Norovirus-specific cd8+ t cell epitope peptide and use thereof

Abstract

This invention belongs to the fields of genetic engineering and protein engineering technology, specifically relating to a norovirus-specific CD8. + T-cell epitope peptides and their applications. This invention focuses on the CD8 specific capsid protein of the NV virus. + The study focused on T cells, using in vitro detection methods to sequentially screen a peptide library and peptide fragments that exhibit dominant immune responses to capsid proteins. Subsequently, based on these dominant immune peptides, a CD8-specific antibody against NV immune dominance was constructed. + The in vitro expansion and culture system for T cells successfully cultured VP1-P23, VP2-P16, and VP2-P26 dominant peptide-specific CD8. + T cells. Furthermore, based on the dominant 18 peptides, epitopes of the core short peptides were sequentially identified. This invention relates to norovirus-specific CD8... + T-cell epitope peptides can be used for the diagnosis of NV infection, and the superior response in previously infected individuals has multiple protective effects, which can be used for the development of antiviral vaccines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering and protein engineering, and particularly relates to a norovirus-specific CD8 + T cell epitope peptide and application thereof. BACKGROUND

[0002] The study on the specific immune response of human natural infection of norovirus (NV) finds that the body has a cross-immune protection effect of NV-specific T cells (NSTs). NV has dominant antigens and various dominant response T cell epitopes, and the NSTs derived from healthy people can effectively recognize the non-structural protein NS6 and capsid protein VP1 of the immune dominant epitope of NV, and present a multifunctional cross-immune protection effect on the clinical isolated mutant strains, so that the NSTs can be used for immune therapy against NV infection in clinic.

[0003] The NSTs (CD4 + and CD8 + T) play an important protective effect in the immune response against NV infection, but no non-CD8 + T lymphocyte can play a role in clearing NV. However, the function of specific CD8 + T cells is a "double-edged sword" in clinic. On the one hand, it plays an important role in the immune response against NV infection that cannot be ignored. It can effectively clear the potential virus in the body and produce immune memory, and plays a cross-protection effect when NV infects again. On the other hand, the specific CD8 + T cells also cause pathological damage to local tissues in the process of clearing viruses, thereby aggravating the occurrence and progression of gastrointestinal diseases. At the same time, the research results of specific T cells show that the occurrence of gastrointestinal diseases or symptoms after NV infection may be closely related to the effective response of specific CD8 + T lymphocytes, but the specific immune damage response mechanism needs to be further explored.

[0004] The capsid proteins VP1 and VP2 of NV together constitute the nucleocapsid component of the virus, which belongs to the structural protein of the virus. Its role is to combine with the susceptible receptors of the host, leading to the first step of virus infection of the host. The NV virus enters the body to infect host cells and replicates in the cells. The first step of T cell immune response is that the TCR on the surface of the virus-specific T cells specifically recognizes the epitope peptide processed and presented by the APC cells infected by the virus, so as to completely clear the virus in the body to achieve the purpose of cure, so screening the T cell epitope peptide will become the best source of vaccine material research and development, and provide a solid research foundation for follow-up norovirus vaccine research and development.

[0005] Epitope, also known as antigenic determinant, is a key amino acid sequence determining adaptive immune response. According to the different recognition receptors, epitope is divided into B cell epitope and T cell epitope. T cell epitope can be recognized by T cell receptor, thereby stimulating the proliferation and differentiation of specific T cells to produce anti-viral immunity. However, the specific cellular response produced by the body after pathogen infection or antigen stimulation is not from all amino acid sequences of the whole antigen molecule, but often a comprehensive embodiment of the response stimulated by one or several dominant peptide segments (epitopes) on the whole antigen molecule, i.e. the "immune dominance" phenomenon. Different individuals have unique immune dominant response spectrum characteristics, which may correspond to different disease processes and outcomes. Therefore, the development of CD8 + T cell dominant epitope peptides for NV capsid proteins VP1 and VP2 undoubtedly has a very broad prospect for the prevention and treatment of NV. SUMMARY

[0006] In order to overcome the deficiencies of the prior art, the present application focuses on the development of NV virus-specific CD8 + T cell epitope peptides, and uses in vitro detection method to sequentially screen the peptide library and peptide segment of the immune dominant response of the capsid protein, and then constructs the CD8 + T cell in vitro expansion culture system of the immune dominant peptide of the capsid protein, which lays a foundation for the determination of the immune dominant core peptide and the elucidation of the characteristics and functions of the immune dominant response epitope spectrum. The present application focuses on the spectrum characteristics of the specific CD8 + T cell dominant response in the gastrointestinal symptom group, which provides a new idea for in-depth understanding of the functions and mechanisms of immune response after NV infection, and provides a new clue for analyzing the pathogenic mechanism of NV infection and related gastrointestinal diseases.

[0007] To achieve the above object, the present application is realized by the following technical solutions:

[0008] The present application provides a norovirus-specific CD8 + T cell epitope peptide, wherein the epitope peptide is selected from at least one of the amino acid sequences shown in SEQ ID NO. 3, SEQ ID NO. 16, SEQ ID NO. 26, SEQ ID NO. 34, SEQ ID NO. 38, SEQ ID NO. 42, SEQ ID NO. 54, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 59, and SEQ ID NO. 71.

[0009] The epitope peptide corresponding to SEQ ID NO. 3 is VP1-P23 "LSPSQVTMFPHIIVDVRQ", the epitope peptide corresponding to SEQ ID NO. 34 is VP1-P23-4 "QVTMFPHIIVDVR", the epitope peptide corresponding to SEQ ID NO. 54 is VP1-P23-4-9 "MFPHIIVDVR", the epitope peptide corresponding to SEQ ID NO. 46 is VP1-P23-4-1 "MFPHIIVDV", and the epitope peptide corresponding to SEQ ID NO. 48 is VP1-P23-4-3 "FPHIIVDV"; the epitope peptide corresponding to SEQ ID NO. 16 is VP2-P16 "FSETDAARGAINAPMTKA", the epitope peptide corresponding to SEQ ID NO. 38 is VP2-P16-3 "TDAARGAINAPMT", and the epitope peptide corresponding to SEQ ID NO. 59 is VP2-P16-3-5 "TDAARGAIN"; the epitope peptide corresponding to SEQ ID NO. 26 is VP2-P26 "PARGPSNKSSNSSTATSV", the epitope peptide corresponding to SEQ ID NO. 42 is VP2-P26-2 "PARGPSNKSSNSS", and the epitope peptide corresponding to SEQ ID NO. 71 is VP2-P26-2-5 "RGPSNKSSN".

[0010] Preferably, the epitope peptide is selected from at least one of the amino acid sequences shown in SEQ ID NO. 48, SEQ ID NO. 59 and SEQ ID NO. 71.

[0011] Firstly, the present application identifies VP1 133-150 (VP1-P23), VP2 91-108 (VP2-P16) and VP2 151-168 (VP2-P26) through in vitro detection and in vitro amplification culture, which are three immunodominant 18mer peptide epitopes, and the specific CD8 + T cell responses to capsid proteins VP1 and VP2 are developed. + It is found that the amount of IFN-γ secreted by specific CD8

[0012] Then, based on the in vitro detection of the capsid protein dominant response peptide segment, the in vitro amplification culture system of the specific CD8 + T cells to the dominant peptide is constructed, and through continuous exploration and optimization of the in vitro culture system, the specific CD8 +T cell lines. On this basis, the immunodominant core short peptide is screened, and the dominant 18mer, 13mer, 10mer, 9mer and 8mer peptides are identified in turn.

[0013] Further identification of a series of short peptides is carried out around the immunodominant epitope VP1-P23 (VP1 133-150 ) of the capsid protein. On the basis of screening the 18mer peptide of VP1-P23 (VP1 133-150 ) "LSPSQVTMFPHIIVDVRQ", the screening of the core short peptide is carried out, and the amino acid sequences of the core 13mer, 10mer, 9mer and 8mer are QVTMFPHIIVDVR, MFPHIIVDVR, MFPHIIVDV and FPHIIVDV in turn. In addition, the most core 9mer and 8mer are identified in turn on the basis of the core 10mer peptide. Theoretically, the epitope recognized by specific CD8 + T cells is 8-12 amino acids, and thus the core 8mer sequence FPHIIVDV identified by the application can be confirmed as the minimum epitope that can be recognized by specific CD8 + T cells. In addition, the application also first discovers and identifies two immunodominant epitopes VP2-P16 (VP2 91-108 ) and P26 (VP2 151-168 ) of VP2. On the basis of the dominant response 18mer, the core 13mer peptide corresponding to the epitope is screened, which are TDAARGAINAPMT and PARGPSNKSSNSS respectively. On this basis, the screening of the core short peptide is carried out, and the sequences of the VP2 core 9mer short peptides are TDAARGAIN and RGPSNKSSN respectively.

[0014] The application provides the amino acid sequences of the above-mentioned norovirus-specific CD8 + T cell epitope peptides.

[0015] The application provides a biological material containing the amino acid sequences of the above-mentioned norovirus-specific CD8 + T cell epitope peptides, characterized in that the biological material comprises synthesis of overlapping peptides or isolation and extraction of host cells.

[0016] The application provides that the above-mentioned norovirus-specific CD8 + T cell epitope peptides exhibit a highly conservative property in different genotypes of norovirus.

[0017] The application provides the application of the above-mentioned norovirus-specific CD8 + T cell epitope peptides in the preparation of reagents or kits for detecting norovirus.

[0018] The application provides the norovirus-specific CD8 + The application provides the norovirus-specific CD8

[0019] The application discloses the most core epitope 8mer "FPHIIVDV" of the VP1-P23-4 dominant epitope-specific CD8 + The application discloses the most core epitope 9mer "TDAARGAIN" of the VP2-P16-3 dominant epitope-specific CD8 + The application discloses the most core epitope 9mer "RGPSNKSSN" of the VP2-P26-2 dominant epitope-specific CD8 + The application discloses the most core epitope 9mer "RGPSNKSSN" of the VP2-P26-2 dominant epitope-specific CD8 + The application discloses the most core epitope 9mer "RGPSNKSSN" of the VP2-P26-2 dominant epitope-specific CD8

[0020] The application provides a polypeptide vaccine, and the vaccine comprises the norovirus-specific CD8 + T cell epitope peptide.

[0021] The application provides a detection kit for norovirus, and the kit comprises the norovirus-specific CD8 + T cell epitope peptide.

[0022] Compared with the prior art, the application has the beneficial effects that:

[0023] The application discloses the most core epitope 8mer "FPHIIVDV" of the VP1-P23-4 dominant epitope-specific CD8 + The application discloses the most core epitope 8mer "FPHIIVDV" of the VP1-P23-4 dominant epitope-specific CD8 + The application discloses the most core epitope 8mer "FPHIIVDV" of the VP1-P23-4 dominant epitope-specific CD8 +T cells. And on the basis of the dominant 18mer peptide, epitopes of core short peptides (13mer, 10mer, 9mer, 8mer peptides) were identified in turn. The NV-specific CD8 + T cell epitope peptides can be used for the diagnosis of NV infection, and the dominant response screened in the previously infected persons can have an immune protective effect, which can be used for the development of anti-viral infection vaccines. The present application focuses on the specific CD8 + The spectrum characteristics of the T cell dominant response provide new ideas for in-depth understanding of the function and mechanism of the immune response after NV infection, and provide new clues for analyzing the pathogenic mechanism of NV infection and related gastrointestinal diseases. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The in vitro screening results of the VP1 peptide library (the left graph is the frequency of the dominant response of the VP1 peptide library, and the right graph is the intensity analysis of the dominant response of the VP1 peptide library; * p<0.05, ** p<0.01, *** p<0.001);

[0025] Figure 2 The in vitro screening results of the VP2 peptide library (the left graph is the frequency of the dominant response of the VP2 peptide library, and the right graph is the intensity analysis of the dominant response of the VP2 peptide library; * p<0.05, ** p<0.01, *** p<0.001);

[0026] Figure 3 The screening results of the 18mer peptide specific to the CD8+ T cells of the VP1-POOL3 peptide library (the left graph is the peptide screening heat map of the VP1-POOL3 peptide library, and the right graph is the statistical analysis graph of the dominant response of the VP1-POOL3 peptide library);

[0027] Figure 4 The screening results of the 18mer peptide specific to the CD8+ T cells of the VP2-POOL2 and VP2-POOL3 peptide libraries (the upper left graph is the peptide screening heat map of the VP2-POOL2 peptide library, the upper right graph is the statistical analysis graph of the dominant response of the VP2-POOL2 peptide library; the lower left graph is the peptide screening heat map of the VP2-POOL3 peptide library, and the lower right graph is the statistical analysis graph of the dominant response of the VP2-POOL3 peptide library);

[0028] Figure 5Figure A shows that two 10mers are dominant responses, Figure B shows the dilution to identify the core 10mer sequence, Figure C shows that on the basis of the dominant 10mer, shorter 9mers and 8mers are identified, and the figure is a representative display of the entire process and logical relationship of the screening of the dominant short peptide segment in the experiment;

[0029] Figure 6 Figure A and Figure B show the results of the step-by-step screening of NV-VP2-P16, Figure C and Figure D show the results of the step-by-step screening of NV-VP2-P26, and the figure shows the results of the step-by-step screening of NV-VP2-P16 and P26 to identify the short peptide sequence of the T cell immune dominant response from 13mer to 9mer.

[0030] Figure 7 Figure C shows the results of the HLA-I molecule antibody blocking experiment of the dominant epitope of VP1-P23-4 (CD8 + T cell responses specifically recognize epitopes and exhibit the restriction characteristics of HLA-I molecules. DETAILED DESCRIPTION

[0031] The specific embodiments of the present application are further described below. It is to be understood that the description of these embodiments is intended to help understand the present application and does not constitute a limitation on the present application. In addition, the technical features involved in the various embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0032] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the experimental materials used in the following examples are all commercially available unless otherwise specified.

[0033] The polypeptides involved in the following examples are all synthesized by Shanghai Qiangyao Company through the overlapping peptide synthesis technology.

[0034] The peripheral whole blood samples of NV infected persons involved in the following examples are numbered as Donor 1-n (n is the total number of samples), and the same number is the same sample source.

[0035] Example 1 Norovirus-specific CD8 + T cell epitope peptide screening

[0036] 1. NV capsid protein epitope-specific CD8 +Response analysis of T cells and screening of dominant response peptide pool (POOL)

[0037] (1) In vitro ex vivo stimulation experiment of freshly isolated PBMC

[0038] ① 319 peripheral blood samples (264 of which were from NV previously infected persons) were collected, centrifuged at 1500 rpm for 5 min, and the plasma was collected into a 1.5 mL Ep tube and stored at -20℃ for subsequent NV infection antibody level detection analysis. The remaining blood sample was diluted with physiological saline, and an equal amount of separation solution (Tianjin Haoyang TBD Biotechnology Co., Ltd.) was added to a 15 mL centrifuge tube. The diluted blood sample was carefully suspended on the surface of the lymphocyte separation solution (Tianjin Haoyang TBD Biotechnology Co., Ltd.) with a 3 mL plastic soft pipette, and the interface of the separation solution was not broken. Then the centrifuge was adjusted to 0g and 800g, and centrifuged for 20 min. First, the clear liquid was discarded with a plastic pipette, and then the white single nucleus cell layer was ringed with a new pipette (the action of the plastic pipette was ringed) into a new 15 mL centrifuge tube. Finally, the cells were washed twice with 5 times the volume of physiological saline. The cells were precipitated by centrifugation at 250g for 10 min, and the freshly isolated PBMC was obtained. The concentration of the cells was adjusted to 5×10 5 cells / well for in vitro stimulation detection experiment.

[0039] ② Nine sub-peptide pools corresponding to VP1 (i.e. VP1-POOL1-9, each sub-peptide pool contains 10 peptide segments) and four sub-peptide pools of VP2 (i.e. VP2-POOL1-4, each sub-peptide pool contains 10 peptide segments) were selected from the NV-VP1 / VP2 total peptide pool (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6321856 / https: / / www.ncbi.nlm.nih.gov / nuccore / NC_039477.1 / ), and 1 μL of the corresponding peptide segment in the peptide pool (final concentration 1 μM) was used as the stimulator for in vitro ex vivo stimulation experiment. DMSO was designed as a control well.

[0040] (2) Intracellular cytokine staining (ICS) experiment

[0041] ①Cell surface marker staining: After 5h of cell stimulation at 37℃, the cells were first centrifuged at 1800rpm for 5min, and the supernatant was discarded. PerCP-Cy5.5 anti-human CD3 antibody, APC anti-human CD8 antibody, and FITC anti-human CD4 antibody (purchased from Biolegend, USA) were diluted with PBS (1:200). Then, the cells were resuspended in 50μL / well, and stained at 4℃ for 30min in the dark.

[0042] ②Cell fixation: First, 150μL / well of PBS was added, and the cells were centrifuged at 1800rpm for 5min, and the free antibody was discarded. Then, 100μL / well of 4% paraformaldehyde was added, and the cells were fixed at 4℃ for 20min in the dark.

[0043] ③Cell membrane breaking and intracellular staining: The cells were washed twice with 0.2% saponin, centrifuged at 1800rpm for 5min, and the paraformaldehyde was removed. The cytokine antibody was PE anti-human IFN-γ antibody (purchased from Biolegend, USA), which was diluted with 0.2% saponin at 1:100, and 50μL per well was added, and the cells were stained at 4℃ for 30min in the dark.

[0044] ④150μL of 0.2% saponin was added per well, and the cells were centrifuged at 1800rpm for 5min, and the free antibody was discarded. Then, 200μL / well of PBS was added to resuspend the cell pellet, and finally, the cells were detected by flow cytometry, and the NV-specific CD8 + T cell response was analyzed by Flowjo.

[0045] ⑤The characteristics of the T cell response specific to the capsid protein antigen were identified in vitro by analyzing the amount of IFN-γ secreted by NV-specific CD8 + T cells.

[0046] ⑥The Tc epitope peptide library corresponding to the dominant T cell immune epitope of the capsid protein was identified ex vivo by analyzing the characteristics of the NV-specific CD8 + T cell response.

[0047] As shown in Table 1, VP1-POOL3 (VP1-P21-30) was the dominant response peptide library in the VP1 peptide library; and as shown in Table 1, VP2-POOL2 (VP2-P11-20) and VP2-POOL3 (VP2-P21-30) were the dominant response peptide libraries in the VP2 peptide library. Figure 1 Figure 2 Table 1 Basic information of NV-VP1 / VP2 immune dominant peptide library

[0048] Table 1 Basic information of NV-VP1 / VP2 immune dominant peptide library

[0049] ​

[0050]

[0051] 2. Screening of 18mer peptides with superior responses by antigen-specific CD8+ T cell detection

[0052] On the basis of the screened superior response peptide libraries (VP1-POOL3, VP2-POOL2 and VP2-POOL3), further screening of 18mer peptides with superior responses by antigen-specific CD8+ T cell detection was carried out, and the specific steps were as follows: + T cell detection, and the specific steps were as follows:

[0053] (1) Screening of NV-VP1 / VP2 superior CTL epitopes

[0054] ① Isolation of peripheral blood mononuclear cells and in vitro ex vivo stimulation test: the specific experimental operation steps are described in detail in the process of screening of the peptide library. Screening of 18mer peptides with superior responses was carried out for individuals against the superior response peptide libraries (VP1-POOL3, VP2-POOL2 and VP2-POOL3). The difference between the two was that the stimulator was changed from the superior peptide library to the corresponding 10 peptides, and the final concentration of the peptides was 1 μM.

[0055] ② Cells were plated in 96-well U-bottom plates (100 μL / well) according to the amount of cells required per well (1 x 10 5 cells / sample). The corresponding 10 18mer peptides in the three superior response peptide libraries were added in turn. Meanwhile, mixed superior peptide library positive control wells and DMSO negative control wells were set up. The final stimulation concentration of the peptides was 1 μM, and 0.15 μL of Golgi stop (prevents protein from being secreted from the cell to the outside of the cell) was added per well. The stimulation culture was carried out in a 37°C, 5% CO2 incubator for 5 h, and finally the RF-10 complete culture medium was supplemented to a final volume of 200 μL per well.

[0056] ③ After the stimulation culture was completed, the proportion of CD8+ T cells secreting IFN-γ among the total CD8+ T cells was observed by ICS experiment to evaluate the strength of the NV-VP1 / VP2 superior peptide-specific signal. + +

[0057] (2) Intracellular factor staining of NV-VP1 / VP2 superior peptide-specific CD8+ T cells +

[0058] ​​​After 5 hours of cell culture at 37°C, specific T cell surface antibodies (PerCP-Cy5.5 anti-human CD3 antibody, APC anti-human CD8 antibody, FITC anti-human CD4 antibody) and cytokines (PE anti-human IFN-γ antibody) were stained, as detailed in the specific ICS experimental procedure. Finally, Flowjo software was used to analyze specific CD8 antibodies. + The response of T cells.

[0059] like Figure 3 As shown, in the dominant response VP1-POOL3 peptide library, VP1-P23 and VP1-P27 are the dominant 18-mer peptides. Figure 4 As shown, in the dominant response VP2-POOL2 peptide library, VP2-P16 is the dominant response 18mer peptide; in the dominant response POOL3 peptide library, VP2-P26 and VP2-P28 are the dominant response 18mer peptides.

[0060] 3. Highly conserved analysis and modeling of NV-VP1 / VP2 immunogenic 18mer peptides

[0061] ① Sequence alignment analysis of the identified norovirus-specific T-cell epitopes was performed on the National Center for Biotechnology Information (NCBI) website to clarify the conservation characteristics of the newly identified epitopes. Nucleotide and amino acid alignments of the target sequences were performed in the NCBI database (https: / / blast.ncbi.nlm.nih.gov / ). Search criteria: no country restrictions, source protein VP1 / VP2, source strain NV-GII. Through alignment analysis, the high conservation of the screened epitopes can be further clarified.

[0062] ② The dominant 18mer peptides screened in ex vivo in vitro experiments were subjected to homology analysis on NCBI. The results indicate that the selected dominant 18mer peptides are highly conserved among viral strains circulating in different regions, located in the shell region of the viral capsid protein, and exhibit high conservation. This provides important theoretical basis for the subsequent development of broad-spectrum anti-NV vaccines (see Table 2 for details).

[0063] Table 2. Amino acid sequence alignment analysis of representative NV-VP1 and VP2 dominant 18mer peptides.

[0064]

[0065]

[0066] Note: * represents the same amino acid.

[0067] ③ The amino acid sequence of the norovirus VP1 protein corresponding to the target sequence GenBank ID: NC_039477 (GII.P16_GII.4 genotype) was imported into the SWISS-MODEL online server (https: / / swissmodel.expasy.org / interactive) to predict the structure of the VP1 protein, and then the immunodominant epitopes were mapped into the three-dimensional crystal structure of the VP1 protein using PyMOL 2.5 software, so as to complete the modeling and positioning of the NV-VP1 immunodominant CD8 + T cell epitopes. It was finally found that the immunodominant epitopes screened were located in the S region, which was a highly conserved sequence of NV.

[0068] 4. Screening of NV-VP1 / VP2 dominant response short peptides

[0069] On the basis of the screened dominant response 18mer peptide segments (VP1-P23, VP2-P16, VP2-P26), further screening of dominant response short peptides was carried out, and the general screening process was as follows:

[0070] S1, stepwise overlapping synthesis of dominant peptide 13 amino acid short peptide sequences

[0071] Taking the initially screened immunodominant 18 amino acid short peptide as a template, 13 amino acid short peptides were synthesized by stepwise overlapping, with 2 amino acids per step and 11 amino acids overlapping.

[0072] S2, stepwise overlapping synthesis of dominant peptide 10, 9, 8 amino acid shorter peptide sequences

[0073] Taking the secondarily screened immunodominant 13 amino acid short peptide as a template, 10, 9, 8 amino acid shorter peptides were synthesized by stepwise overlapping, with 1 amino acid per step and 9 amino acids overlapping for 10mer, and then 9mer and 8mer short peptides were synthesized in turn.

[0074] S3, synthesis of series of short peptides

[0075] All NV-VP1 / VP2 synthetic peptides were initially purified, with a purity of more than 90%. The quality of each short peptide synthesized was 5 mg (2 mg + 2 mg + 1 mg), and 2 mg of short peptide was uniformly taken out for dilution and dissolution of the peptide to avoid repeated freeze-thawing of the stored peptide, which would affect the function of the peptide.

[0076] S4, the specific screening method of the dominant response short peptide is as follows:

[0077] (1) Screening of NV-VP1 / VP2 dominant CTL epitopes

[0078] ① Collection of cells: the specific CD8 + T cells (which are enriched in specific CD8 + T cells after stimulation with specific dominant peptides) from the isolated peripheral blood mononuclear cells (PBMCs) of a patient are inoculated into RF-10 complete medium (10% FCS 1640 medium) at a cell concentration of about 2-2.5 x 10 6 / mL, and cultured in vitro for 10 days at 37°C in a 5% CO2 incubator. The cells are collected by centrifugation at 1500 rpm for 8 min, and the supernatant is discarded to remove the old culture medium containing rhIL-2.

[0079] ② The specific T cells collected after in vitro culture for 10 days are resuspended in fresh RF-10 (1 x 10 6 / mL), and the cells are plated in a 96-well U-bottom plate (100 μL / well) at the required cell amount (1 x 10 5 cells / sample) per well. The corresponding peptide segments in the three dominant response peptide libraries are sequentially added. Meanwhile, a mixed dominant peptide library positive control well and a DMSO negative control well are set up. The final stimulation concentration of the peptides is 1 μM, and 0.15 μL of Golgi stop (to prevent protein secretion from the cells to the outside) is added per well. The stimulation culture is performed at 37°C in a 5% CO2 incubator for 5 h, and finally, RF-10 complete medium is added to a final volume of 200 μL per well.

[0080] ③ After the stimulation culture is completed, the proportion of CD8 + T cells secreting IFN-γ among the total CD8 + T cells is observed by ICS to evaluate the strength of the NV-VP1 / VP2 dominant peptide-specific signal.

[0081] (2) Factor staining of NV-VP1 / VP2 dominant peptide-specific CD8 + T cells

[0082] After 5 h of stimulation culture at 37°C, the specific T cell surface antibodies (PerCP-Cy5.5 anti-human CD3 antibody, APC anti-human CD8 antibody, and FITC anti-human CD4 antibody) and the cytokines (PE anti-human IFN-γ antibody) are stained, as described in detail in the specific operation steps of the ICS experiment. Finally, the Flowjo software is used to analyze the response of the specific CD8 + T cells.

[0083] (3) Screening of VP1-P23, VP2-P16, and VP2-P26 dominant response core 13mer peptides

[0084] As shown in Tables 3 and Figure 5 ,Figure 6 As shown in Table 3 and Table 4, on the basis of the VP1-P23-4, VP2-P16-3 and VP2-P26-2 dominant 13mer short peptides, further screening of the dominant 10mer short peptide VP1-P23-4-9, 9mer short peptide VP1-P23-4-1, 8mer short peptide VP1-P23-4-3, 9mer short peptide VP2-P16-3-5 and 9mer short peptide VP2-P26-2-5 was carried out.

[0085] Table 3 Basic information of NV-VP1 / VP2 immune dominant 18mer overlapping synthesis 13mer short peptides

[0086]

[0087]

[0088] (4) Screening of VP1-P23-4, VP2-P16-3 and VP2-P26-2 dominant response core series short peptides

[0089] As shown in Table 3 and Table 4, on the basis of the VP1-P23-4, VP2-P16-3 and VP2-P26-2 dominant 13mer short peptides, further screening of the dominant 10mer short peptide VP1-P23-4-9, 9mer short peptide VP1-P23-4-1, 8mer short peptide VP1-P23-4-3, 9mer short peptide VP2-P16-3-5 and 9mer short peptide VP2-P26-2-5 was carried out. Figure 5 、 Figure 6

[0090] Table 4 Basic information of NV-VP1 / VP2 immune dominant 13mer overlapping synthesis 10, 9, 8mer short peptides

[0091]

[0092]

[0093] ​As can be seen from the comprehensive embodiment 1, the application first adopts an in vitro detection method to screen and identify a peptide library of NV-VP1 / VP2 specific CD8+T cell immune dominant response, which are VP1-POOL3, VP2-POOL2 and VP2-POOL3; further screening obtains the dominant epitope peptides VP1-P23 "LSPSQVTMFPHIIVDVRQ", VP1-P23-4 "QVTMFPHIIVDVR", VP1-P23-4-9 "MFPHIIVDVR", VP1-P23-4-1 "MFPHIIVDV", VP1-P23-4-3 "FPHIIVDV" (in turn, 18mer, 13mer, 10mer, 9mer, 8mer peptide amino acid sequences corresponding to the screened and identified NV-VP1-P23); VP2-P16 "FSETDAARGAINAPMTKA", VP2-P16-3 "TDAARGAINAPMT", VP2-P16-3-5 "TDAARGAIN" (in turn, 18mer, 13mer, 9mer peptide amino acid sequences corresponding to the screened and identified NV-VP2-P16); VP2-P26 "PARGPSNKSSNSSTATSV", VP2-P26-2 "PARGPSNKSSNSS", VP2-P26-2-5 "RGPSNKSSN" (in turn, 18mer, 13mer, 9mer peptide amino acid sequences corresponding to the screened and identified NV-VP2-P26), which are all located in the highly conserved shell region of the NV virus genome.

[0094] Example 2: judging the HLA restriction characteristics of the dominant response core short peptides by HLA-I (A, B, C) class molecule antibody blocking experiment

[0095] Collecting the specific T cells after culture (resuspend the specific CD8 + After resuspending the T cells with RF-10, the concentration of cell culture is about 2-2.5×10 6 / mL, and the T cells are cultured in vitro for 10 days), centrifuging at 1500 rpm for 8 min, resuspending in fresh RF-10 culture medium after discarding the supernatant, and the concentration is 5×10 6Cells were seeded in 96-well U-type plates (100 μL / well), with 4 wells. 2 μL of HLA-A antibody, 3 μL of HLA-B antibody, and 2 μL of HLA-C antibody were added to each well. The control wells were treated with 1 μL of DMSO instead of the specific HLA class I antibody. The cells were incubated at 37°C for 30 min to allow the specific antibodies to block HLA molecules on the cell surface. The cells were then washed twice with RF-10 complete medium to remove free antibodies. 1 μM of a specific epitope peptide with a identified dominant response (the 8-mer peptide VP1-P23-4-3, and the 9-mer peptides VP2-P16-5 and VP2-P26-5, whose HLA restriction characteristics were verified by subsequent next-generation sequencing) and 0.15 μL / well Golistop RF-10 were added to each well to make a culture volume of 200 μL per well. After incubation at 37°C for 5 h, ICS staining was performed. By observing CD8 in each antibody well + Whether the secretion of IFN-γ by T cells is blocked is used to determine the HLA-restricted main type of the dominant peptide.

[0096] like Figure 7 As shown, the addition of HLA-A antibody to different individuals with a dominant response to the VP1-P23-4-3 epitope resulted in a decrease in the amount of INF-γ secreted by T cells, suggesting that this epitope exhibits HLA-A-restrictive characteristics.

[0097] Example 3: Next-generation sequencing to determine the HLA-I genotype of the dominant response core short peptide in NV samples.

[0098] Peripheral whole blood samples from NV-infected individuals were compared with norovirus-specific CD8. + The samples used for T-cell epitope peptides (8-mer short peptide VP1-P23-4-3, 9-mer short peptide VP2-P16-5, and VP2-P26-5) belonged to the same patient. The bottom layer, consisting of granulocytes and erythrocytes, obtained by Ficoll density gradient centrifugation was sent to Guangzhou Bofurui Medical Laboratory Co., Ltd. for HLA-I genotyping analysis using next-generation sequencing. Genotyping focused on the HLA-I gene, employing high-resolution genotyping detection targeting multiple alleles at three loci: HLA-A, B, and C.

[0099] Based on PCR amplification, library construction, data reading, and analysis, HLA typing results are obtained. NGS can simultaneously detect exon and intron regions, and when the distance between heterozygous sites is small, NGS can effectively identify the allele to which the heterozygous site belongs. This effectively reduces ambiguity and increases the resolution of the results.

[0100] As shown in Table 5, the VP1-P23-4 dominant response epitope presents the restriction characteristics of HLA-A*11:01, in which Donor3 is a homozygous individual of this genotype. At the same time, it can be seen that the genotyping detection results of this embodiment are consistent with the results of antibody blocking experiment. That is, the successful culture of NST-CD8 + T cells in vitro expansion culture, respectively, were analyzed for their HLA-I class molecule restriction. First, the cell surface expression of HLA-I class molecules was blocked by HLA-I (A, B, C) antibodies, respectively, and then VP1-P23-4-3 (8mer) was used to stimulate in vitro after culture, and ICS experiment was performed to identify the corresponding HLA-I gene restriction characteristics, which were all HLA-A. Then, the HLA-I genotype of all patients was detected by next-generation sequencing method, and it was found that 4 of the 8 NV infected patients with successful culture identification had HLA-A*11:01 genotype, and Donor3 was a homozygous genotype of HLA-A*11:01.

[0101] As shown in Table 6, 8 of the 21 samples responding to the VP2-P16-3-5 dominant response epitope have HLA-A*24:02 genotype characteristics, of which sample Donor10 is a homozygous individual of HLA-A*24:02 genotype, suggesting that the VP2-P16-3-5 dominant response epitope presents the restriction characteristics of HLA-A*24:02. As shown in Table 7, 8 of the 15 samples responding to the VP2-P26-2-5 dominant response epitope have HLA-C*07:02 genotype characteristics, of which samples Donor17 and Donor18 are both homozygous individuals of HLA-C*07:02 genotype, suggesting that the VP2-P16-3-5 dominant response epitope presents the restriction characteristics of HLA-C*07:02.

[0102] Table 5 HLA-I class molecule genotyping determination results of VP1-P23-4 dominant response epitope

[0103]

[0104] Table 6 HLA-I class molecule genotyping determination results of VP2-P16-3-5 dominant response epitope

[0105]

[0106] Table 7 HLA-I class molecule genotyping determination results of VP2-P26-2-5 dominant response epitope

[0107]

[0108]

[0109] As is well known, each individual possesses a specific HLA genotype, which determines the unique characteristics and patterns of the body's immune response, and is closely related to the occurrence, development, and prognosis of diseases. NV infection can stimulate the body to produce significant CD8+. + T lymphocyte response. This invention focuses on patients with a history of NV infection and gastrointestinal symptoms, specifically targeting the capsid protein-specific CD8. + Research was conducted on the dominant T-cell response profile and dominant epitope profile, revealing the specific CD8 dominant epitope of the capsid protein VP1-P23-4. + The core epitope of the T cell response, 8mer “FPHIIVDV”, was revealed to have a restriction characteristic of “HLA-A*11:01”; the capsid protein VP2-P16-3 dominant epitope specific CD8 + The core epitope of the T cell response, 9mer "TDAARGAIN," was revealed to have a restriction characteristic of "HLA-A*24:02"; the capsid protein VP2-P26-2 dominant epitope specific to CD8 + The core epitope of the T cell response, 9mer “RGPSNKSSN”, was identified, and the limiting characteristic of this epitope was revealed to be “HLA-C*07:02”.

[0110] Example 4 Norovirus-specific CD8 + Application of T-cell epitope peptides in clinical practice

[0111] Serum antibody levels (NV-VP-IgG were detected using the Norovirus ELISA Kit (DEIA2068S, purchased from CD Creative Diagnostics)) and viral nucleic acid (detected using the Norovirus Nucleic Acid Detection Kit (product standard number: YZB / LDQS003-2013, purchased from Hubei Langde Medical Technology Co., Ltd.) via PCR-fluorescent probe method) were analyzed. Finally, samples from 264 previously infected individuals who were negative for viral nucleic acid and positive for antibodies were included in the NV-specific CD8 assay. + A screening system for T-cell epitope peptides was used. Of the 264 samples, 152 were from patients presenting with significant gastrointestinal symptoms, while the remaining 112 samples came from healthy individuals without gastrointestinal symptoms. CD8 was found in patients with gastrointestinal symptoms. + The positive rate of T cell response was significantly higher in the two groups than in healthy individuals (44.74% vs. 9.82%, χ² = 37.49, p < 0.0001). Specifically, the CD8+ response rate differed significantly between the two groups. + The mean T cell response intensity was respectively ( The mean values ​​were 2.87±4.46 vs 2.33±1.78. Pairwise comparisons revealed that the number of individuals with gastrointestinal symptoms was significantly higher than that of healthy individuals (t=10.59, p<0.0001).

[0112] Comparison of specific CD8 in different populations of individuals previously infected with NV + Differences in T cell responses (experimental methods detailed in Example 1, CD8) + The in vitro stimulation assay and intrinsic factor staining assay used in T cell epitope screening analyze specific CD8+. + Differences in the amount of IFN-γ produced by T cells represent differences in their response. It was found that, compared to the healthy control group, the gastrointestinal symptom group showed higher levels of specific CD8+. + The positive rate of T cell responses was higher, and the intensity of the response was stronger. This CD8 + Differential T-cell responses may play different functions and effects. In the healthy control group, they exhibit anti-infective immune protection, while in the gastrointestinal symptom group, they may be related to the occurrence of gastrointestinal symptoms and the mechanism of local mucosal damage. Further analysis of the capsid protein-specific CD8... + This study investigates the response characteristics and patterns of T cells in previous infections based on dominant epitope profiles, explores their potential functions and roles, and further explores NV-specific CD8. + Study on the response characteristics of T cell dominant response epitopes in different populations (experimental methods are detailed in Example 1, CD8). + The in vitro stimulation assay and intrinsic factor staining assay used in T cell epitope screening analyze the specific CD8+ of cells stimulated by different peptide epitopes. + The changes in IFN-γ production by T cells were used to characterize the response features of their corresponding epitope profiles. Results showed that in 14 randomly selected healthy individuals exhibiting dominant responses to VP1-POOL3, VP2-POOL2, and VP2-POOL3, the 18-mer positive rates for the three dominant responses were VP1-P23 29% (4 / 14), VP2-P16 71% (10 / 14), and VP2-P26 50% (7 / 14), respectively. In contrast, in 19 randomly selected healthy individuals exhibiting dominant responses to VP1-POOL3, VP2-POOL2, and VP2-POOL3, the 18-mer positive rates were VP1-P23 63% (12 / 19), VP2-P16 26% (5 / 19), and VP2-P26 16% (3 / 19), respectively. Pairwise comparisons revealed that the positive rate of VP1-P23 was higher in the gastrointestinal symptom group than in the healthy control group (χ²). 2= 3.860, p = 0.049), which may be related to the damage caused by NV infection, and can be used for the diagnosis of NV infection and treatment; VP2-P16 and VP2-P26 have higher positive rates in the healthy examination group (χ 2 = 6.617, p = 0.015; χ 2 = 4.467, p = 0.035), which suggests that it is related to the immune protection of the body after NV infection, and can be used for the development of anti-NV infection vaccine.

[0113] The existing research on NV infection is only limited to the number and intensity of the responding cells, and the specific differences and response spectrum characteristics of the specific antigen (peptide) specific response, and the correlation and mechanism of NV infection and gastrointestinal diseases are still unclear. The research of the present application finds that the HLA-restricted CD8 + T cell response or functional differences may promote or cause NV infection-related gastrointestinal diseases, and even accelerate the process and other adverse outcomes. The present application provides a new clue for explaining the possible pathogenesis of NV infection and related gastrointestinal diseases, provides candidate antigen molecules and theoretical basis for the development of anti-NV infection vaccine, and provides a diagnostic basis for the evaluation of different processes of post-infection diseases.

[0114] In summary, the present research explains the strength of T cell response from the perspective of antigen and epitope. The dominant response in the acute and chronic infection can be used for the diagnosis of NV infection, and the dominant response screened in the previously infected persons has a protective effect, which can be used for the development of anti-viral infection vaccine.

[0115] The above describes the embodiments of the present application in detail, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of the embodiments can be made without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application. SEQUENCE LISTING <110> The Eighth Affiliated Hospital of Sun Yat-Sen University (Futian, Shenzhen) <120> A Norovirus-specific CD8+ T cell epitope peptide and application thereof <160> 79 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> PRT <213> VP1-P21 (Norovirus) <400> 1 Phe Ala Ala Val Pro Pro Asn Phe Pro Thr Glu Gly Leu Ser Pro Ser Gln Val Thr Met Phe Pro His lie lie Val Asp Val Arg Gin Leu Glu Pro Val Leu lie 1 5 10 15 Gln Val <210> 2 <211> 18 <212> PRT <213> VP1-P22 (Norovirus) <400> 2 Asn Phe Pro Thr Glu Gly Leu Ser Pro Ser Gln Val Thr Met Phe Pro 1 5 10 15 His Ile <210> 3 <211> 18 <212> PRT <213> VP1-P23 (Norovirus) <400> 3 Leu Ser Pro Ser Gln Val Thr Met Phe Pro His Ile Ile Val Asp Val 1 5 10 15 Arg Gln <210> 4 <211> 18 <212> PRT <213> VP1-P24 (Norovirus) <400> 4 Thr Met Phe Pro His Ile Ile Val Asp Val Arg Gln Leu Glu Pro Val 1 5 10 15 Leu Ile <210> 5 <211> 18 <212> PRT <213> VP1-P25 (Norovirus) <400> 5 Ile Val Asp Val Arg Gin Leu Glu Pro Val Leu lie Pro Leu Pro Asp 1 5 10 15 Val Arg <210> 6 <211> 18 <212> PRT <213> VP1-P26 (Norovirus) <400> 6 Leu Glu Pro Val Leu lie Pro Leu Pro Asp Val Arg Asn Asn Phe Tyr 1 5 10 15 His Tyr <210> 7 <211> 18 <212> PRT <213> VP1-P27 (Norovirus) <400> 7 Pro Leu Pro Asp Val Arg Asn Asn Phe Tyr His Tyr Asn Gin Ser Asn 1 5 10 15 Asp Ser <210> 8 <211> 18 <212> PRT <213> VP1-P28 (Norovirus) <400> 8 Asn Asn Phe Tyr His Tyr Asn Gin Ser Asn Asp Ser Thr lie Lys Leu 1 5 10 15 Ile Ala <210> 9 <211> 18 <212> PRT <213> VP1-P29 (Norovirus) <400> 9 Asn Gln Ser Asn Asp Ser Thr Ile Lys Leu Ile Ala Met Leu Tyr Thr 1 5 10 15 Pro Leu <210> 10 <211> 18 <212> PRT <213> VP1-P30 (Norovirus) <400> 10 Thr Ile Lys Leu Ile Ala Met Leu Tyr Thr Pro Leu Arg Ala Asn Asn 1 5 10 15 Ala Gly <210> 11 <211> 18 <212> PRT <213> VP2-P11 (Norovirus) <400> 11 Lys Glu Met Leu Gln Ala Gln Ile Glu Ala Thr Lys Lys Leu Gln Gln 1 5 10 15 Glu Met <210> 12 <211> 18 <212> PRT <213> VP2-P12 (Norovirus) <400> 12 Gln Ile Glu Ala Thr Lys Lys Leu Gln Gln Glu Met Met Lys Val Lys 1 5 10 15 Gln Ala <210> 13 <211> 18 <212> PRT <213> VP2-P13 (Norovirus) <400> 13 Lys Leu Gln Gln Glu Met Met Lys Val Lys Gln Ala Met Leu Leu Glu 1 5 10 15 Gly Gly <210> 14 <211> 18 <212> PRT <213> VP2-P14 (Norovirus) <400> 14 Met Lys Val Lys Gln Ala Met Leu Leu Glu Gly Gly Phe Ser Glu Thr 1 5 10 15 Asp Ala <210> 15 <211> 18 <212> PRT <213> VP2-P15 (Norovirus) <400> 15 Met Leu Leu Glu Gly Gly Phe Ser Glu Thr Asp Ala Ala Arg Gly Ala 1 5 10 15 Ile Asn <210> 16 <211> 18 <212> PRT <213> VP2-P16 (Norovirus) <400> 16 Phe Ser Glu Thr Asp Ala Ala Arg Gly Ala Ile Asn Ala Pro Met Thr 1 5 10 15 Lys Ala <210> 17 <211> 18 <212> PRT <213> VP2-P17 (Norovirus) <400> 17 Ala Arg Gly Ala Ile Asn Ala Pro Met Thr Lys Ala Leu Asp Trp Ser Gly Thr Arg Tyr Trp Ala Pro Asp Ala Arg Thr Thr Thr Tyr Asn Ala Gly Arg Phe Ser 1 5 10 15 Gly Thr <210> 18 <211> 18 <212> PRT <213> VP2-P18 (Norovirus) <400> 18 Ala Pro Met Thr Lys Ala Leu Asp Trp Ser Gly Thr Arg Tyr Trp Ala 1 5 10 15 Pro Asp <210> 19 <211> 18 <212> PRT <213> VP2-P19 (Norovirus) <400> 19 Leu Asp Trp Ser Gly Thr Arg Tyr Trp Ala Pro Asp Ala Arg Thr Thr 1 5 10 15 Thr Tyr <210> 20 <211> 18 <212> PRT <213> VP2-P20 (Norovirus) <400> 20 Arg Tyr Trp Ala Pro Asp Ala Arg Thr Thr Thr Tyr Asn Ala Gly Arg 1 5 10 15 Phe Ser <210> 21 <211> 18 <212> PRT <213> VP2-P21 (Norovirus) <400> 21 Ala Arg Thr Thr Thr Tyr Asn Ala Gly Arg Phe Ser Thr Pro Gin Pro Ser Gly Ala Leu Pro Gly Arg Ala Asn Leu Arg Asp Ala Val Pro Ala Arg Gly Pro Ser 1 5 10 15 Ser Gly <210> 22 <211> 18 <212> PRT <213> VP2-P22 (Norovirus) <400> 22 Asn Ala Gly Arg Phe Ser Thr Pro Gin Pro Ser Gly Ala Leu Pro Gly Arg Ala Asn Leu Arg Asp Ala Val Pro Ala Arg Gly Pro Ser 1 5 10 15 Arg Ala <210> 23 <211> 18 <212> PRT <213> VP2-P23 (Norovirus) <400> 23 Thr Pro Gin Pro Ser Gly Ala Leu Pro Gly Arg Ala Asn Leu Arg Asp Ala Val Pro Ala Arg Gly Pro Ser 1 5 10 15 Ala Val <210> 24 <211> 18 <212> PRT <213> VP2-P24 (Norovirus) <400> 24 Ala Leu Pro Gly Arg Ala Asn Leu Arg Asp Ala Val Pro Ala Arg Gly Pro Ser 1 5 10 15 Pro Ser <210> 25 <211> 18 <212> PRT <213> VP2-P25 (Norovirus) <400> 25 Asn Leu Arg Asp Ala Val Pro Ala Arg Gly Pro Ser Asn Lys Ser Ser 1 5 10 15 Asn Ser <210> 26 <211> 18 <212> PRT <213> VP2-P26 (Norovirus) <400> 26 Pro Ala Arg Gly Pro Ser Asn Lys Ser Ser Asn Ser Ser Thr Ala Thr 1 5 10 15 Ser Val <210> 27 <211> 18 <212> PRT <213> VP2-P27 (Norovirus) <400> 27 Asn Lys Ser Ser Asn Ser Ser Thr Ala Thr Ser Val Tyr Ser Asn Gln 1 5 10 15 Thr Ile <210> 28 <211> 18 <212> PRT <213> VP2-P28 (Norovirus) <400> 28 Ser Thr Ala Thr Ser Val Tyr Ser Asn Gln Thr Ile Ser Thr Arg Leu 1 5 10 15 Gly Ser <210> 29 <211> 18 <212> PRT <213> VP2-P29 (Norovirus) <400> 29 Tyr Ser Asn Gln Thr lie Ser Thr Arg Leu Gly Ser Thr Ala Gly Ser Gly Thr Ser Val Ser Ser 1 5 10 15 Gly Thr <210> 30 <211> 18 <212> PRT <213> VP2-P30 (Norovirus) <400> 30 Ser Thr Arg Leu Gly Ser Thr Ala Gly Ser Gly Thr Ser Val Ser Ser 1 5 10 15 Leu Pro <210> 31 <211> 13 <212> PRT <213> VP1-P23-1 (Norovirus) <400> 31 Glu Gly Leu Ser Pro Ser Gln Val Thr Met Phe Pro His 1 5 10 <210> 32 <211> 13 <212> PRT <213> VP1-P23-2 (Norovirus) <400> 32 Leu Ser Pro Ser Gln Val Thr Met Phe Pro His lie lie 1 5 10 <210> 33 <211> 13 <212> PRT <213> VP1-P23-3 (Norovirus) <400> 33 Pro Ser Gln Val Thr Met Phe Pro His lie lie Val Asp 1 5 10 <210> 34 <211> 13 <212> PRT <213> VP1-P23-4 (Norovirus) <400> 34 Gln Val Thr Met Phe Pro His lie lie Val Asp Val Arg 1 5 10 <210> 35 <211> 13 <212> PRT <213> VP1-P23-5 (Norovirus) <400> 35 Thr Met Phe Pro His lie lie Val Asp Val Arg Gln Leu 1 5 10 <210> 36 <211> 13 <212> PRT <213> VP2-P16-1 (Norovirus) <400> 36 Gly Phe Ser Glu Thr Asp Ala Ala Arg Gly Ala lie Asn 1 5 10 <210> 37 <211> 13 <212> PRT <213> VP2-P16-2 (Norovirus) <400> 37 Ser Glu Thr Asp Ala Ala Arg Gly Ala lie Asn Ala Pro 1 5 10 <210> 38 <211> 13 <212> PRT <213> VP2-P16-3 (Norovirus) <400> 38 Thr Asp Ala Ala Arg Gly Ala lie Asn Ala Pro Met Thr 1 5 10 <210> 39 <211> 13 <212> PRT <213> VP2-P16-4 (Norovirus) <400> 39 Ala Ala Arg Gly Ala Ile Asn Ala Pro Met Thr Lys Ala Leu Asp 1 5 10 <210> 40 <211> 13 <212> PRT <213> VP2-P16-5 (Norovirus) <400> 40 Arg Gly Ala Ile Asn Ala Pro Met Thr Lys Ala Leu Asp 1 5 10 <210> 41 <211> 13 <212> PRT <213> VP2-P26-1 (Norovirus) <400> 41 Ala Val Pro Ala Arg Gly Pro Ser Asn Lys Ser Ser Asn Ser Ser 1 5 10 <210> 42 <211> 13 <212> PRT <213> VP2-P26-2 (Norovirus) <400> 42 Pro Ala Arg Gly Pro Ser Asn Lys Ser Ser Asn Ser Ser 1 5 10 <210> 43 <211> 13 <212> PRT <213> VP2-P26-3 (Norovirus) <400> 43 Arg Gly Pro Ser Asn Lys Ser Ser Asn Ser Ser Thr Ala 1 5 10 <210> 44 <211> 13 <212> PRT <213> VP2-P26-4 (Norovirus) <400> 44 Pro Ser Asn Lys Ser Ser Asn Ser Ser Thr Ala Thr Ser 1 5 10 <210> 45 <211> 13 <212> PRT <213> VP2-P26-5 (Norovirus) <400> 45 Asn Lys Ser Ser Asn Ser Ser Thr Ala Thr Ser Val Tyr 1 5 10 <210> 46 <211> 9 <212> PRT <213> VP1-P23-4-1 (Norovirus) <400> 46 Met Phe Pro His Ile Ile Val Asp Val 1 5 <210> 47 <211> 9 <212> PRT <213> VP1-P23-4-2 (Norovirus) <400> 47 Thr Met Phe Pro His Ile Ile Val Asp 1 5 <210> 48 <211> 8 <212> PRT <213> VP1-P23-4-3 (Norovirus) <400> 48 Phe Pro His lie lie Val Asp Val 1 5 <210> 49 <211> 8 <212> PRT <213> VP1-P23-4-4 (Norovirus) <400> 49 Met Phe Pro His lie lie Val Asp 1 5 <210> 50 <211> 8 <212> PRT <213> VP1-P23-4-5 (Norovirus) <400> 50 Thr Met Phe Pro His lie lie Val 1 5 <210> 51 <211> 10 <212> PRT <213> VP1-P23-4-6 (Norovirus) <400> 51 Gln Val Thr Met Phe Pro His lie lie Val 1 5 10 <210> 52 <211> 10 <212> PRT <213> VP1-P23-4-7 (Norovirus) <400> 52 Val Thr Met Phe Pro His lie lie Val Asp 1 5 10 <210> 53 <211> 10 <212> PRT <213> VP1-P23-4-8 (Norovirus) <400> 53 Thr Met Phe Pro His lie lie Val Asp Val 1 5 10 <210> 54 <211> 10 <212> PRT <213> VP1-P23-4-9 (Norovirus) <400> 54 Met Phe Pro His lie lie Val Asp Val Arg 1 5 10 <210> 55 <211> 9 <212> PRT <213> VP2-P16-3-1 (Norovirus) <400> 55 Gly Phe Ser Glu Thr Asp Ala Ala Arg 1 5 <210> 56 <211> 9 <212> PRT <213> VP2-P16-3-2 (Norovirus) <400> 56 Phe Ser Glu Thr Asp Ala Ala Arg Gly 1 5 <210> 57 <211> 9 <212> PRT <213> VP2-P16-3-3 (Norovirus) <400> 57 Ser Glu Thr Asp Ala Ala Arg Gly Ala 1 5 <210> 58 <211> 9 <212> PRT <213> VP2-P16-3-4 (Norovirus) <400> 58 Glu Thr Asp Ala Ala Arg Gly Ala lie 1 5 <210> 59 <211> 9 <212> PRT <213> VP2-P16-3-5 (Norovirus) <400> 59 Thr Asp Ala Ala Arg Gly Ala Ile Asn 1 5 <210> 60 <211> 9 <212> PRT <213> VP2-P16-3-6 (Norovirus) <400> 60 Asp Ala Ala Arg Gly Ala Ile Asn Ala 1 5 <210> 61 <211> 9 <212> PRT <213> VP2-P16-3-7 (Norovirus) <400> 61 Ala Ala Arg Gly Ala Ile Asn Ala Pro 1 5 <210> 62 <211> 9 <212> PRT <213> VP2-P16-3-8 (Norovirus) <400> 62 Ala Arg Gly Ala Ile Asn Ala Pro Met 1 5 <210> 63 <211> 9 <212> PRT <213> VP2-P16-3-9 (Norovirus) <400> 63 Arg Gly Ala Ile Asn Ala Pro Met Thr 1 5 <210> 64 <211> 9 <212> PRT <213> VP2-P16-3-10 (Norovirus) <400> 64 Gly Ala Ile Asn Ala Pro Met Thr Lys 1 5 <210> 65 <211> 9 <212> PRT <213> VP2-P16-3-11 (Norovirus) <400> 65 Ala Ile Asn Ala Pro Met Thr Lys Ala 1 5 <210> 66 <211> 9 <212> PRT <213> VP2-P16-3-12 (Norovirus) <400> 66 Ile Asn Ala Pro Met Thr Lys Ala Leu 1 5 <210> 67 <211> 10 <212> PRT <213> VP2-P26-2-1 (Norovirus) <400> 67 Ala Val Pro Ala Arg Gly Pro Ser Asn Lys 1 5 10 <210> 68 <211> 9 <212> PRT <213> VP2-P26-2-2 (Norovirus) <400> 68 Val Pro Ala Arg Gly Pro Ser Asn Lys 1 5 <210> 69 <211> 9 <212> PRT <213> VP2-P26-2-3 (Norovirus) <400> 69 Pro Ala Arg Gly Pro Ser Asn Lys Ser 1 5 <210> 70 <211> 9 <212> PRT <213> VP2-P26-2-4 (Norovirus) <400> 70 Ala Arg Gly Pro Ser Asn Lys Ser Ser 1 5 <210> 71 <211> 9 <212> PRT <213> VP2-P26-2-5 (Norovirus) <400> 71 Arg Gly Pro Ser Asn Lys Ser Ser Asn 1 5 <210> 72 <211> 9 <212> PRT <213> VP2-P26-2-6 (Norovirus) <400> 72 Gly Pro Ser Asn Lys Ser Ser Asn Ser 1 5 <210> 73 <211> 9 <212> PRT <213> VP2-P26-2-7 (Norovirus) <400> 73 Pro Ser Asn Lys Ser Ser Asn Ser Ser 1 5 <210> 74 <211> 9 <212> PRT <213> VP2-P26-2-8 (Norovirus) <400> 74 Ser Asn Lys Ser Ser Asn Ser Ser Thr 1 5 <210> 75 <211> 9 <212> PRT <213> VP2-P26-2-9 (Norovirus) <400> 75 Asn Lys Ser Ser Asn Ser Ser Thr Ala 1 5 <210> 76 <211> 9 <212> PRT <213> VP2-P26-2-10 (Norovirus) <400> 76 Lys Ser Ser Asn Ser Ser Thr Ala Thr 1 5 <210> 77 <211> 9 <212> PRT <213> VP2-P26-2-11 (Norovirus) <400> 77 Ser Ser Asn Ser Ser Thr Ala Thr Ser 1 5 <210> 78 <211> 9 <212> PRT <213> VP2-P26-2-12 (Norovirus) <400> 78 Ser Asn Ser Ser Thr Ala Thr Ser Val 1 5 <210> 79 <211> 9 <212> PRT <213> VP2-P26-2-13 (Norovirus) <400> 79 Asn Ser Ser Thr Ala Thr Ser Val Tyr 1 5

Claims

1. A norovirus-specific CD8 + T-cell epitope peptide, characterized in that... The epitope peptide is selected from at least one of the amino acid sequences shown in SEQ ID NO.16, SEQ ID NO.38, and SEQ ID NO.

59.

2. The norovirus-specific CD8 according to claim 1 + T-cell epitope peptide, characterized in that... The amino acid sequence of the epitope peptide is shown in SEQ ID NO.

59.

3. The norovirus-specific CD8 according to claim 1 or 2 + T-cell epitope peptide, characterized in that... It is prepared by synthesizing overlapping peptides or by isolating and extracting from host cells.

4. The norovirus-specific CD8 as described in claim 1 or 2 + Application of T-cell epitope peptides in the preparation of vaccines to prevent norovirus infection.

5. A polypeptide vaccine, characterized in that, Including the norovirus-specific CD8 as described in claim 1 or 2 + T-cell epitope peptides.

6. A polypeptide vaccine according to claim 5, characterized in that, It also includes pharmaceutically acceptable adjuvants.

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