Method for high-throughput screening of viral ctl epitopes based on immunopeptidomics, restricted epitope peptides, nucleic acid molecules and applications

Abstract

The application belongs to the field of biology, and discloses a method for high-throughput screening of viral CTL epitopes based on immunopeptidomics, which comprises the following steps: transfecting mammalian cells with eukaryotic expression plasmids having MHC I molecules in series with the alpha chain and the beta2m chain to establish a mammalian cell line expressing animal MHC I molecules; using a virus to infect the mammalian cell line expressing MHC I molecules to prepare MHC I-peptide complexes; and obtaining antigen peptides existing in the MHC I-peptide complexes. The method takes chicken MHC I allele BF2*1901 molecules as the research object, takes H9N2 subtype avian influenza virus as the model virus, comprehensively characterizes chicken MHC I restricted H9N2 antigen peptide groups, and identifies immunodominant CTL epitope immunity, and is suitable for CTL epitope screening of different chicken MHC I molecules and different subtypes of avian influenza viruses, provides a fast, efficient and economical technical means for studying the specific binding of chicken MHC I molecules and antigen peptides, provides a favorable reference for T cell epitope screening of major animal pathogens, and provides a scientific basis for poultry disease-resistant breeding, development of new vaccines and immune evaluation strategies. Meanwhile, the application also discloses restricted epitope peptides, nucleic acid molecules and applications.

Description

Technical Field

[0001] This 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, restriction epitope peptides, nucleic acid molecules, and applications. Background Technology

[0002] MHC I molecules are central to the adaptive immune response. During viral infection, viral proteins are recognized and degraded by the host's proteasome system via an endogenous pathway, generating short peptides. These peptides are then transported to the endoplasmic reticulum by TAP proteins and bind to newly assembled MHC I molecules. The MHC I molecules loaded with viral peptides are subsequently transported to the cell surface, forming peptide-MHC I complexes. The pHLA complex formed by the binding of MHC I molecules to antigenic peptides is recognized by the TCR, highly specifically activating the CD8+ T lymphocyte (CTL) response, thereby precisely recognizing and eliminating pathogen-infected cells without harming normal cells. Traditional inactivated vaccines-mediated humoral immunity is less effective against pathogens with rapid mutation and recombinant properties, such as avian influenza virus (AIV), where the vaccine strain is not matched to currently circulating strains, making it difficult to provide sufficient protection for the host. Antigenic epitopes derived from viral proteins presented by MHC I molecules can effectively activate T cell immune responses and induce cross-protection, improving cellular immunity and effectively controlling pathogen infection, thus enhancing the effectiveness of inactivated vaccines. Therefore, novel vaccines that target and induce cellular immunity have become a research hotspot. Studying the interaction between MHC I molecules and viral antigen peptides and identifying immune-active T-cell epitopes (CTL epitopes) are crucial for the design of novel vaccines.

[0003] The main methods for screening T-cell epitopes include computer prediction, synthetic peptide libraries, and cell-mediated immunopeptidomics. Computer prediction is currently the fastest method, relying on known epitope data to train and validate the predictive model. However, due to the scarcity of raw data on animal MHC-restricted epitopes, training machine learning models is difficult, resulting in high false positive rates and difficulty in screening effective immune peptides. Synthetic peptide libraries allow for high-throughput screening of large numbers of peptides in a short time; however, for rapidly mutating and multi-subtype co-circulating influenza viruses, this method may face the risk of rapid obsolescence and struggles to accurately and comprehensively capture the peptide characteristics presented by MHC molecules in all subtypes and strains. Cell-mediated immunopeptidomics provides experimentally validated epitope data by directly identifying MHC-presented peptides from host cells, revealing the actual peptides presented by MHC molecules in vivo that may elicit T-cell responses. However, it requires a large number of haplotype MHC experimental animals and expensive MHC monoclonal antibodies, and the peptide separation is low with the potential for contamination during extraction, posing challenges to basic research on CTL epitopes and subsequent epitope vaccine design. Liquid chromatography-mass spectrometry (LC-MS) makes it possible to study MHC I molecule restriction epitopes in vitro. However, cell immunopeptidomics faces challenges such as difficulty in obtaining tissue samples, low MHC coverage, low accuracy in allocating highly polymorphic peptides in individual MHC molecules, and the need for large amounts of starting samples, which further hinders the development of cell lines expressing single alleles.

[0004] Chicken MHC I molecules exhibit a single dominant expression characteristic, simplifying the genetic background of the immune response. By analyzing the sequence characteristics of the antigen-presenting peptides encoded by a single allele of the MHC I molecule, the binding groove's preference for amino acid residues can be inferred, which can also help understand the immune response mechanism of chickens to specific pathogens. Currently, information on chicken MHC-restricted CTL epitopes is still limited. Only 24 CTL epitopes of chicken MHC I molecules restricting AIV have been recorded, of which only 4 are BF2*1901 allele-restricted epitopes. The BF2*1901 allele is a major expressed gene of the B19 haplotype, which is prevalent in chicken flocks. Avian influenza virus is an important animal infectious disease threatening poultry and public health.

[0005] Existing T-cell epitope screening strategies mainly fall into three categories: computer prediction, synthetic peptide libraries, and cell-washing peptide methods. Computer prediction is currently the fastest method for T-cell epitope screening; however, it relies on known epitope data to train and validate the prediction model. Because raw data on chicken MHC-restricted epitopes is very limited, training machine learning models is difficult, resulting in high false positive rates and difficulty in screening effective peptides. Synthetic peptide libraries allow for high-throughput screening of large numbers of peptides in a short time; however, for rapidly mutating and multi-subtype co-circulating influenza viruses, this method may face the risk of rapid obsolescence, making it difficult to accurately and comprehensively capture peptides presented by MHC molecules in all subtypes and strains. Cell-washing peptide methods primarily involve directly detaching peptides from the surface of host immune cell membranes. This method typically produces a small number of peptides, and directly disrupting the cell membrane can easily introduce non-MHC-presented impurities, resulting in low sensitivity and insufficient specificity.

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

[0007] The problem with the above method is that although it can verify the peptides that bind to MHC I, it is uncertain whether these peptides will be cleaved out of the cells. In other words, the results of this method need to be verified by subsequent experiments, and its success rate is not very high.

[0008] Professor Jim Kaufman of the University of Cambridge used a peptide-washing method in his research on chicken MHC I (BF) molecules. They captured BF complex molecules in chickens with antibodies, then eluted the peptides bound to the BF molecules with a weak acid, determined the peptide sequences using mass spectrometry, selected suitable peptides for synthesis, and finally conducted crystallographic studies. This method can directly obtain the functional epitopes of BF molecules, and the information is comprehensive. However, this method is difficult to apply to other animals because chickens mainly express one type of BF molecule, while most species simultaneously express multiple MHC I subtypes. The MHC I complex molecules captured by antibodies using this method are a mixture, and the correspondence cannot be clearly defined after peptide-washing sequencing.

[0009] In other words, this method requires a large number of haplotype chicken immune cells for experiments, and in reality, obtaining a large number of homozygous chicken MHC I molecule haplotype chicken immune cells requires a lot of manpower and resources.

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

[0011] The main objective of this invention is to provide a high-throughput screening method for viral CTL epitopes based on immunopeptidomics. This method uses the chicken MHC I allele BF2*1901 molecule as the research object and H9N2 subtype avian influenza virus as the model virus to comprehensively characterize the chicken MHC I-restricted H9N2 antigenic peptide group and identify immunogenic CTL epitopes. It is applicable to the screening of CTL epitopes for different chicken MHC I molecules and different subtypes of avian influenza viruses, providing a rapid, efficient, and economical technical means for studying the specific binding of chicken MHC I molecules and antigenic peptides. It provides a valuable reference for T cell epitope screening of major animal pathogens and provides a scientific basis for poultry disease resistance breeding, new vaccine development, and immune evaluation strategies.

[0012] This method is not limited by the peptide length in the scheme represented by CN109669043B, and can avoid misjudgment or missed detection of weakly binding peptides that do not conform to the motif and ultrashort peptides of atypical length.

[0013] This method uses mammalian cells as the transfection target to construct cell lines, avoiding the problem of requiring a large number of haplotype chicken immune cells in the approach represented by Professor Jim Kaufman. Through verification, this invention has shown that using mammalian cells for transfection, infection, and peptide washing yields results with very strong targeting.

[0014] In addition, the present invention also discloses restriction epitope peptides, nucleic acid molecules, and applications.

[0015] According to a first aspect of the present invention, the present invention provides a method for high-throughput screening of viral CTL epitopes based on immunopeptidomics, comprising transfecting mammalian cells with a eukaryotic expression plasmid containing the α chain and β2m chain of an MHC I molecule in tandem to establish a mammalian cell line expressing animal MHC I molecules; preparing an MHC I-peptide complex by infecting the mammalian cell line expressing MHC I molecules with a virus; and obtaining the antigenic peptide present in the MHC I-peptide complex.

[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 expressing the plasmid with the α chain and β2m chain of the MHC I molecule in series, the extracellular region of the α chain is connected to the β2m chain with 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 the 293F cell line.

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

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

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

[0021] In addition, the present invention also discloses a nucleic acid molecule that encodes a gene for a restriction epitope peptide as described above.

[0022] Meanwhile, this invention also discloses the application of the AIV MHC-BF2*1901 restricted epitope peptide as described above in the preparation of anti-influenza virus vaccines.

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

[0024] Finally, this invention also discloses the application of the AIV MHC-BF2*1901 restricted epitope peptide as described above in the preparation of influenza virus vaccine diagnostic kits.

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

[0026] This 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 antigenic peptide group and identify immune-dominant CTL epitopes. It is applicable to the screening of CTL epitopes for different chicken MHC I molecules and different subtypes of avian influenza viruses, providing a rapid, efficient, and economical technical means for studying the specific binding of chicken MHC I molecules and antigenic peptides. It provides a valuable reference for the screening of T cell epitopes for major animal pathogens and provides a scientific basis for poultry disease-resistant breeding, new vaccine development, and immune evaluation strategies.

[0027] This invention successfully screened 23 chicken MHC I-restricted avian influenza virus CTLs (Cellular Tracts) of molecule BF2*1901, of which 21 peptides were newly identified CTL epitopes in this study. This invention can identify weakly binding peptides that do not conform to the motif and ultrashort peptides of atypical length. These peptides are easily misidentified or missed by traditional methods, but they possess immunogenicity. Furthermore, this invention overcomes the limitation of traditional screening methods requiring a large number of haplotype chicken immune cells, and the use of tagged antibodies instead of chicken MHC I monoclonal antibodies significantly reduces the cost of CTL epitope screening. This provides a rapid, economical, and effective CTL epitope screening technology for avian influenza virus vaccine design and research on cellular immune mechanisms. Attached Figure Description

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

[0029] Figure 1 A schematic diagram of the structural design for expressing genes;

[0030] Figure 2 Western blot identification results of chicken MHC I molecules;

[0031] Figure 3A Western blot identification results of purified chicken pMHC-Strep protein;

[0032] Figure 3B Western blot identification results of purified chicken pMHC-Strep protein;

[0033] Figure 4 The matching results of the H9N2 virus antigen peptide library were searched by LC-MS;

[0034] Figures 5A to 5X The molecular sieve purification results show the binding of BF2*1901 to the peptide.

[0035] Figure 6 Statistical results of spot count for identifying potential AIV epitopes for ELISpot;

[0036] Figure 7 A schematic diagram of a spot used by ELISpot to identify potential AIV epitopes. Detailed Implementation

[0037] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote 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 should not be construed as limiting the present invention.

[0038] Example 1: Conditions for screening AIV antigenic peptides using 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 was designed for the chicken MHC I α chain (GenBank ID: Z54360.1) and β2m chain (GenBank ID: M84767.1) genes. The extracellular region of the α chain (excluding the transmembrane and intracellular regions) was linked to the β2m chain using a (G4S)4-linker to form a chicken MHC I dimer. A Twin-Strep tag was inserted at the 3' end of the β2m chain (the amino acid sequence of the Twin-Strep tag (SEQ ID NO:31) is: SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK). The design of the intended expression gene is described in [link to gene design]. Figure 1 Its sequence can be seen in SEQ ID NO.24.

[0042] Table 1 Primer sequence list

[0043]

[0044] The above three primer pairs were used for PCR amplification.

[0045] Using cDNA from chicken PBMCs of haplotype B19 as a template, the extracellular region of the α chain was amplified by MHC Iα-F / R, and the β2m chain was amplified by 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 products were purified by agarose gel electrophoresis, the first-round amplified fragment was used as a template. Using MHC Iα-F and Strep-R as primers, fusion PCR was performed to recover the MHC Iα-β2m-Strep fragment, which was cloned into the pRK5 vector, transformed into DH5α, and single colonies were picked for bacterial culture PCR identification. Positive colonies were sent for sequencing, and plasmids were extracted from correctly sequenced colonies and 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 h after transfection, and protein expression was detected by Western blotting.

[0050] Western blot (WB) detection of protein expression: After culture, the cell suspension was centrifuged at 800g for 10 min. 200 μL of the cell supernatant was carefully transferred to a new EP tube. 50 μL of 6× Protein Loading was added, and the mixture was thoroughly mixed. The tube was then boiled at 100℃ for 10 min, followed by SDS-PAGE electrophoresis. The membrane was then transferred at a constant current of 200 mA for 1 h, blocked with 5% skim milk at room temperature for 1 h, washed, and incubated with a 1:1000 dilution of mouse anti-chicken MHC class I antibody overnight at 4℃. The membrane was washed again, and a 1:5000 dilution of rabbit anti-mouse IgG antibody was prepared and incubated on a shaker at room temperature for 1 h. The membrane was washed, excess liquid was blotted with absorbent paper, and exposure buffer was added to the membrane surface for 1 min before development and photographing. (See attached image) Figure 2 ; Figure 2 In the table, the meanings of each number are as follows: M: protein molecular weight standard; 1-2: expression of chicken MHC I molecular protein in the supernatant and cell lysate of transfected recombinant plasmid cells; 3-4: expression of chicken MHC I molecular protein in the supernatant and cell lysate of transfected well vector cells.

[0051] 1.2 Preparation and purification of chicken pMHC protein

[0052] Preparation of pMHC protein: After transfecting 293F cells with the pRK5 / MHC Iα-β2m-Strep recombinant plasmid, H9N2 cells were infected with MOI=1 36 h later (A / chicken / Hebei / m0530-1 / 2017(HB17) isolated and preserved in our laboratory). At the time of infection, virus dilution solution was added to the cell shake flask and the culture flask was gently shaken to ensure that the virus solution was evenly distributed in the cells.

[0053] Purification of pMHC protein: Supernatant was harvested 48 hours post-infection, and affinity purification of pMHC-Strep protein was performed according to the STarm Streptactin Beads 4FF instructions. Protein expression and purification levels at each step were detected by SDS-PAGE and Western blotting. The purified target protein was collected and concentrated to 1 mL using a 10 kDa ultrafiltration tube; this yielded chicken pMHC protein. (See details...) Figure 3A and Figure 3B ; Figure 3A The meanings of each number in the table are as follows: M. Protein standard molecular weight, 1. Unpurified cell supernatant, 2. Purified effluent, 3. Biotin elution buffer, 4. Concentrated biotin elution buffer, 5. NaOH elution buffer, 6. Concentrated NaOH elution buffer, 7. Blank control cell supernatant; Figure 3B The meanings of the labels are as follows: M. Standard molecular weight of protein, 1. Unpurified cell supernatant, 2. Purified effluent, 3. Biotin elution buffer, 4. Concentrated biotin elution buffer, 5. NaOH elution buffer, 6. Concentrated NaOH elution buffer, 7. Blank control cell supernatant.

[0054] 1.3 Isolation and Mass Spectrometry Identification of Antigenic Peptides

[0055] Gentle acid washing separation of antigenic peptides: Add two volumes of 0.02N acetic acid to pMHC-Strep and mix well. Incubate at 65℃ for 25 min to dissociate the antigenic peptides. Centrifuge at 12000 rpm for 10 min and collect the supernatant. Filter all the supernatant after acid washing through a 3kDa ultrafiltration tube and collect the filtrate, which is the antigenic peptide fragment presented by the chicken MHC I monoallelic molecule.

[0056] Mass spectrometry identification and data processing: Protein samples were analyzed by liquid chromatography-LC-MS / MS and de novo analysis was performed. PEAKS Studio8 was used for database search, searching human protein databases and a self-constructed influenza virus protein database (derived from the viral protein sequences of the H9N2 strain used in the experiment). Parameters were set as follows: Peptide-10lgP≥15, PTMAscore≥0, Protein-10lgP≥20, Proteins unique peptides≥0, Denovo score(%)≥50%, Parent Mass Error Tolerance: 7.0ppm, Fragment Mass Error Tolerance: 0.02Da, Enzyme: None. Search results are referenced. Figure 4 .

[0057] Example 2: Peptide profiling and immunological identification of chicken MHC I-restricted AIV antigens

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

[0059] Table 2 presents the information on H9N2 subtype avian influenza virus antigenic peptides presented by the chicken MHC I allele BF2*1901 molecule (antigenic peptides that match more than twice are included in the analysis). The known binding motif of the BF2*1901 molecule is XR-XXXXXX-Y / P / L / F, with anchor residues at positions 2 and 9. Positions 1 and 2 are typically positively charged amino acids R, and positions 8 and 9 are typically hydrophobic amino acids. High-abundance peptides with repeated matching were identified through the binding motif. Based on the length and number of matching viral antigenic peptides, 23 peptides were selected as representative potential epitopes and sent to Genscript Biotech Co., Ltd. (HPLC processing, purity ≥98%) for synthesis. Specific information is shown in Table 2.

[0060] Table 2. Antigen Peptide Information Table

[0061]

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

[0063] 2.2.1 Refolding verification of the in vitro binding efficiency of latent epitopes to MHC:

[0064] Prokaryotic expression and inclusion body extraction of BF2*1901α and β2m chains

[0065] (1) Induction: Prepare a certain amount of ampicillin LB culture medium. Add 200 μL of positive bacterial culture (two positive bacterial cultures with PET-21a as the prokaryotic expression vector, with 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 in a shaker for 2 h until the OD600 value is about 0.6. Then add 1 mL of IPTG (1:1000) and induce culture in a shaker at 37℃ for 8 h.

[0066] (2) Collecting bacterial cells: The induced bacterial solution was centrifuged at low temperature to obtain bacterial cell precipitate. The precipitate was then resuspended in 25 mL of ultrapure water and sonicated on ice for 12 s, 18 s interval, and 45 min. The lysed bacterial solution was centrifuged at low temperature at 6000 rpm for 15 min. The supernatant was discarded, and bacterial cell impurities on the surface of the inclusion bodies were gently removed with a pipette tip.

[0067] (3) Washing: Vortex rinse the inclusion bodies with the prepared washing solution, centrifuge at low temperature and discard the supernatant, repeat washing until the inclusion bodies are relatively pure in appearance.

[0068] (4) Resuspension: Vortex inclusion bodies in an appropriate amount of resuspension solution until they are in a suspended state. Take a portion of the sample for identification 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 body and calculate the amount required to dissolve the inclusion body. Adjust the final concentration to 30 mg / mL, add the dissolving solution, and stir at low temperature until there is no obvious solid. Centrifuge multiple times to remove bottom impurities, then dispense, label and freeze for storage.

[0070] BF2*1901 refolding and concentration with antigenic peptides:

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

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

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

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

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

[0076] (6) Change the liquid: When the volume of the refolded liquid 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, centrifuge at 4℃ for 7000 rpm for 5 min and take the supernatant.

[0077] (7) Concentrate again: Prepare a 15mL, 10kDa ultrafiltration tube in advance, centrifuge at low temperature to concentrate the liquid to less than 1mL, transfer it to an EP tube, centrifuge again to collect the supernatant, filter with a 0.22μm filter membrane, centrifuge at 12000rpm to remove air bubbles, and store in a 4℃ refrigerator 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 (approximately 5.7%) remained constant. The maximum column pressure (Alarm Pressure = 0.33 MPa), flow rate (1 mL / min), and collection parameters (Peak Frac Parameters UV = 20 mAU; Peak Size = 1.2 mL) were then set. After zeroing the UV value, the sample to be tested was slowly aspirated into the loop for purification. The sample loading was observed periodically, and samples were collected based on the peak position and size. The sample at the peak tip was used for SDS-PAGE analysis to examine the binding peptide of the BF2*1901 molecule. Specific results are shown in [link to results]. Figures 5A to 5X ;

[0080] After in vitro refolding of chicken MHC I α-chain, β2m chain inclusion body proteins and potential epitope peptides, the samples were purified by molecular sieve. The samples showed three main protein peaks in sequence: α-chain polymer, α-chain-β2m chain-peptide complex, and β2m chain polymer. The MHC I-peptide complex (pBF2*1901) generally appeared between 80-100 mL.

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

[0082] Molecular sieving results showed that 18 peptides could form stable MHC I-peptide complexes with BF2*1901, including 8 motif peptides with P2-anchored residues: IRV8, ARM8, MRP8, IRL9, RRN9, QRD9, FRM11, and KRP12; 3 non-motif peptides that do not conform to the motif: AYP8, TTE9, and ISR11; and 7 ultrashort peptides: TTR7, ARA7, RQM7, RTP7, MGR7, AGK7, and MID7.

[0083] Four peptides, LPT8, KAF9, QNQ9, and MAC12, showed the desired peak shape, but the complex peak was below 50 mAU, indicating insufficient protein quantity for SDS-PAGE identification. This may be due to weak or no binding to BF2*1901. GRK8 did not show the ideal peak shape, and SDS-PAGE analysis revealed no complex peak, classifying it as an unbound peptide.

[0084] 2.2.2 ELISPOT validation of the immunomodulatory activity of potential epitopes

[0085] B19 haplotype chicken virus infection

[0086] Six 4-week-old SPF B19 haplotype White Leghorn chickens were used, and each chicken was intranasally infected with 106 EID50 / 0.1 mL of the H9N2 strain (A / chicken / Hebei / M0530-1 / 2017). Fresh peripheral blood anticoagulated by heparin was collected from the chicken peripheral vein 14 days post-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 (PBMCs)

[0088] Chicken peripheral blood was diluted with an equal volume of PBS. The centrifuge tube containing the separation buffer was then slowly tilted, and the diluted peripheral blood was carefully added along the tube wall above the separation buffer. The tube was centrifuged horizontally at 500g for 20 minutes. After centrifugation, distinct layers formed. The second layer, the white membrane layer of lymphocytes, was aspirated into a clean centrifuge tube. An appropriate amount of cell washing buffer was added, and the tube was gently inverted to mix the cells. The tube was centrifuged horizontally at 250g for 10 minutes, and the supernatant was discarded. The washing process was repeated once. Finally, the cells were resuspended in RPMI 1640 medium containing 10% FBS for later use.

[0089] ELISPOT testing

[0090] (1) First, take out the 96-well ELISpot plate, add 200 μL of sterile PBS to each well, wash 4 times, and aspirate the residual liquid after the last wash.

[0091] (2) Add 200 μL of RPMI 1640 culture medium containing 10% FBS to each well and incubate at 37°C for 2 hours;

[0092] (3) Wash the plate 4 times with PBS, add 100 μL of a single peptide stimulation (final concentration 1 mg / 0.1 mL), and then add 106 chicken PBMC lymphocytes, for a total volume of 200 μL / well. Two replicate wells were set up for each peptide, and two wells without peptide stimulation were used as negative controls. The cells were incubated at 37°C and 5% CO2 for 48 hours.

[0093] (4) After the culture is completed, discard the cell suspension, wash the plate 3 times with PBS, dilute the antibody MT7C10-biotin to a concentration of 1 μg / mL with 0.5% FBS-PBS buffer, 100 μL per well, and incubate at room temperature for 2 hours;

[0094] (5) Discard the antibody, wash the plate 3 times with PBS, dilute Streptavidin-HRP with 0.5% FBS-PBS at a ratio of 1:1000, 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 the color for about 15 min until obvious spots appear. Stop the color development reaction with deionized water and air 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 affinity for chicken MHC I molecule BF2*1901 that underwent in vitro refolding were selected, along with two weak-binding peptides, MAC12 and QNQ9, which showed multiple matching. The level of IFN-γ secreted by AIV-specific T lymphocytes after stimulation by these peptides was further detected using ELISPOT. Two replicate wells were set up for each stimulating peptide, and negative control wells without peptides were also included. The results showed that, compared with the negative control without peptides, except for MAC12, all 19 potential epitope peptides could stimulate T cells to secrete IFN-γ and produce immunospots to varying degrees.

Claims

1. The application of the AIV MHC-BF2*1901 restriction epitope peptide with the amino acid sequence shown in SEQ ID NO.2 in the preparation of an anti-H9N2 influenza virus vaccine, wherein the anti-H9N2 influenza virus vaccine is an epitope vaccine.

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