An SLA I allele and its application

Abstract

This invention discloses an SLA I allele and its applications. The allele expresses the amino acid sequences shown in SEQ ID NO:1 and / or SEQ ID NO:2. The resulting SLA I heavy chain polypeptide, upon the addition of a random peptide, can form a stable complex with β2-microglobulin in vitro, exhibiting broad-spectrum / high-frequency presentation capabilities. Based on this allele, stable "empty slot" complexes can be constructed for loading and identifying antigenic peptides, thereby achieving high-throughput discovery of high-frequency presented polypeptides.

Description

Technical Field

[0001] This invention belongs to the field of immunology and protein engineering, specifically relating to an SLA I allele and its applications. Background Technology

[0002] The MHC-I molecule consists of a heavy chain α chain and a light chain β2-microglobulin (β2m). The α chain includes five regions: a polypeptide-binding region α1, a polypeptide-binding region α2, an immunoglobulin-like region α3, a transmembrane region, and a cytoplasmic region. α1 and α2 are located at the outer ends of the MHC-I molecule, forming the MHC-I antigen-binding groove. Typically, the antigen-binding groove has six pockets (A to F) from the N-terminus to the C-terminus, and each pocket is composed of different amino acids.

[0003] In their natural state, the heavy chain and β2-microglobulin in MHC-I molecules need to form a tight complex with the peptide to maintain stability. Once the peptide dissociates, the heavy chain and β2-microglobulin rapidly denature and aggregate, causing the complex to fail. The preparation of conventional MHC-I complexes usually requires the addition of a high-affinity reference peptide to stabilize the groove formed by α1 and α2; otherwise, it is difficult to obtain a single, separable complex. Summary of the Invention

[0004] The purpose of this invention is to provide an SLA I allele and its application. The polypeptide expressed by this allele exhibits an exceptionally stable "empty slot / low occupancy" phenotype and broad-spectrum peptide binding ability, solving the problems of dependence on high-affinity reference peptides and loading bias in the prior art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of the present invention provides an SLA I allele comprising an amino acid sequence encoding the sequence shown in SEQ ID NO:1.

[0006] SEQ ID NO:1: LGDSRFIEVGYVDDTQFVRFDSDALNPRMEPRAPWIEKEGQDYWDEETRKVKETAQINRVDLKTLRGYYNQSEAGSHTLQSMFGCYLGPDGLLLRGYRQDAYDGADYIALNEDLRSWTAADTAAQITKRKWEAADEAEQWRSYLQGLCVESLRE.

[0007] A second aspect of the present invention provides an SLA I allele comprising an amino acid sequence encoding an amino acid sequence having ≥90% sequence identity with the amino acid sequence shown in SEQ ID NO:1, and maintaining the same amino acid sequence at positions 1, 25 and 154.

[0008] As a further embodiment of the present invention, the allele comprises an amino acid sequence that has ≥95% sequence identity with the amino acid sequence shown in SEQ ID NO:1, and maintains the same amino acid sequence at positions 1, 25 and 154.

[0009] As a further embodiment of the present invention, the allele comprises an amino acid sequence that has ≥99% sequence identity with the amino acid sequence shown in SEQ ID NO:1, and maintains the same amino acid sequence at positions 1, 25 and 154.

[0010] A third aspect of the present invention provides an SLA I allele comprising an amino acid sequence encoding the amino acid sequence described in the first or second aspect, with 1 to 100 amino acid residues appended to the N-terminus and / or C-terminus, and the appended sequence having an antigen peptide binding function.

[0011] As a further embodiment of the present invention, the allele encodes the amino acid sequence shown in SEQ ID NO:2.

[0012] SEQ ID NO:2: GPHSLSYFYTAVSRPDLGDSRFIEVGYVDDTQFVRFDSDALNPRMEPRAPWIEKEGQDYWDEETRKVKETAQINRVDLKTLRGYYNQSEAGSHTLQSMFGCYLGPDGLLLRGYRQDAYDGADYIALNEDLRSWTAADT AAQITKRKWEAADEAEQWRSYLQGLCVESLREYLEMGKDTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWD.

[0013] As a further embodiment of the present invention, the allele encodes an amino acid sequence with ≥90% sequence identity to the amino acid sequence shown in SEQ ID NO:2, and retains amino acid sequences of homologous residues at the B pocket and F pocket sites.

[0014] As a further embodiment of the present invention, the allele encodes an amino acid sequence with ≥95% sequence identity to the amino acid sequence shown in SEQ ID NO:2, and retains amino acid sequences of homologous residues at the B pocket and F pocket sites.

[0015] As a further embodiment of the present invention, the allele encodes an amino acid sequence with ≥99% sequence identity to the amino acid sequence shown in SEQ ID NO:2, and retains amino acid sequences of homologous residues at the B pocket and F pocket sites.

[0016] As a further embodiment of the present invention, the allele encoding the amino acid sequence shown in SEQ ID NO:2, after substitution, deletion and / or addition of amino acid residues, has ≥80% sequence identity with the sequence shown in SEQ ID NO:2, and maintains the same amino acid sequence at positions 17, 41 and 170.

[0017] As a further embodiment of the present invention, the allele encoding the amino acid sequence shown in SEQ ID NO:2, after substitution, deletion and / or addition of amino acid residues, has ≥90% sequence identity with the sequence shown in SEQ ID NO:2, and still maintains the same amino acid sequence at positions 17, 41 and 170.

[0018] As a further embodiment of the present invention, the allele encoding the amino acid sequence shown in SEQ ID NO:2, after substitution, deletion and / or addition of amino acid residues, has ≥95% sequence identity with the sequence shown in SEQ ID NO:2, and still maintains the same amino acid sequence at positions 17, 41 and 170.

[0019] As a further embodiment of the present invention, the allele encoding the amino acid sequence shown in SEQ ID NO:2, after substitution, deletion and / or addition of amino acid residues, has ≥99% sequence identity with the sequence shown in SEQ ID NO:2, and still maintains the same amino acid sequence at positions 17, 41 and 170.

[0020] A fourth aspect of the present invention provides an SLA I allele comprising the nucleotide sequence shown in SEQ ID NO:3.

[0021] As a further embodiment of the present invention, the allele comprises a nucleotide sequence as shown in SEQ ID NO:3.

[0022] SEQ ID NO:3: ATGGGACCCCACAGTCTATCATATTTTTACACAGCGGTTAGCCGTCCGGACTTGGGCGATTCTCGCTTCATCGAGGTTGGTTACGTGGATGATACCCAGTTTGTTCGTTTCGACTCCGACGCTCTGAACCCGCGTATGGAACCGCGTGCACCGTGGATTGAGAAGGAGGGCCAGGACTACTGGGACGAAGAGACTCGCAAGGTGAAAGAGACAGCCCAAATCAACCGTGTTGACCTGAAAACCCTGCGTGGCTACTATAACCAGAGTGAAGCGGGTAGCCATACCTTGCAAAGCATGTTCGGCTGCTACTTAGGTCCGGATGGTTTGTTACTGCGTGGTTACCGCCAGGACGCGTATGATGGCGCAGATTACATTGCGCTGAATGAAGATCTGCGTAGCTGGACCGCGGCGGATACTGCGGCTCAGATTACCAAACGTAAATGGGAAGCTGCTGACGAGGCCGAGCAATGGCGTTCCTATCTGCAAGGTTTGTGCGTCGAGAGCCTGAGGGAGTATCTGGAAATGGGTAAAGATACCCTCCAACGTGCGGAGCCGCCTAAGACGCACGTGACCCGCCACCCGTCTAGCGATCTGGGGGTGACCTTGCGCTGCTGGGCACTGGGCTTTTATCCGAAGGAGATCAGCCTGACGTGGCAGAGAGAGGGTCAGGACCAATCGCAAGACATGGAACTGGTTGAAACGCGTCCGTCTGGTGACGGCACCTTTCAGAAGTGGGCAGCCTTGGTAGTCCCGCCAGGTGAAGAACAGTCCTACACCTGTCATGTGCAGCACGAAGGCCTTCAAGAACCGCTGACCCTGCGCTGGGAC。

[0023] In the fifth aspect of the present invention, there is provided an SLA I allele, comprising a nucleotide sequence having ≥ 80% sequence identity with the nucleotide sequence shown in SEQ ID NO: 3 and having the same function.

[0024] As a further embodiment of the present invention, the allele comprises a nucleotide sequence having ≥90% sequence identity with the nucleotide sequence shown in SEQ ID NO:3 and having the same function.

[0025] As a further embodiment of the present invention, the allele comprises a nucleotide sequence having ≥95% sequence identity with the nucleotide sequence shown in SEQ ID NO:3 and having the same function.

[0026] As a further embodiment of the present invention, the allele comprises a nucleotide sequence having ≥99% sequence identity with the nucleotide sequence shown in SEQ ID NO:3 and having the same function.

[0027] The sixth aspect of the present invention provides a polypeptide encoded by an allele as described in any of the preceding claims.

[0028] The seventh aspect of the present invention provides a polypeptide comprising the amino acid sequence shown in SEQ ID NO:1.

[0029] The eighth aspect of the present invention provides a polypeptide comprising a nucleotide sequence having ≥80% sequence identity with the amino acid sequence shown in SEQ ID NO:1 and having the same function.

[0030] As a further embodiment of the present invention, the polypeptide comprises a nucleotide sequence having ≥90% sequence identity with the amino acid sequence shown in SEQ ID NO:1 and having the same function.

[0031] As a further embodiment of the present invention, the polypeptide comprises a nucleotide sequence having ≥95% sequence identity with the amino acid sequence shown in SEQ ID NO:1 and having the same function.

[0032] As a further embodiment of the present invention, the polypeptide comprises a nucleotide sequence having ≥99% sequence identity with the amino acid sequence shown in SEQ ID NO:1 and having the same function.

[0033] The ninth aspect of the present invention provides a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.

[0034] The tenth aspect of the present invention provides a polypeptide comprising a nucleotide sequence having ≥80% sequence identity with the amino acid sequence shown in SEQ ID NO:2 and having the same function.

[0035] As a further embodiment of the present invention, the polypeptide comprises a nucleotide sequence having ≥90% sequence identity with the amino acid sequence shown in SEQ ID NO:2 and having the same function.

[0036] As a further embodiment of the present invention, the polypeptide comprises a nucleotide sequence having ≥95% sequence identity with the amino acid sequence shown in SEQ ID NO:2 and having the same function.

[0037] As a further embodiment of the present invention, the polypeptide comprises a nucleotide sequence having ≥99% sequence identity with the amino acid sequence shown in SEQ ID NO:2 and having the same function.

[0038] As a further embodiment of the present invention, the polypeptide is an SLA I heavy chain polypeptide.

[0039] As a further embodiment of the present invention, the polypeptide is a chimeric heavy chain polypeptide, which is obtained by replacing the nucleotide fragments encoding the B pocket and / or F pocket in the allele with another SLA I / HLA I genomic backbone for expression.

[0040] The eleventh aspect of the present invention provides a complex comprising the polypeptide as described in any of the above claims.

[0041] As a further aspect of the present invention, the complex also includes β2-microglobulin and short peptides.

[0042] As a further aspect of the present invention, the complex is prepared by the following method: adding the polypeptide and β2-microglobulin to a solution containing a short peptide for co-refolding to obtain the complex.

[0043] As a further aspect of the present invention, the short peptide has a length of 8 to 20 amino acids, preferably 9 amino acids.

[0044] As a further aspect of the present invention, the molar ratio of the polypeptide to β2-microglobulin is 1:(1.0~1.5), preferably 1:(1.0~1.2).

[0045] As a further aspect of the present invention, the solution is a refolding buffer solution.

[0046] As a further embodiment of the present invention, the refolding buffer contains Tris-HCl, L-arginine, reduced glutathione, oxidized glutathione, and EDTA.

[0047] As a further aspect of the present invention, the preparation process of the composite also includes a denaturation step.

[0048] As a further aspect of the present invention, the denaturation step uses guanidine hydrochloride.

[0049] As a further embodiment of the present invention: the composite is subjected to 10~20 mM Tris-HCl solution, 140~150 mM NaCl solution and pH=7~8, and after standing at 4℃ for 24~72 h, the monomer peak area ratio is measured by size exclusion chromatography to be ≥80%.

[0050] The twelfth aspect of the present invention provides a biological material comprising a nucleic acid molecule having alleles as described in any one of the above claims.

[0051] The thirteenth aspect of the present invention provides a biological material comprising at least one of cDNA having the nucleic acid molecule described in the twelfth aspect, a synthetic gene, an expression cassette, a recombinant vector, and a recombinant microorganism.

[0052] The fourteenth aspect of the present invention provides the use of alleles, peptides, complexes or biological materials as described in any of the above claims in constructing MHC monomer libraries and / or peptide presentation databases.

[0053] As a further embodiment of the present invention: the MHC monomer library is an MHC-I monomer library.

[0054] As a further embodiment of the present invention: the presented peptide database includes a high-frequency presented peptide database classified by genotype.

[0055] As a further aspect of the present invention, the high-frequency / broad-spectrum presentation capability of the presented peptide database is determined in the following manner: 8-11 peptide sets are randomly selected or derived from ≥N proteins (N≥10) for loading, and evaluated by fluorescence polarization / competition experiments or SEC-DSF combined.

[0056] The fifteenth aspect of the present invention provides the use of alleles, polypeptides, complexes or biological materials as described in any of the above claims in screening and / or optimizing vaccine candidate peptides and / or diagnostic marker peptides.

[0057] The sixteenth aspect of the present invention provides an application of alleles, peptides, complexes or biological materials as described in any of the above claims in immunogenicity assessment and / or epitope mapping.

[0058] The seventeenth aspect of the present invention provides an application of alleles, polypeptides, complexes or biomaterials as described in any of the above claims in the preparation of MHC tetramer tools.

[0059] The MHC monomer library described in this invention refers to a library composed of multiple single MHC-peptide complexes (MHC monomers), each monomer containing an MHC molecule and a peptide bound to it.

[0060] The vaccine candidate peptides described in this invention refer to peptide segments that have potential immunogenicity and can induce T cell or B cell immune responses, obtained through immunoinformatics prediction or experimental screening, and are used as active ingredients for developing vaccines.

[0061] The diagnostic marker peptides described in this invention refer to peptide segments that are specifically expressed or presented in a disease state and can serve as biomarkers for disease diagnosis, classification, or prognosis assessment.

[0062] The immunogenicity assessment described in this invention refers to evaluating the ability of a peptide to induce an immune response through in vitro or in vivo experimental methods (such as T cell activation assay, ELISPOT, MHC binding assay, etc.) in order to determine its potential as a vaccine or therapeutic peptide.

[0063] The epitope mapping described in this invention refers to determining, through experimental or computational methods, the specific peptide regions in an antigen protein that are recognized by T cell receptors or antibodies.

[0064] The MHC tetramer tool described in this invention refers to a polymer formed by linking four MHC-peptide monomers through molecules such as streptavidin.

[0065] The present invention has at least the following technical effects: This invention provides an SLA I allele that can refold into a stable complex without requiring a high-affinity reference peptide and exhibits broad-spectrum peptide presentation capability. This allele can be expressed in a standard *E. coli* / mammalian cell system, and the resulting complex can be prepared using a universal buffer system and chromatography equipment. The complex provided by this invention possesses broad-spectrum peptide binding capability, expands the peptide coverage of MHC-I in vitro refolding, avoids loading bias, facilitates the statistical discovery of high-frequency peptide presentation sites, and can also be directly used for front-end screening in vaccine / diagnostic development, immunogenicity assessment, epitope mapping, and as a tool for preparing MHC tetramers. Attached Figure Description

[0066] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the accompanying drawings.

[0067] Figure 1 The composite remodeling SEC result obtained in Example 1 of this invention; Figure 2 The results of polypeptide presentation ability tests by different alleles in Example 2 of this invention; Figure 3 This is a summary analysis of the polypeptide presentation capabilities of different alleles in Example 2 of the present invention; Figure 4 This is the result of a preference analysis of random peptides for pockets in Example 3 of the present invention.

[0068] Figure label: pSLA-I_R9Ps: A complex formed by the heavy chain polypeptide obtained from the expression of the SLA-1*12:01 allele, β2-microglobulin, and random peptide. Detailed Implementation

[0069] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0070] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0071] Example 1: In vitro refolding and stability study of SLA I heavy chain with β2-microglobulin and random peptides 1.1 Expression Carrier The SLA I allele was cloned into the pET-21a(+) vector for expression (with XhoⅠ and NdeⅠ restriction sites) to obtain the SLA I heavy chain polypeptide; β2-microglobulin (β2m) was also expressed in the pET-21a(+) vector (with XhoⅠ and NdeⅠ restriction sites).

[0072] The amino acid sequence of the SLA I allele is shown in SEQ ID NO:2.

[0073] SEQ ID NO:2: GPHSLSYFYTAVSRPDLGDSRFIEVGYVDDTQFVRFDSDALNPRMEPRAPWIEKEGQDYWDEETRKVKETAQINRVDLKTLRGYYNQSEAGSHTLQSMFGCYLGPDGLLLRGYRQDAYDGADYIALNEDLRSWTAADT AAQITKRKWEAADEAEQWRSYLQGLCVESLREYLEMGKDTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWD.

[0074] The nucleotide sequence of the SLA I allele is shown in SEQ ID NO:3.

[0075] SEQ ID NO:3: ATGGGACCCCACAGTCTATCATATTTTTACACAGCGGTTAGCCGTCCGGACTTGGGCGATTCTCGCTTCATCGAGGTTGGTTACGTGGATGATACCCAGTTTGTTCGTTTCGACTCCGACGCTCTGAACCCGCGTATGGAACCGCGTGCACCGTGGATTGAGAAGGAGGGCCAGGACTACTGGGACGAAGAGACTCGCAAGGTGAAAGAGACAGCCCAAATCAACCGTGTTGACCTGAAAACCCTGCGTGGCTACTATAACCAGAGTGAAGCGGGTAGCCATACCTTGCAAAGCATGTTCGGCTGCTACTTAGGTCCGGATGGTTTGTTACTGCGTGGTTACCGCCAGGACGCGTATGATGGCGCAGATTACATTGCGCTGAATGAAGATCTGCGTAGCTGGACCGCGGCGGATACTGCGGCTCAGATTACCAAACGTAAATGGGAAGCTGCTGACGAGGCCGAGCAATGGCGTTCCTATCTGCAAGGTTTGTGCGTCGAGAGCCTGAGGGAGTATCTGGAAATGGGTAAAGATACCCTCCAACGTGCGGAGCCGCCTAAGACGCACGTGACCCGCCACCCGTCTAGCGATCTGGGGGTGACCTTGCGCTGCTGGGCACTGGGCTTTTATCCGAAGGAGATCAGCCTGACGTGGCAGAGAGAGGGTCAGGACCAATCGCAAGACATGGAACTGGTTGAAACGCGTCCGTCTGGTGACGGCACCTTTCAGAAGTGGGCAGCCTTGGTAGTCCCGCCAGGTGAAGAACAGTCCTACACCTGTCATGTGCAGCACGAAGGCCTTCAAGAACCGCTGACCCTGCGCTGGGAC。

[0076] 1.2 Host cell line For prokaryotic expression, E. coli BL21 competent cells were used. After conventional heat shock transformation (ice bath → heat shock at 42 °C for 45 s → recovery → plating → picking monoclonal colonies for sequencing confirmation), they were used for expression. 1.3 Expression and Inclusion Body Extraction 1.3.1 Large-scale culture of microbial strains Take 100 μL / 10 mL of sequencing-positive bacterial culture (100 μL / 10 mL A). + LB (ampicillin-resistant liquid Luria-Bertani, cultured at 37°C and 180 rpm for 11–13 h), inoculated at a volume ratio of 1:20 to 1 LA + LB, stays before induction at OD600 = 0.4~0.6.

[0077] Add IPTG (1 mol / L stock solution added at a volume ratio of 1:1000) and induce at 37 ℃ and 170 rpm for 6-8 h. 1.3.2 Harvesting and Lysis Collect bacterial cells by centrifugation at 4000 r / min for 10 min at 4 ℃; resuspend in 30 mL of water.

[0078] Ultrasonic disruption on ice (60W power, 2 s on / 4 s off, 60 min total).

[0079] Centrifuge at 4 ℃, 7500 r / min, for 30 min, and discard the supernatant; the lower layer is milky white (inclusion bodies) and the upper layer is brown (impurities) separated into layers, and the upper layer is removed.

[0080] The inclusion bodies were washed three times with washing buffer, rinsed with resuspension buffer, and weighed dry.

[0081] 1.4 In vitro dilution and refolding 1.4.1 Inclusion body degeneration Inclusion bodies were placed in EP tubes, denaturing buffer (DTT added immediately before use) was added, and the solution was dissolved at 4°C for 2 h using a magnetic stirrer to obtain a denatured inclusion body solution. SLA I heavy chain inclusion bodies and β2m inclusion bodies were denatured separately according to this method for subsequent renaturation operations.

[0082] 1.4.2 Refolding System and Sampling Order Prepare 500 mL of refolding buffer in a beaker and add the target peptide (a random short peptide synthesized by Zhongke Yaguang, with a length of 9 amino acids, each amino acid represented by X, and the peptide sequence being XXXXXXXXX, where X excluding Cys) dissolved in DMSO. Fix a syringe containing 2 mL of 30 mg / mL β2m inclusion body solution vertically at the mouth of the beaker and slowly drip it in over approximately 1 hour. After stirring at 4°C and 370 rpm for 12–18 hours, add 6 mL of 30 mg / mL SLA I heavy chain inclusion body solution and continue stirring at 4°C and 370 rpm for 24–36 hours to obtain a soluble pMHC complex solution.

[0083] 1.5 Concentration and Chromatographic Purification 1.5.1 Pressure Ultrafiltration Concentration / Liquid Replacement Transfer the pMHC complex solution to a 10 kDa membrane stirred concentrator, purge with nitrogen to stabilize the system pressure at 40 MPa, concentrate to 20-30 mL, add 3 times the volume of molecular sieve buffer to replace the solution, and continue concentrating to a volume <20 mL; centrifuge at 4 ℃ to clarify and filter at 0.22 μm for later use. 1.5.2 Molecular sieve chromatography (SEC) / binding capacity verification Chromatographic column: Superdex 200 pg; molecular sieve buffer; after equilibration of 2 column volumes at a flow rate of 1 mL / min, the sample was loaded, separated, and collected. The collected peaks were identified by SDS-PAGE (boiling for 10 min, then constant pressure at 160 V for 60 min). SEC results are as follows. Figure 1 As shown.

[0084] Figure 1 This is a molecular sieve chromatogram. The horizontal axis represents the elution volume (mL), and the vertical axis represents the UV absorbance at 280 nm. The separation follows the size exclusion principle, with larger molecules eluting first and smaller molecules eluting later. During sample refolding, heavy chain aggregates, heavy chain-light chain-random polypeptide aggregates (target protein), and light chain aggregates can be formed. The elution peaks of heavy chain aggregates, target protein, and light chain aggregates are located in the ranges of 40–50 mL, 80–90 mL, and 100–115 mL, respectively, consistent with their molecular weight order.

[0085] 1.6 Buffer Formulation and Preparation Method Washing buffer (500 mL): NaCl 8.766 g, Tris-HCl pH 8.0 (1 mol / L) 25 mL, EDTA-Na2 (0.5 mol / L) 10 mL, DTT (1 mol / L) 5 mL (added immediately before use), Triton X-100 0.5% (2.5 mL), bring the volume to 500 mL.

[0086] Resuspension buffer (500 mL): NaCl 2.922 g, Tris-HCl pH 8.0 (1 mol / L) 25 mL, EDTA-Na2 (0.5 mol / L) 10 mL, DTT (1 mol / L) 5 mL, bring to a final volume of 500 mL.

[0087] Denaturing buffer (500 mL): NaCl 2.922 g, Tris-HCl pH 8.0 (1 mol / L) 25 mL, EDTA-Na2 (0.5 mol / L) 10 mL, DTT (1 mol / L) 5 mL (added immediately before use), guanidine hydrochloride 286.59 g, glycerol 50 mL, bring the volume to 500 mL.

[0088] Refolding buffer (1 L): 50 mL glycerol, 100 mL Tris-HCl pH 8.0 (1 mol / L), 84.264 g L-arginine-HCl, 4 mL EDTA-Na2 (0.5 mol / L), 1.5306 g reduced glutathione, 0.3064 g oxidized glutathione, bring to a final volume of 1 L, filter through a 0.22 μm filter, and store at 4 °C.

[0089] Molecular sieve buffer (1 L): 20 mL Tris-HCl pH 8.0 (1 mol / L), 2.922 g NaCl, bring to a final volume of 1 L, and filter through a 0.22 μm filter.

[0090] Example 2: In vitro refolding and stability study of heavy chain polypeptides, β2-microglobulin, and random peptides expressed by high-frequency alleles in porcine herds. The high-frequency alleles in the pig population obtained from the statistics were expressed as proteins. The high-frequency alleles included SLA-1*04:01, SLA-1*07:03, SLA-1*08:08, SLA-1*08:19, SLA-1*12:01, SLA-1*13:01, SLA-2*01:01, SLA-2*04:10, SLA-2*10:01, SLA-2*10:05, SLA-2*16:03, SLA-3*01:01, SLA-3*04:05, SLA-3*05:02, SLA-3*05:07, and SLA-3*06:01. The above alleles were expressed to obtain heavy chain inclusion bodies. The detailed operation procedure is shown in Example 1. In this refolding system, a commercially synthesized random peptide was used as a uniform loading substrate. This random peptide does not contain cysteine ​​residues and is composed of 19 randomly combined amino acids to unbiasedly evaluate the ability of different SLA I alleles to form complexes.

[0091] The specific refolding procedure is as follows: the random peptide is pre-dissolved in DMSO and added to 500 mL of refolding buffer at a final mass of 10 mg of peptide; then, a β2-microglobulin denaturing solution with a concentration of 30 mg / mL is slowly added dropwise to the refolding system containing the random peptide over 1 h using a fixed syringe, allowing it to gradually fold and form an intermediate that can bind to the random peptide under stirring conditions of 4 ℃ and 370 rpm. After the addition is completed, stirring is continued for 12~18 h to ensure that the β2-microglobulin reaches a stable folded state. Next, a denaturing solution of the same concentration of heavy chain was slowly added to the system under the same conditions, maintaining a molar ratio of heavy chain to β2-microglobulin of 1:1, and stirring was continued at 4 °C for 24-36 h, so that the heavy chain could complete the correct folding under the occupancy of random peptides and bind to β2-microglobulin to generate refolded products.

[0092] The refolded product was concentrated by 10 kDa ultrafiltration and replaced with buffer solution, then separated by size exclusion chromatography (SEC), such as... Figure 2 As shown, the main peaks of the complexes corresponding to different alleles were observed at an elution volume of approximately 80 mL. The differences in peak area and peak shape reflect the loading capacity and renaturation efficiency of each allele under the condition of uniform random peptide loading.

[0093] Figure 3 This is the statistical analysis result of the ability of different porcine alleles to form complexes under random peptide conditions in Example 2. By integrating the area of ​​the SEC elution curve and combining the results of multiple parallel renaturation replicate experiments, it can be seen that there are significant differences in peptide presentation capabilities among the alleles. The heavy chain encoded by the SLA-1*12:01 allele showed the highest complex formation efficiency, with a peak area much higher than other alleles, indicating that it has broad-spectrum and strong peptide loading capabilities. Other alleles showed moderate or low renaturation performance, verifying the inherent differences in structure and function of alleles.

[0094] Example 3: Pocket Preference Analysis of SLA-1*12:01 The complex formed by the heavy chain polypeptide encoded by the allele SLA-1*12:01 in Example 2 was concentrated and then further purified by anion exchange chromatography.

[0095] In anion exchange chromatography (AEX), the AEX buffer is prepared as follows: Solution A (Tris-HCl pH 8.0 10 mL + NaCl 0.5844 g / L); Solution B (Tris-HCl pH 8.0 10 mL + NaCl 58.44 g / L).

[0096] A ResourceQ column was used. The column was first rinsed with 3 column volumes of solution B, and then equilibrated with 3 column volumes of solution A. After loading the sample, the column was linearly increased to 50% of solution B after 60 min. The sample was collected according to the peak and identified by SDS-PAGE. The target peak was ultrafiltered and replaced with SEC buffer to obtain the purified pMHC complex. Heavy chain peptides (from pMHC complex) were eluted with acetic acid. (1) Concentration: The purified pMHC complex was concentrated to 200 μL by ultrafiltration at 10 kDa; (2) Acid elution: Mix evenly with twice the volume of acetic acid solution (approximately 400 μL, 0.02 equivalents) and incubate in a metal bath at 65 °C for 30 min; (3) Decomposition of complex proteins: Centrifuge at 4 ℃, 12000 r / min for 30 min, and collect the supernatant; (4) Retention of heavy chain peptides: The supernatant was passed through a 3 kDa ultrafiltration device, and the heavy chain peptide fraction obtained by acid elution was collected; (5) Mass spectrometry was used to identify the heavy chain peptides using Denovo parameter-free analysis.

[0097] The data results were analyzed using Weblogo graphics software. The pocket preference analysis results generated from the heavy chain polypeptide set are as follows: Figure 4 As shown.

[0098] The results showed that the heavy chain polypeptide expressed by the SLA-1*12:01 allele exhibited a significant preference for hydrophobic amino acids at the P2 site (with valine V being the most enriched) and a significant preference for positively charged residues (mainly arginine R) at the P9 site. This indicates that the B pocket and F pocket of the heavy chain polypeptide expressed by SLA-1*12:01 tend to bind hydrophobic and positively charged amino acid residues, respectively, consistent with the typical structural chemistry of MHC-I molecules. This further explains the molecular basis for its high stability and broad-spectrum peptide binding ability.

[0099] Example 4: Amino acid sequence study of high-frequency allele expression in various pig herds The amino acid sequence of allele SLA-1*12:01 in Example 2 was compared with the amino acid sequences of alleles SLA-1*04:01, SLA-1*07:03, SLA-1*08:08, SLA-1*08:19, SLA-1*13:01, SLA-2*01:01, SLA-2*04:10, SLA-2*10:01, SLA-2*10:05, SLA-2*16:03, SLA-3*01:01, SLA-3*04:05, SLA-3*05:02, SLA-3*05:07 and SLA-3*06:01.

[0100] The amino acid sequence of the allele SLA-1*12:01 is shown in SEQ ID NO:2.

[0101] The amino acid sequence of the allele SLA-1*04:01 is shown in SEQ ID NO:01-04-01.

[0102] SEQ ID NO:01-04-01: MGPGALFLLLSGTLALTGTQAGPHSLSYFYTAVSRPDRGDSRFIAVGYVDDTQFVRFDNYAPNPRMEPRVPWIQQEGQEYWDRETRNVKETAQTYGVGLNTLRGYYNQSEAGSHTLQSMYGCYLGPDGLLLHGYRQDAYDGADYIALNEDLRSWTAADMAAQITKRKWEAADEAERRRSYL QGLCVESLRRYLEMGKDTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWDPAQPPVPIVGIIVGLVLVLVAGAMVAGVVIWRKTRSGEKGGSYTQAAGSDSDQGSDVSLTKDPRV.

[0103] The amino acid sequence of the allele SLA-1*07:03 is shown in SEQ ID NO:01-07-03.

[0104] SEQ ID NO:01-07-03: MGPRGLFLLLSGTLALTGTQAGPHSLSYFYTAVSRPDRGDSRFIAVGYVDDTQFVRFDSDAPNPREEPRAPWIQQEGQEYWDRNTQIYKETAQTYRVSLNNLRGYYNQSEAGSHTLQSMYGCYLGPDGLLLRGYRQYAYDSADYIALNEDLRSWTAADTAAQITKRKWEAANVAERRRSYLQGLCVESLRRYLEMGKDTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWDPPQTPVPIVGIIVGLVLVLVAGAVVAGVVIWRKKRSGEKGGSYTQAAGSDSDQGSDVSLTKGPRV。

[0105] The amino acid sequence expressed by allele SLA-1*08:08 is as shown in SEQ ID NO:01-08-08.

[0106] SEQ ID NO:01-08-08: MGPGALFLLLSGALALTGTQAGPHSLSYFYTAVSRPDRGDSRFFIVGYVDDTQFVRFDSDAPNAKMEPRAQWIQQEGPEYWDRETQISKETAQTYRVSLNNLRGYYNQSEAGSHTYQNMYGCYLGPDGLLLRGYSQYGYDGADYIALNEDLRSWTAADMAAQISKRKWEAADAAEQWRSYLQGRCVEWLQKYLEMGKDTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDHSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWDPAQPPVPIVGIIVVLVLVLVAGAVVAGVVIWRKKRSGEKGGSYTQAAGSDSDQGSDVSLTKDPRV。

[0107] The amino acid sequence expressed by allele SLA-1*08:19 is as shown in SEQ ID NO:01-08-19.

[0108] SEQ ID NO:01-08-19: GPHSLRYFYTAVSRPDRGDSRFFIVGYVDDTQFVRFDSDAPNAKMEPRAQWIKQEGPEYWDRETQISKETAQNYRVGLKTLRGYYNQSEAGSHTIQVMYGCDVGPDGLLLRGYRQDAYDGADYIALNEDLRSWTAADMAAQITKRKWEAADEAEGERSYLQGLCVEGLRRYLQMGKDTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWD。

[0109] The amino acid sequence expressed by allele SLA-1*13:01 is as shown in SEQ ID NO:01-13-01.

[0110] SEQ ID NO:01-13-01: MGPGALFLLLSGTLALTGTQAGPHSLSYFYTAVSRPDRGDSRFIAVGYVDDTQFVRFDNYAPNPRMEPRVPWIQQEGQDYWDEETRKVKDNAQTYGVGLNTLRGYYNQSEAGSHTLQSMFGCYLGPDGLLLHGYRQDAYDGADYIALNEDLRSWTAADMAAQITKRKWEAANVAERRRSYLQGLCVESLRRYLEMGKDTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWDPAQPPVPIVGIIVGLVLVLVAGAVVAGVVIWRKTRSGEKGGSYTQAAGSDSDQGSDVSLTKDPRV。

[0111] The amino acid sequence expressed by allele SLA-2*01:01 is as shown in SEQ ID NO:02-01-01.

[0112] SEQ ID NO:02-01-01: MRVRGPQAILILLSGALALTGTWAGPHSLSYFSTAVSRPDRGEPRFIAVGYVDDTQFVRFDSDAPNPRMEPRAPWIQQEGQDYWDRETRNVMGSAQTDRVNLKTLRGYYNQSEAGSHTIQSMYGCDVGPDGLLLRGYSQDAYDGADYIALNEDLRSWTAADTAAQITKRKWEAANVAERMRSYLQGLCVEGLQKYLQMGKDTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWDPPQPPIPIVGIIVGLVLVLVAGAMVAGVVIWRKKRSGEKGGSYTQAAGSDSAQGSDVSLTKDPRV。

[0113] The amino acid sequence expressed by allele SLA-2*04:10 is as shown in SEQ ID NO:02-04-10.

[0114] SEQ ID NO:02-04-10: GPHSLRYFDTAVSRPDRGEPRFIEVGYVDDTQFVRFDSDAPNPRMEPRAPWIQQEGQEYWDRNTRNAMGNAQIYRGNLRTALGYYNQSEAGSHTLQIMYGCDVGPDGLLLRGYSQDAYDGADYIALNEDLRSWTAADTAAQITKRKWEAANVAEQWRSYLQGTCVEWLQKYLQMGKDTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWD.

[0115] The amino acid sequence expressed by allele SLA-2*10:01 is as shown in SEQ ID NO:02-10-01.

[0116] SEQ ID NO:02-10-01: MRVRGPQAILILLSGALALTGTQAGPHSLSYFYTAVSRPDRGDSRFFIVGYVDDTQFVRFDSDAPNAKMEPRAQWIQQEGQEYWDRETQISKDNAQINRVNLNTLRGYYNQSEAGSHTLQRMYGCYLGPDGLLLRGYDQDAYDGADYIALNEDLRSWTAADMAAQISKRKREAADEAERMRSYLQGRCVEWLQKYLEMGKDTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLSWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWDPAQPPVPMVGIIVGLVLVLVAGAMVAGVVIWRKKRSGEKGGSYTQAAGSDSAQGSDVSLTKDPRV。

[0117] The amino acid sequence expressed by the allele SLA-2*10:05 is as shown in SEQ ID NO:02-10-05.

[0118] SEQ ID NO:02-10-05: MRVRGPQAILILLSGALALTGTQAGPHSLSYFYTAVSRPDRGDSRFFIVGYVDDTQFVRFDSDAPNAKMEPRAQWIQQEGQEYWDRETQISKETAQNYRVDLNTLRGYYNQSEAGSHTYQWMYGCYLGPDGLLLRGYDQDAYDGADYLALNEDLRSWTAADMAAQISKRKREAADEAERMRSYLQGRCVEWLQKYLEMGKDTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLSWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWDPAQPPVPMVGIIVGLVLVLVAGAMVAGVVIWRKKRSGEKGGSYTQAAGSDSAQGSDVSLTKDPRV。

[0119] The amino acid sequence expressed by the allele SLA-2*16:03 is as shown in SEQ ID NO:02-16-03.

[0120] SEQ ID NO:02-16-03: MRVRGPQAILILLSGALALTGTQAGSHSLSYFYTAVSRPDRGDSRFLTVGYVDDTQFVRFDSDAPNPREEPRAPWIQQEGQDYWDRNTQIYKETAQIYRVGLNNLRGYYNQSEAGSHTFQSMYGCDVGPDGLFLRGYSQDAYDGADYVALNEDLRSWTAADTAAQITKRKREAADAAEQWRSYLQGTCVEGLRRYLEMGKDTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWDPPQPPVPIVGIIVGLVLVLVTGAVVTGVVIWRKKRSGEKGGSYTQAAGSDSAQGSDVSLTKDPRV。

[0121] The amino acid sequence expressed by allele SLA-3*01:01 is shown as SEQ ID NO:03-01-01 below.

[0122] SEQ ID NO:03-01-01: MGPRALFLLLSGTLALTGTREGPHSLRYFDTAVSRPDRGKPRFISVGYVDDTQFVRFDSDAPNPRMEPRAPWIEQEGQEYWDEETRNAMGSAQTFRVNLNNLRGYYNQSEAGSHTFQSMYGCDVGPDGLLLRGYSQFGYDGADYIALNEDLRSWTAADTAAQITKRKREAADAAEQMRSYLEGACVVWLQKYLEMGNNTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWDPPQPPVPIVGITVGLVLVLVAGAVVAGVVIWRKKRSGEKGGSYTQAAGSDSAQGSDVSLTKDPRV。

[0123] The amino acid sequence expressed by allele SLA-3*04:05 is shown as SEQ ID NO:03-04-05 below.

[0124] SEQ ID NO:03-04-05: MGPRALFLLLSGALALTGTRAGPHSLRYFDTAVSRPDRGKPRFIEVGYVDDTQFVRFDSDAPNPRMEPRVPWIEKEGQEYWDKETENAMGSAQTFRVNLKNLRGYYNQSEAGSHTLQSMYGCDVGPDGLLLRGYSQDAYDGADYIALNEDLRSWTAADTAAQITKRKREAADAAEQMRSYLEGACVEWLQKYLQMGKDTLQHAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWDPPQPPVPIVGIIIGLVLVLVAGAVVTGVVIWRKKRSGEKGGSYTQAAGSDSAQGSDVSLTKDPRV。

[0125] The amino acid sequence expressed by allele SLA-3*05:02 is as shown in SEQ ID NO:03-05-02.

[0126] SEQ ID NO:03-05-02: MGPRALVLLLSGTLALTGTRAGPHSLRYFDTAVSRPDRGKPRFIEVGYVDDTQFVWFDSDAPNPRMEPRAPWIQQEGQEYWDRETQISKDNAQTYRVNLNNLRGYYNQSEAGSHTLQSMYGCDVGPDGLFLRGYSQFAYDGADYIALNEDLRSWTAADTAAQITKRKREAADAAEQMRSYLEGACVEGLQKYLEMGKDTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLSWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWDPPQPPVPIVGIIVGLVLVLVAGAMVAGVVIWRKKRSGEKGGSYTQAAGSDSAQGSDVSLTQDPRV。

[0127] The amino acid sequence expressed by allele SLA-3*05:07 is as shown in SEQ ID NO:03-^{}05-07.

[0128] SEQ ID NO:03-05-07: MGPRALFLLLSGTLALTGTRAGPHSLRYFDTAVSRPDRGKPRFIEVGYVDDTQFVWFDSDAPNPRMEPRAPWIEKEGQEYWDRETQISKDNAQTYRVNLNNLRGYYNQSEAGSHTLQSMYGCDVGPDGLFLRGYSQFAYDGADYIALNEDLRSWTAADTAAQITKRKREAADAAEQMRSYL EGACVEGLQKYLEMGKDTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWDPAQPPVPIVGIIVGLVLVLVAGAVVTGVVIWRKKRSGEKGGSYTQAAGSDSAQGSDVSLTKDPRV.

[0129] The amino acid sequence of the allele SLA-3*06:01 is shown in SEQ ID NO:03-06-01.

[0130] SEQ ID NO:03-06-01: MGPRALFLLLSGTLALTGTREGPHSLRYFDTAVSRPDRGKPRFISVGYVDDTQFVRFDSDAPNPRMEPRAPWIEQEGQEYWDEETRNAMGSAQTFRVNLNNLRGYYNQSEAGSHTLQSMYGCDVGPDWLFLRGYSQDAYDGADYIALNEDLRSWTAADTAAQITKRKWEAADEAEQMRSYL EGACVEWLQKYLQMGNNTLQRAEPPKTHVTRHPSSDLGVTLRCWALGFYPKEISLTWQREGQDQSQDMELVETRPSGDGTFQKWAALVVPPGEEQSYTCHVQHEGLQEPLTLRWDPPQPPVPIVGIIVGLVLVLVAGAVVAGVVIWRKKRPGEKGGSYTQAAGSDSAQGSDVSLTKDPRV.

[0131] The results showed that the amino acid sequence SEQ ID NO:2 differed significantly from other amino acid sequences at positions 17, 41, and 170. Specifically: In SEQ ID NO:2, position 17 is L, while the other amino acid sequences are R at the corresponding positions; In SEQ ID NO:2, position 41 is L, while the other amino acid sequences are P at the corresponding positions; In SEQ ID NO:2, position 170 is E, while the other amino acid sequences are K or R at the corresponding positions.

[0132] This indicates that the amino acid encoding at the aforementioned sites is a key factor affecting the efficiency of heavy chain peptides in forming complexes. The presence of the amino acid sequence shown in SEQ ID NO:1 in the SLAI heavy chain peptide gives it a broad spectrum of peptide loading capacity, allowing it to form a stable complex with β2-microglobulin in vitro after the addition of random peptides.

[0133] SEQ ID NO:1: LGDSRFIEVGYVDDTQFVRFDSDALNPRMEPRAPWIEKEGQDYWDEETRKVKETAQINRVDLKTLRGYYNQSEAGSHTLQSMFGCYLGPDGLLLRGYRQDAYDGADYIALNEDLRSWTAADTAAQITKRKWEAADEAEQWRSYLQGLCVESLRE.

[0134] The SLA I allele provided by this invention encodes amino acid sequences as shown in SEQ ID NO:1 and / or SEQ ID NO:2. The resulting heavy-chain polypeptide, upon the addition of a random peptide, can form a stable complex with β2-microglobulin in vitro, exhibiting broad-spectrum / high-frequency presentation capabilities. Based on this allele, stable "empty slot" complexes can be constructed for loading and identifying antigenic peptides, thereby achieving high-throughput discovery of high-frequency presented polypeptides.

[0135] The above description is merely an example and illustration of the structure of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the structure of the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. An SLA I allele, characterized in that, A nucleotide sequence containing any of the following amino acid sequences (a1) to (a3): (a1) The amino acid sequence shown in SEQ ID NO:1; (a2) It has ≥90% sequence identity with the amino acid sequence shown in SEQ ID NO:1, and maintains the same amino acid sequence at positions 1, 25 and 154; (a3) An amino acid sequence having 1 to 100 amino acid residues appended to the N-terminus and / or C-terminus of the amino acid sequence described in (a1) or (a2), wherein the appended sequence has an antigen peptide binding function.

2. The allele according to claim 1, characterized in that, The nucleotide sequence encodes any of the following amino acid sequences (b1) to (b3): (b1) The amino acid sequence shown in SEQ ID NO:2; (b2) An amino acid sequence that has ≥90% sequence identity with the amino acid sequence shown in SEQ ID NO:2 and retains homologous residues at the B pocket and F pocket sites; (b3) After substitution, deletion and / or addition of amino acid residues in the amino acid sequence shown in SEQ ID NO:2, the sequence has ≥80% sequence identity with the sequence shown in SEQ ID NO:2, and the same amino acid sequence is maintained at positions 17, 41 and 170.

3. An SLA I allele, characterized in that, It contains any of the following nucleotide sequences (c1) to (c2): (c1) The nucleotide sequence shown in SEQ ID NO:3; (c2) A nucleotide sequence that has ≥80% sequence identity with the nucleotide sequence shown in SEQ ID NO:3 and has the same function.

4. A polypeptide, characterized in that, Encoded by the allele as described in any one of claims 1 to 3.

5. A polypeptide, characterized in that, It contains any of the following amino acid sequences (d1) to (d4): (d1) The amino acid sequence shown in SEQ ID NO:1; (d2) A nucleotide sequence that has ≥80% sequence identity with the amino acid sequence shown in SEQ ID NO:1 and has the same function; (d3) The amino acid sequence shown in SEQ ID NO:2; (d4) A nucleotide sequence that has ≥80% sequence identity with the amino acid sequence shown in SEQ ID NO:2 and has the same function.

6. A composite, characterized in that, It comprises the polypeptide described in any one of claims 4 to 5.

7. The composite according to claim 6, characterized in that, The complex was prepared by adding the polypeptide and β2-microglobulin to a solution containing a short peptide for co-refolding.

8. The composite according to claim 7, characterized in that, The short peptide has a length of 8 to 20 amino acids.

9. A biomaterial, characterized in that, Satisfying any of the following features (e1) to (e6): (e1) A nucleic acid molecule having the alleles described in any one of claims 1 to 3; (e2) cDNA containing the nucleic acid molecule described in (e1); (e3) contains a gene for synthesizing the nucleic acid molecule described in (e1); (e4) An expression cassette containing the nucleic acid molecule described in (e1); (e5) A recombinant vector containing the nucleic acid molecule described in (e1); (e6) Recombinant microorganisms containing the nucleic acid molecules described in (e1).

10. The use of the allele as described in any one of claims 1 to 3, the polypeptide as described in any one of claims 4 to 5, the complex as described in any one of claims 6 to 8, or the biomaterial as described in claim 9 in any one or a combination of the following (f1) to (f4): (f1) Construct an MHC monomer library and / or a peptide presentation database; (f2) Screening and / or optimizing vaccine candidate peptides and / or diagnostic marker peptides; (f3) Perform immunogenicity assessment and / or epitope mapping; (f4) Preparation of MHC tetramer tool.

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