidate peptides. Subsequently, antigenicity prediction analysis was performed on the above preliminary candidate peptides, and peptides with high antigenicity were screened as candidate recognition peptides for subsequent chemical synthesis and cellular immune function verification. The obtained candidate peptides were artificially synthesized according to the needs of subsequent experiments. The purity of the synthesized candidate peptides was preferably not less than 95%. The synthesized candidate peptides were dissolved in dimethyl sulfoxide and prepared into stock solutions, aliquoted, and stored at -80°C for later use. Specific epitope information is shown in Table 1.

[0055] Table 1. Candidate peptide sequence information KSLKLAVGLRNVPSRSSRGIFGAIAGFIE HA 321-349 14 GWYGFQHSNDQGVGMAA HA 358-374 15 LGENMAPEKVDFEDCKDVNDLKQYNSDEPEP PA 370-400 16 LTDSSWVELDEIGEDVAP PA 417-434 17 TPGGEVRNDDVD PB2 245-256 18 LPFAAAPPEQSRM PB2 619-631 19 GKEDKRYGPALSINELSNLAKGE PB2 698-720 20 KRKRDSSILTDSQTATKRIRMAIN PB2 736-759 21

[0056] 2.5 TCRγδ + CD8α + Screening for T cell recognition of dominant antigenic epitopes To further clarify TCRγδ + CD8α + T-cell-recognized H9N2 AIV immunodominant epitopes were analyzed using ICS (Intracytoplasmic Stromaling). TCRγδ-specific dominant protein was collected. + CD8α + T cells associate candidate epitopes with TCRγδ + CD8α +T cells were co-cultured for 6 h with BFA added, and a negative control group (DMSO) was set up. After co-culturing at 39℃ for 6 h, flow cytometry staining was performed and the cells were analyzed.

[0057] To screen TCRγδ + CD8α + The dominant antigenic epitope for T cell recognition of H9N2 AIV was first identified through candidate peptide region prediction for H9N2 AIV-related structural proteins. By analyzing the spatial structure and surface exposed regions of the target proteins, candidate peptide regions located in relatively exposed areas of the protein with potential for immune recognition were screened, and candidate peptides were designed and synthesized accordingly. Subsequently, the synthesized candidate peptides were used in T cells cultured in 2.5 μm to target the dominant protein-specific TCRγδ. + CD8α + T cells were used, and the ability of candidate peptides to induce cellular immune responses was detected using IFN-γ ICS.

[0058] The test results showed: TCRγδ + CD8α + IFN-γ in T cells + Cell ratio, results showed HA 321-349 PA 417-434 PB2 698-720 and PB2 736-759 Stimulation can increase IFN-γ + TCRγδ + CD8α + The T cell ratio (P < 0.05) indicates that the above candidate peptides can be activated by chicken TCRγδ. + CD8α + T cells recognize and induce a functional response. Figures 6A to 6C ).

[0059] Based on this, the positive candidate peptides were further truncated using a stepwise process to further pinpoint the core epitope region capable of inducing T cell responses. ICS analysis revealed that HA... 330-338 PA 417-427 and PB2 698-706 It can induce significant IFN-γ responses (P < 0.05, P < 0.01, or P < 0.001) and promote TCRγδ. + CD8α + T cells produce IFN-γ, indicating that this type of truncated peptide contains IFN-γ that can be absorbed by chicken TCRγδ. + CD8α + Candidate core epitopes recognized by T cells ( Figures 6D to 6F (Table 2).

[0060] The specific information on positive epitopes is shown in Table 2.

[0061] Table 2 Positive Epitope Sequence Information RNVPSRSSR HA 330-338 1 LTDSSWVELDE PA 417-427 2 GKEDKRYGP PB2 698-706 3

[0062] Example 3 Conservatism analysis of positive epitopes The positive epitope HA identified by screening 330-338 PA 417-427 PB2 698-706 Conservation analysis was performed, and the HA, PA, and PB2 protein sequences of H5N1, H5N6, H5N8, H7N9, and H9N2 AIV strains reported from 2005 to 2026 were compared. The results showed that among the three positive epitopes, PA... 417-427 With PB2 698-706 All five subtypes of avian influenza strains are highly conserved, PA 417-427 The conserved proportion among the five subtypes of avian influenza strains was 89.4%–99.48%, PB2 698-706 The conserved proportion among the five subtypes of avian influenza strains was 83.35%–99.35%, PA 417-427 With PB2 698-706 The epitopes showed a small number of variant strains in the compared sequences, and each mutant sequence involved only a single or two amino acid sites; HA 330-338 It is highly conserved only in the H9N2 AIV strain, accounting for 87.82%, but its conservation is lower in H5N1, H5N6, H5N8, and H7N9 AIV strains, with a high proportion of variant strains present. Figures 7A to 7C ).

[0063] The above results indicate that the three positive epitopes identified have broad protective potential against H9N2 AIV, and can provide a target basis for the design and development of subsequent T-cell epitope vaccines.

[0064] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A type of avian influenza virus TCRγδ + CD8α + T-cell recognition epitope polypeptide, characterized in that... The amino acid sequence of the epitope polypeptide is shown in any one of SEQ ID NO.1 to SEQ ID NO.

3.

2. The avian influenza virus TCRγδ according to claim 1 + CD8α + T-cell recognition epitope polypeptide, characterized in that... The avian influenza virus mentioned is one of H5N1, H5N6, H5N8, H7N9, or H9N2.

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