Method for high-throughput screening of virus CTL epitopes based on immunopeptidomics, restrictive epitope peptide, nucleic acid molecule and application

Abstract

The invention belongs to the field of biology, and discloses a method for high-throughput screening of virus CTL epitopes based on immunopeptidomics, which comprises the following steps: transfecting mammalian cells by adopting eukaryotic expression plasmids connected with alpha chains and beta 2m chains of MHC I molecules in series, and establishing a mammalian cell line for expressing animal MHC I molecules; preparing an MHC I-peptide complex by using a mammalian cell line expressing an MHC I molecule by virus infection; and obtaining the antigen peptide existing in the MHC I-peptide complex. According to the method, chicken MHC I allele BF2 * 1901 molecules are used as research objects, H9N2 subtype avian influenza viruses are used as model viruses, chicken MHC I restrictive H9N2 antigen peptide groups are comprehensively represented, CTL epitope immunization of immunodominance is identified, the method is suitable for CTL epitope screening of different chicken MHC I molecules and different subtype avian influenza viruses, and the method is suitable for clinical application. A rapid, efficient and economic technical means is provided for researching specific binding of chicken MHC I molecules and antigen peptides, a favorable reference is provided for screening of T cell epitopes of major animal pathogens, and a scientific basis is provided for poultry breeding for disease resistance, novel vaccine development and immune evaluation strategies. Meanwhile, the invention further discloses the restrictive epitope peptide, a nucleic acid molecule and application.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to a method for high-throughput screening of viral CTL epitopes based on immunopeptidomics, a restricted epitope peptide, a nucleic acid molecule and an application thereof. Background Art

[0002] MHC class I molecules are central to the adaptive immune response. During viral infection, viral proteins are recognized and degraded by the host's proteasome system through endogenous pathways, generating short peptide fragments. These peptide fragments are then transported to the endoplasmic reticulum by the TAP protein, where they bind to newly assembled MHC class I molecules. The MHC class I molecules loaded with viral peptides are then transported to the cell surface, forming a peptide-MHC class I complex. The pHLA complex formed by the binding of MHC class I molecules to antigenic peptides is recognized by the TCR, highly specifically activating CD8+ T lymphocytes (CTLs), thereby precisely identifying and eliminating pathogen-infected cells in the body while sparing normal cells. Traditional inactivated vaccine-mediated humoral immunity struggles to provide adequate protection against pathogens with rapidly mutating and recombining properties, such as avian influenza virus (AIV), when the vaccine strain does not match the currently circulating strain. Presenting viral protein-derived epitopes via MHC class I molecules can effectively activate T cell immune responses and induce cross-protection. Boosting cellular immunity can effectively control pathogen infection and improve the effectiveness of inactivated vaccines. Therefore, targeting new vaccines that can induce cellular immunity has become a research hotspot. Studying the interaction between MHC I molecules and viral antigen peptides and identifying T cell epitopes (CTL epitopes) with immune activity are crucial for the design of new vaccines.

[0003] Methods for screening T cell epitopes primarily include computer prediction, synthetic peptide libraries, and cellular immunopeptidomics. Currently, computer prediction is the most rapid method for T cell epitope screening. It relies on known epitope data to train and validate prediction models. However, due to the limited availability of raw data on animal MHC-restricted epitopes, machine learning models are difficult to train, and the high false-positive rate makes it difficult to screen for effective immunogenic peptides. Synthetic peptide libraries allow for high-throughput screening of a large number of peptides in a short period of time. However, for rapidly mutating and multi-subtype co-circulating influenza viruses, this approach faces the risk of rapid obsolescence and struggles to accurately and comprehensively capture the peptide signatures presented by MHC molecules across all subtypes and strains. Cellular immunopeptidomics provides experimentally validated epitope data by directly identifying peptides presented by MHC molecules in host cells, revealing authentic peptides that are presented by MHC molecules in vivo and potentially stimulating T cell responses. However, it requires a large number of haplotyped MHC test animals and expensive MHC monoclonal antibodies. The peptide yield is low, and contaminating peptides are easily generated during the extraction process, complicating basic research on CTL epitopes and subsequent epitope vaccine design. Liquid chromatography-mass spectrometry technology makes it possible to study MHC I molecule-restricted antigen epitopes in vitro. However, tissue samples for cellular immune peptidomics are difficult to obtain, MHC coverage is low, the accuracy of assigning highly polymorphic peptide segments of individual MHC molecules is low, and a large number of starting samples are required, making it difficult to develop cell lines expressing single alleles.

[0004] The single dominant expression of chicken MHC I molecules simplifies the genetic context of immune responses. By analyzing the sequence characteristics of antigenic peptides presented by MHC I molecules encoded by a single allele, it is possible to infer the amino acid residue preferences of the binding groove, which can also help understand the immune response mechanisms of chickens to specific pathogens. Currently, little is known about chicken MHC-restricted CTL epitopes. Only 24 CTL epitopes have been documented for AIVs restricted by chicken MHC I molecules, of which only four are restricted by the BF2*1901 allele. The BF2*1901 allele is the predominant gene expressed by the B19 haplotype in chickens, which is prevalent in chickens. Avian influenza virus (AIV) is a significant zoonotic threat to poultry and public health.

[0005] Existing T cell epitope screening strategies can be mainly divided into computer prediction, synthetic peptide library, and cell-eluted peptide method. Computer prediction is currently the fastest T cell epitope screening method, but it relies on known epitope data to train and validate the prediction model. However, due to the very limited raw data of chicken MHC-restricted epitopes, machine learning models are difficult to train, and the high false positive rate makes it difficult to screen out effective peptides. The synthetic peptide library method allows for high-throughput screening of a large number of peptides in a short period of time. However, for rapidly mutating and multi-subtype co-circulating influenza viruses, this method may face the risk of rapid obsolescence and it is difficult to accurately and comprehensively capture peptides presented by MHC molecules in all subtypes and strains. The cell-eluted peptide method mainly removes peptides directly from the surface of the host immune cell membrane. This method usually produces a small number of peptides and directly destroys the cell membrane, which easily introduces impurity small molecules that are not presented by MHC. It has low sensitivity and insufficient specificity.

[0006] The applicant previously filed an invention patent application CN109669043B, which discloses a method for identifying MHC I binding polypeptide motifs, comprising the following steps: (1) synthesizing a random polypeptide with a random sequence length of 8-12; (2) performing De Novo analysis on the random polypeptide to obtain an amino acid distribution; (3) expressing the α chain and β2m chain of MHC I; (4) forming an MHC I-polypeptide complex with the random polypeptide, the α chain and the β2m chain in a solution; (5) heat-denaturing the MHC I-polypeptide complex, separating and purifying the MHC I binding peptide; (6) performing De Novo analysis on the MHC I binding polypeptide to obtain an amino acid distribution of the MHC I binding polypeptide, and by comparing it with the amino acid distribution in step (2), obtaining the amino acid preference and main anchor position of the corresponding MHC I binding polypeptide at different sites.

[0007] The problem with the above method is that although it can verify the peptide binding to MHC I, it is uncertain whether this peptide will be cleaved out in the cell. In other words, the results of this method need to be verified by subsequent experiments, and its success rate cannot meet very high requirements.

[0008] Professor Jim Kaufman of the University of Cambridge in the UK employed a peptide elution method in his research on chicken MHC I (BF) molecules. They used antibodies to capture BF complex molecules in chickens, then eluted the peptides bound to the BF molecules with a weak acid. Mass spectrometry was then used to sequence the peptides, and suitable peptides were selected for synthesis and ultimately crystallographic analysis. This method directly and comprehensively identifies functional epitopes on the BF molecule. However, this method is difficult to apply to other animals because chickens primarily express a single BF molecule, whereas most species express multiple MHC I subtypes. The MHC I complex molecules captured by this method are a mixture, and clear correspondence cannot be determined after peptide elution sequencing.

[0009] That is to say, this method requires a large number of haploid chicken immune cells for experiments, and in actual experiments, obtaining a large number of haploid chicken immune cells with homozygous chicken MHC I molecules requires a lot of manpower and material resources.

[0010] Therefore, it is necessary to establish a new method for efficient screening of avian influenza virus CTL epitopes, enrich the basic data background of chicken MHC-restricted epitopes, and provide a scientific basis for poultry disease-resistant breeding, new vaccine development and immune evaluation strategies. Summary of the Invention

[0011] The main purpose of the present invention is to provide a method for high-throughput screening of viral CTL epitopes based on immunopeptidomics. The method uses the chicken MHC I allele BF2*1901 molecule as the research object and the H9N2 subtype avian influenza virus as the model virus, comprehensively characterizes the chicken MHC I-restricted H9N2 antigen peptide group, and identifies immunodominant CTL epitope immunity. The method is suitable for screening CTL epitopes of different chicken MHC I molecules and different subtypes of avian influenza viruses, provides a rapid, efficient and economical technical means for studying the specific binding of chicken MHC I molecules and antigen peptides, provides a favorable reference for the screening of T cell epitopes of major animal pathogens, and provides a scientific basis for poultry disease-resistant breeding, new vaccine development and immune evaluation strategies.

[0012] This method is not limited by the length of the polypeptide in the solution represented by CN109669043B, and can avoid the misjudgment or omission of weak binding peptides that do not conform to the motif and ultra-short peptides of atypical length;

[0013] By using mammalian cells as transfection targets to construct cell lines, this method avoids the requirement for large numbers of haploid chicken immune cells, a problem exemplified by the approach presented by Professor Jim Kaufman. The present invention has demonstrated that the transfection, infection, and peptide elution using mammalian cells yields highly specific results.

[0014] At the same time, the invention also discloses a restricted epitope peptide, a nucleic acid molecule and an application.

[0015] According to a first aspect of the present invention, a method for high-throughput screening of viral CTL epitopes based on immunopeptidomics is provided, wherein a eukaryotic expression plasmid containing the α chain and β2m chain of an MHC I molecule in series is used to transfect mammalian cells to establish a mammalian cell line expressing animal MHC I molecules; the mammalian cell line expressing MHC I molecules is infected with a virus to prepare an MHC I-peptide complex; and the antigenic peptide present in the MHC I-peptide complex is obtained.

[0016] In the above method, the preparation method of the eukaryotic expression plasmid containing the α chain and β2m chain of the MHC I molecule in series is as follows: when the α chain and β2m chain of the MHC I molecule are expressed in series in the plasmid, the extracellular region of the α chain is linked to the β2m chain using a flexible linker (G4S)4 to form an MHC I molecule dimer, and a Twin-Strep tag is inserted at the 3' end of the β2m chain.

[0017] In the above method, the mammalian cell is a 293F cell line.

[0018] In the above method, the virus is H9N2 virus.

[0019] In the above method, 2×10 6 / mL was the starting cell density at the time of transfection, MOI=1 was the multiplicity of virus infection, and the supernatant 48 hours after infection was used as the harvest sample.

[0020] At the same time, the present invention also discloses an AIV MHC-BF2*1901 restricted epitope peptide, the amino acid sequence of the epitope is shown as SEQ ID NO.1 to SEQ ID NO.18.

[0021] In addition, the present invention also discloses a nucleic acid molecule, which encodes the gene of the restricted epitope peptide as described above.

[0022] At the same time, the present invention also discloses the use of the above-mentioned AIV MHC-BF2*1901 restricted epitope peptide in the preparation of anti-influenza virus vaccines.

[0023] In the above application, the anti-influenza virus vaccine is an immune adjuvant, a vector vaccine, an epitope vaccine or an mRNA vaccine.

[0024] Finally, the present invention also discloses the use of the above-mentioned AIV MHC-BF2*1901 restricted epitope peptide in the preparation of an influenza virus vaccine diagnostic kit.

[0025] One of the above technical solutions of the present invention has at least one of the following advantages or beneficial effects:

[0026] The present invention uses the chicken MHC I allele BF2*1901 molecule as the research object and the H9N2 subtype avian influenza virus as the model virus to comprehensively characterize the chicken MHC I-restricted H9N2 antigen peptide group and identify immunodominant CTL epitope immunity. The present invention is suitable for screening CTL epitopes of different chicken MHC I molecules and different subtypes of avian influenza viruses, provides a rapid, efficient and economical technical means for studying the specific binding of chicken MHC I molecules and antigen peptides, provides a favorable reference for the screening of T cell epitopes of major animal pathogens, and provides a scientific basis for poultry disease-resistant breeding, new vaccine development and immune evaluation strategies.

[0027] The present invention successfully screened 23 chicken MHC I molecule BF2*1901-restricted avian influenza virus CTLs, 21 of which were newly identified CTL epitopes. This method can identify weakly binding peptides that do not conform to the motif and ultrashort peptides of atypical lengths, which are easily misidentified or missed by traditional methods but are immunogenic. Furthermore, this method overcomes the limitation of traditional screening methods, which require large numbers of haplotyped chicken immune cells. By replacing chicken MHC I monoclonal antibodies with labeled antibodies, the cost of CTL epitope screening is significantly reduced, providing a rapid, economical, and effective CTL epitope screening technology for avian influenza virus vaccine design and research on cellular immune mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments;

[0029] Figure 1 Design a schematic diagram for the structure of expressed genes;

[0030] Figure 2 This is the Western blot identification result of chicken MHC I molecules;

[0031] Figure 3A Western blot analysis of the purified chicken pMHC-Strep protein.

[0032] Figure 3B Western blot analysis of the purified chicken pMHC-Strep protein.

[0033] Figure 4 Search the matching results of H9N2 virus antigen peptide library for LC-MS;

[0034] Figures 5A to 5X This is the molecular sieve purification result of BF2*1901 combined with peptide;

[0035] Figure 6 The statistical results of the number of spots for ELISpot identification of AIV potential epitopes;

[0036] Figure 7 Schematic diagram of the spots used for ELISpot identification of potential epitopes of AIV. DETAILED DESCRIPTION

[0037] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention.

[0038] Example 1 Conditions for Screening AIV Antigen Peptides in Chicken MHC I Monoallelic Expression Cells

[0039] 1.1 Eukaryotic expression of chicken MHC I monoallelic recombination

[0040] 1.1.1 Construction of chicken MHC I monoallelic recombinant plasmid

[0041] A tandem expression plasmid for the chicken MHC I α chain (GenBank No.: Z54360.1) and β2m chain (GenBank No.: M84767.1) genes was designed. The extracellular region of the α chain (excluding the transmembrane and intracellular regions) was connected to the β2m chain using a (G4S)4 linker to form a chicken MHC I dimer. A Twin-Strep tag (amino acid sequence of the Twin-Strep tag (SEQ ID NO: 31): SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK) was inserted at the 3' end of the β2m chain. The design of the genes to be expressed can be found in [ 15 ]. Figure 1 , its sequence can be seen in SEQ ID NO.24.

[0042] Table 1 Primer sequence list

[0043]

[0044] The above three pairs of primers were used for PCR amplification respectively.

[0045] The cDNA of B19 haplotype chicken PBMC was used as a template to amplify the extracellular region of the α chain using MHC Iα-F / R, and the β2m chain using MHC Iβ2m-F / R.

[0046] The Puc57 / Twin-Strep tag plasmid was used as a template to amplify the Strep fragment using Strep-F / R.

[0047] After the amplified product was recovered and purified by agarose gel electrophoresis, the first-round amplified fragment was used as a template and MHC Iα-F and Strep-R were used as primers to perform fusion PCR to recover the MHC Iα-β2m-Strep fragment. The fragment was cloned into the pRK5 vector and transformed into DH5α. Monoclonal colonies were picked for bacterial liquid PCR identification and positive colonies were sent for sequencing. The plasmid extracted from the colony with correct sequencing was named pRK5 / MHC Iα-β2m-Strep.

[0048] 1.1.2 Expression of chicken MHC I monoallelic recombinant plasmid

[0049] The recombinant plasmid pRK5 / MHC Iα-β2m-Strep was transfected according to the instructions of the Polyethylenimine MAX, MW 40000 transfection kit. Cell supernatant samples were harvested 48 hours after transfection, and protein expression was detected by Western Blot.

[0050] WB detection of protein expression: aspirate the cell suspension after culture, centrifuge at 800g for 10 minutes, carefully aspirate 200μL of cell supernatant into a new EP tube, add 50μL 6× Protein Loading, mix thoroughly, boil at 100℃ for 10 minutes, and then perform SDS-PAGE electrophoresis, then transfer to the membrane at a constant current of 200mA for 1 hour, block with 5% skim milk at room temperature for 1 hour, wash the membrane, prepare mouse anti-chicken MHC class I antibody at a concentration of 1:1000, incubate at 4℃ overnight, wash the membrane, prepare rabbit anti-mouse IgG antibody at a concentration of 1:5000, incubate on a shaker at room temperature for 1 hour, wash the membrane, absorb excess liquid with absorbent paper, add exposure solution to the membrane surface for 1 minute, and then develop and take pictures. Figure 2 ; Figure 2 The meanings of the numbers are as follows: M: protein molecular weight standard, 1-2: expression of chicken MHC I molecule protein in the supernatant and cell lysate of cells transfected with recombinant plasmids, 3-4: expression of chicken MHC I molecule protein in the supernatant and cell lysate of cells transfected with vectors.

[0051] 1.2 Preparation and purification of chicken pMHC protein

[0052] Preparation of pMHC protein: 293F cells were transfected with the pRK5 / MHC Iα-β2m-Strep recombinant plasmid and infected with H9N2 (A / chicken / Hebei / m0530-1 / 2017 (HB17), isolated and stored in our laboratory) at an MOI of 1 36 h after infection. During infection, virus dilution was added dropwise to the cell shake flask, and the culture flask was gently shaken to ensure uniform distribution of the virus solution in the cells.

[0053] Purification of pMHC protein: 48 hours after infection, the supernatant was harvested and affinity purified using STarm Streptactin Beads 4FF according to the instructions. Protein expression and purification were assessed at each step using SDS-PAGE and Western blotting. The purified target protein was collected and concentrated to 1 mL using a 10 kDa ultrafiltration tube, which is the chicken pMHC protein. See the instructions for details. Figure 3A and Figure 3B ; Figure 3A The meanings of the numbers in the table are: M. protein standard molecular weight, 1. unpurified cell supernatant, 2. purified effluent, 3. biotin eluate, 4. concentrated biotin eluate, 5. NaOH eluate, 6. concentrated NaOH eluate, 7. blank control cell supernatant; Figure 3B The meanings of the labels are: M. protein standard molecular weight, 1. unpurified cell supernatant, 2. purified effluent, 3. biotin eluate, 4. biotin eluate after concentration, 5. NaOH eluate, 6. NaOH eluate after concentration, 7. blank control cell supernatant.

[0054] 1.3 Separation and mass spectrometry identification of antigenic peptides

[0055] Mild acid wash to separate antigenic peptides: Add two volumes of 0.02N acetic acid to pMHC-Strep and mix thoroughly. Incubate at 65°C for 25 minutes to dissociate the antigenic peptides. Centrifuge at 12,000 rpm for 10 minutes and collect the supernatant. Filter the entire supernatant through a 3 kDa ultrafiltration tube. Collect the filtrate, which represents the antigenic peptides presented by the chicken MHC I monoallelic molecule.

[0056] Mass spectrometry identification and data processing: The protein samples were subjected to liquid chromatography LC-MS / MS detection and De Novo analysis. PEAKS Studio8 was used for library search analysis, searching the human protein database and the self-constructed influenza virus protein database (derived from the viral protein sequence of the H9N2 strain used in the experiment). The parameters were set as follows: Peptide-101gP ≥ 15, PTMAscore ≥ 0, Protein-101gP ≥ 20, Proteins unique peptides ≥ 0, Denovo score (%) ≥ 50%, Parent Mass Error Tolerance: 7.0ppm, Fragment Mass Error Tolerance: 0.02Da, Enzyme: None. Search results reference Figure 4 .

[0057] Example 2 Peptide Spectrum Analysis and Immunological Identification of Chicken MHC I-Restricted AIV Antigens

[0058] 2.1 Chicken MHC I-restricted AIV antigen peptide spectrum

[0059] Table 2 provides information on antigenic peptides presented by the chicken MHC I allele BF2*1901 molecule for the H9N2 subtype avian influenza virus (peptides matching more than two times were included in the analysis). The binding motif of the BF2*1901 molecule is known to be XR-XXXXXX-Y / P / L / F. Its anchor residues at positions 2 and 9, positions 1 / 2 typically contain positively charged amino acids (R), and positions 8 / 9 typically contain hydrophobic amino acids. This binding motif and multiple repeats are often used to identify high-abundance peptides. Based on the length of the viral antigenic peptides and the number of matches, 23 peptides were ultimately selected as representative potential epitopes and sent to GenScript Biotech for synthesis (HPLC treatment, purity ≥98%). Detailed information is shown in Table 2.

[0060] Table 2 Antigen peptide information

[0061]

[0062] 2.2 Immunological identification of avian influenza virus CTL epitopes

[0063] 2.2.1 Refolding to verify the in vitro binding efficiency of potential epitopes and MHC:

[0064] Prokaryotic Expression and Inclusion Body Extraction of BF2*1901α and β2m Chains

[0065] (1) Induction: Prepare some ampicillin LB culture medium, add 200 μL of positive bacterial solution (two positive bacterial solutions with PET-21a as the prokaryotic expression vector and BF2*1901α chain fragment and galussβ2m chain fragment inserted between the Nde1 and xho1 restriction sites, respectively) to 100 mL of A+LB and shake for 6 h. Then, add 1 L of LB solution and culture on a shaker for 2 h until the OD600 value is around 0.6. Then, add 1 mL of IPTG (1:1000) and induce culture on a shaker at 37°C for 8 h.

[0066] (2) Collecting bacteria: The induced bacterial solution was centrifuged at low temperature to obtain a bacterial pellet. The pellet was then resuspended in 25 mL of ultrapure water and ultrasonically disrupted on ice. The program was ultrasonication for 12 seconds, 18 seconds interval, and 45 minutes. The lysed bacterial solution was centrifuged at low temperature at 6000 rpm for 15 minutes. The supernatant was discarded and the bacterial impurities on the surface of the inclusion bodies were gently removed with a pipette tip.

[0067] (3) Washing: Use the prepared washing solution to vortex and rinse the inclusion bodies, centrifuge at low temperature and discard the supernatant, and repeat the washing process until the inclusion bodies appear relatively pure.

[0068] (4) Resuspension: Vortex the inclusion bodies with an appropriate amount of resuspension solution until they are suspended. Take a portion of the sample and identify it by SDS-PAGE electrophoresis. Then centrifuge at low temperature and discard the supernatant as much as possible;

[0069] (5) Dissolution: Weigh the net weight of the inclusion bodies and calculate the amount required to dissolve the inclusion bodies. Adjust the final concentration to 30 mg / mL, add the dissolution solution, and stir and dissolve at low temperature until no obvious solids are present. Centrifuge several times to remove impurities at the bottom, then aliquot, label, and store frozen.

[0070] Refolding and concentration of BF2*1901 and antigenic peptide:

[0071] (1) Preparation of refolding system: Prepare an appropriate amount of refolding solution according to the number of peptides to be identified. Add 200 mL of refolding solution to a beaker of appropriate size and add a stirrer. Cover the bottle mouth with plastic film and fix the syringe vertically on the film.

[0072] (2) Adding β2m chain: Add 1 mL of chicken β2m inclusion body solution (refer to the preparation process of patent CN 109669043A) to the syringe, slowly drip it into the beaker, and place it on a magnetic stand and stir at 4°C for 8 h;

[0073] (3) Adding peptide: Dissolve the synthetic peptide powder in DMSO, vortex to mix, add about 5 mg of peptide to the refolding solution, and stir for 5 minutes;

[0074] (4) Adding α chain: Add 3 mL of chicken BF2*1901 α chain inclusion body solution (refer to the preparation process of patent CN109669043A) to the syringe, place on a magnetic stand and stir at 4°C for renaturation for more than 24 hours;

[0075] (5) Concentration: Clean and install the 10 kDa concentration cup in advance, transfer the renatured liquid into the concentration cup, and concentrate at 4 °C;

[0076] (6) Liquid exchange: After the refolding solution is concentrated to about 15 mL, add 60 mL of molecular sieve solution and continue to concentrate to about 10 mL-15 mL. Transfer to a 15 mL centrifuge tube and centrifuge at 7000 rpm for 5 min at 4°C to obtain the supernatant.

[0077] (7) Reconcentration: Prepare a 15 mL, 10 kDa ultrafiltration tube in advance, centrifuge at low temperature to concentrate the liquid to less than 1 mL, transfer it to an EP tube, centrifuge again to obtain the supernatant, filter it with a 0.22 μm filter membrane, centrifuge at 12,000 rpm to remove bubbles, and store it in a refrigerator at 4°C for later use.

[0078] Molecular Sieve Purification of BF2*1901 Antigen-Peptide Complex

[0079] The Superdex 200 gel chromatography column was pre-equilibrated with one column volume of molecular sieve buffer until the salt concentration (about 5.7%) remained constant. The maximum column pressure (Alarm Pressure = 0.33 MPa), flow rate (1 mL / min), collection parameters (Peak Frac Parameters UV = 20 mAU; Peak Size = 1.2 mL), etc. were set. After zeroing the UV value, the sample to be tested was slowly aspirated into the Loop for purification. The loading situation was observed regularly. Samples were collected according to the peak position and peak size. The sample at the peak tip was taken and tested for BF2*1901 molecule binding to the peptide using SDS-PAGE. The specific results are shown in Figures 5A to 5X ;

[0080] The chicken MHC I α chain, β2m chain inclusion body protein and potential epitope peptide were renatured in vitro and purified by molecular sieve. The sample showed three main protein peaks: α chain multimer, α chain-β2m chain-peptide complex, and β2m chain multimer. MHC I-peptide complex (pBF2*1901) generally appeared between 80-100 mL.

[0081] In order to determine the affinity of the potential AIV epitope to BF2*1901, the target peak of the complex was collected and identified by SDS-PAGE. An α chain of about 32 kDa and a β2m chain of 12 kDa were detected at the corresponding positions.

[0082] Molecular screening results showed that 18 peptides could form stable MHC I-peptide complexes with BF2*1901, including 8 motif peptides with R anchor residues at the P2 position: IRV8, ARM8, MRP8, IRL9, RRN9, QRD9, FRM11, and KRP12, as well as 3 non-motif peptides: AYP8, TTE9, and ISR11, and 7 ultrashort peptides: TTR7, ARA7, RQM7, RTP7, MGR7, AGK7, and MID7.

[0083] Four peptides, LPT8, KAF9, QNQ9, and MAC12, exhibited the desired peak pattern, but the complex peak was below 50 mAU, indicating too little protein to be identified by SDS-PAGE. These peptides may have weakly bound or not bound to BF2*1901. GRK8 did not exhibit the desired peak pattern, and SDS-PAGE analysis revealed no complex peak, indicating it was a non-binding peptide.

[0084] 2.2.2 ELISPOT verification of the immunoreactivity of potential epitopes

[0085] B19 haplotype virus infection in chickens

[0086] Six four-week-old SPF B19 haplotype White Leghorn chickens were infected intranasally with 10⁶ EID⁵ / 0.1 mL of the H9N2 strain (A / chicken / Hebei / M0530-1 / 2017). Fresh heparinized blood was collected from the peripheral vein of the chicken 14 days after infection, and lymphocytes were isolated according to the instructions of the Chicken Peripheral Blood Lymphocyte Isolation Kit.

[0087] Isolation of Chicken Peripheral Blood Mononuclear Cells (PBMC)

[0088] Dilute chicken peripheral blood with an equal volume of PBS. Gently tilt the centrifuge tube containing the separation buffer and carefully add the peripheral blood dilution solution along the tube wall onto the separation buffer. Centrifuge horizontally at 500g for 20 minutes. At the end of the centrifugation, distinct stratification will form. Transfer the buffy coat lymphocytes from the second layer to a clean centrifuge tube. Add an appropriate amount of cell wash buffer and gently invert to mix the cells. Centrifuge horizontally at 250g for 10 minutes. Discard the supernatant. Repeat the washing process once. Finally, add RPMI1640 medium containing 10% FBS to resuspend the cells for later use.

[0089] ELISPOT assay

[0090] (1) First, take out the 96-well ELISpot plate and add 200 μL of sterile PBS to each well for 4 washes. After the last wash, discard the remaining liquid;

[0091] (2) Add 200 μL of RPMI1640 culture medium containing 10% FBS to each well and incubate at 37°C for 2 hours;

[0092] (3) Wash the plate four times with PBS, add 100 μL of a single peptide (final concentration 1 mg / 0.1 mL), and then add 106 chicken PBMC lymphocytes in a total volume of 200 μL / well. Set up two replicate wells for each peptide, and take two wells without peptide stimulation as negative controls. Incubate in a cell culture incubator at 37°C, 5% CO2 for 48 hours;

[0093] (4) After the incubation period, the cell suspension was discarded, the plate was washed three times with PBS, and the antibody MT7C10-biotin was diluted to a concentration of 1 μg / mL using 0.5% FBS-PBS buffer. 100 μL was added to each well and incubated at room temperature for 2 hours.

[0094] (5) Discard the antibody, wash the plate three times with PBS, dilute Streptavidin-HRP with 0.5% FBS-PBS at a ratio of 1:1000, add 100 μL per well, and incubate at room temperature in the dark for 1 hour;

[0095] (6) Discard the liquid, wash the plate 5 times with PBS, add 100 μl of TMB substrate solution to each well and develop color for about 15 min until obvious spots appear. Terminate the color development reaction with deionized water and let it dry at room temperature.

[0096] (7) Analyze the number of spots in each well using an ELISpot plate reader;

[0097] Results: Reference Figure 6 and Figure 7 Eighteen strong binding peptides with in vitro affinity for the chicken MHC class I molecule BF2*1901 were selected, along with two weakly binding peptides, MAC12 and QNQ9, that were repeatedly matched. ELISPOT assays were then used to measure the levels of IFN-γ secretion by AIV-specific T lymphocytes following peptide stimulation. Two replicate wells were set for each stimulating peptide, along with negative control wells without peptide. The results showed that, with the exception of MAC12, all 19 potential epitope peptides stimulated T cells to secrete IFN-γ and produce immune spots to varying degrees, compared to the negative control without peptide.

Claims

1. A method for high-throughput screening of viral CTL epitopes based on immunopeptidomics, characterized in that: Mammalian cells are transfected with a eukaryotic expression plasmid containing the α chain and β2m chain of the MHC I molecule in series to establish a mammalian cell line expressing animal MHC I molecules; the mammalian cell line expressing the MHC I molecule is infected with a virus to prepare an MHC I-peptide complex; and the antigenic peptide present in the MHC I-peptide complex is obtained.

2. The method according to claim 1, characterized in that The preparation method of the eukaryotic expression plasmid containing the α chain and β2m chain of the MHC I molecule in series is as follows: when the α chain and β2m chain of the MHC I molecule are expressed in series in the plasmid, the extracellular region of the α chain is connected to the β2m chain via a flexible linker (G4S)4 to form an MHC I molecule dimer, and a Twin-Strep tag is inserted into the 3' end of the β2m chain.

3. The method according to claim 1, characterized in that The mammalian cell line is 293F.

4. The method according to claim 1, characterized in that The virus is H9N2 virus.

5. The method according to claim 1, characterized in that: 2×10 6 / mL was the starting cell density at the time of transfection, MOI=1 was the multiplicity of virus infection, and the supernatant 48 hours after infection was used as the harvest sample.

6. An AIV MHC-BF2*1901 restricted epitope peptide, characterized in that: The amino acid sequences of the epitopes are shown in SEQ ID NO.1 to SEQ ID NO.

18.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the gene of the restricted epitope peptide according to claim 6.

8. Use of the AIV MHC-BF2*1901 restricted epitope peptide according to claim 6 in the preparation of an anti-influenza virus vaccine.

9. The use according to claim 8, characterized in that The anti-influenza virus vaccine is an immune adjuvant, a vector vaccine, an epitope vaccine or an mRNA vaccine.

10. Use of the AIVMHC-BF2*1901 restricted epitope peptide according to claim 6 in preparing an influenza virus vaccine diagnostic kit.

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