Chicken Marek's virus MHC II type molecule restriction epitope peptide, gene, primer group, screening method and application

Abstract

The invention provides a chicken Marek's virus (MHC) II type molecule restriction epitope peptide, a gene, a primer group, a screening method and application, and belongs to the technical field of immunology. The restrictive epitope peptide of the II-type molecule BLB2 * 21 of the chicken Marek's virus MHC is obtained through AF3 structure prediction and screening, the deviation of traditional bioinformatics prediction is avoided, and the binding activity of the epitope peptide is guaranteed through accurate structure analysis and crystallization verification; the gene for coding the chicken Marek's virus MHC II type molecule restrictive epitope peptide can be used for preparing Marek's virus epitope vaccines and T cell activators, or can be used as a detection target for evaluating the immune state of chicken flocks, and technical support is provided for Marek's disease prevention and control, disease resistance breeding and novel vaccine research and development.

Description

Technical Field

[0001] This invention relates to the field of immunology, and more particularly to chicken Marek's virus MHC class II restriction epitope peptides, genes, primer sets, screening methods, and applications. Background Technology

[0002] Marek's disease (MD) is caused by a herpesvirus and is the first known disease in chickens where resistance and susceptibility are determined by the MHC (metastatic conjugate markers). Despite routine vaccination, Marek's disease remains a significant burden on the poultry industry, with outbreaks continuing to spread. In fact, while current vaccines can control the occurrence of the disease, they are not effective in controlling the spread of the virus, leading to continuous mutations and increased virulence of the virus strain, thus placing higher demands on vaccines. Similar to other herpesviruses, the Marek's disease virus (MDV) has a large genome, containing over 100 genes, and a complex life cycle. Therefore, many genes may play a role at different stages of infection, tumorigenesis, and transmission, influencing the formation of resistance.

[0003] Within the MHC region, several polymorphic genes have been proposed as candidate genes for determining Marek's disease resistance. These genes include the dominantly expressed classical class I gene (BF2), the NK receptor gene (B-NK), a gene similar to mammalian lactobacillusin (BG1), and classical class II B genes (BLB1 and BLB2). Compared to MHC class I molecules, the academic community has paid less attention to chicken MHC class II molecules.

[0004] The physiological function of classic MHC class II molecules is to bind exogenous peptides and present them to the surface of antigen-presenting cells (APCs) for recognition by CD4+ T cells, thereby inducing cytokine secretion, antibody production, and regulation of the immune response. The structures of classic MHC class II molecules in humans and mice have been described in detail. Generally, MHC class II molecules are heterodimeric complexes composed of two distinct polypeptide chains (α and β chains). Each chain (α and β chains) consists of two domains: α1 and α2, and β1 and β2. α1 and β1 together form the peptide-binding groove (PBG), while two IgC1-like domains (α2 and β2) located proximal to the membrane support the base of the α1 / β1 structure. Unlike MHC class I molecules, the PBG structure of MHC class II molecules is open, allowing for more flexible binding of peptides of varying lengths, typically 13 to 25 amino acid residues. This open structural design enhances the adaptability of MHC-II molecules to antigenic peptides, enabling the presentation of a wider range of peptides. Antigenic peptides bound to MHC-II molecules typically exist in an extended polyproline II conformation, stabilized by a network of hydrogen bonds involved in the interaction between the peptide backbone and MHC-II molecule residues. Structural studies of the peptide-MHC-II (pMHC-II) complex have revealed four main peptide-binding pockets: P1, P4, P6, and P9, which play a decisive role in peptide binding. In addition, P3 and P7 pockets also contribute, collectively controlling the selective binding of antigenic peptides. The specific structure and chemical properties of these binding pockets determine the affinity of MHC-II molecules for different antigenic peptides. In particular, polymorphic residues of MHC-II molecules, located in the regions forming these binding pockets, are key factors influencing T cell epitope immunogenicity.

[0005] Currently, bioinformatics algorithms have driven the development of antigen peptide binding prediction to MHC molecules. Predictions of MHC-I class antigen peptides, using methods such as binding motifs and artificial neural networks, are highly accurate because their binding cores are mostly 9-peptides, requiring no further determination. However, prediction accuracy for MHC-II class molecules is insufficient because the lengths of their bound natural peptides vary greatly, limiting sequence alignment. While experimentally obtained quantitative matrices are effective, they are costly and lack the iterative flexibility of machine learning methods. Existing methods also largely rely on experimental data from specific alleles, while MHC-II molecule prediction requires at least 200 characteristic binding peptides. Current screening of MHC-II class antigen peptides relies on antibody IP and mass spectrometry, with only human and mouse data available. Prediction websites also have species limitations, which restricts most research. Summary of the Invention

[0006] The purpose of this invention is to provide restriction epitope peptides, genes, primer sets, screening methods, and applications for chicken Marek's virus MHC class II molecules. The restriction epitope peptide of chicken Marek's virus MHC class II molecule BLB2*21 is obtained through AF3 structure prediction screening. This avoids the bias of traditional bioinformatics predictions and ensures the binding activity of the epitope peptide through precise structural analysis and crystallization verification. The screening method and application can provide material and technical support for the prevention and control of chicken Marek's disease.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a chicken Marek's virus MHC class II restricted epitope peptide, the amino acid sequence of which is shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4.

[0008] The present invention also provides a gene encoding the restriction epitope peptide of the chicken Marek's virus MHC class II molecule, the nucleotide sequence of which is shown in SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 or SEQ ID No. 8.

[0009] The present invention also provides primer sets for amplifying the aforementioned genes, including primer set 1 for amplifying SEQ ID No. 5, primer set 2 for amplifying SEQ ID No. 6, primer set 3 for amplifying SEQ ID No. 7, or primer set 4 for amplifying SEQ ID No. 8; The primer set 1 includes an upstream primer 1 with a nucleotide sequence as shown in SEQ ID No. 9 and a downstream primer 1 with a nucleotide sequence as shown in SEQ ID No. 10; The primer set 2 includes an upstream primer 2 with a nucleotide sequence as shown in SEQ ID No. 11 and a downstream primer 2 with a nucleotide sequence as shown in SEQ ID No. 12; The primer set 3 includes an upstream primer 3 with a nucleotide sequence as shown in SEQ ID No. 13 and a downstream primer 3 with a nucleotide sequence as shown in SEQ ID No. 14; The primer set 4 includes an upstream primer 4 with a nucleotide sequence as shown in SEQ ID No. 15 and a downstream primer 4 with a nucleotide sequence as shown in SEQ ID No. 16.

[0010] The present invention also provides a method for screening complexes of restricted epitope peptides of chicken Marek's virus MHC class II molecules, comprising the following steps: (1) The gE protein of Marek's virus was truncated in 15 amino acid lengths using a step-by-step method to form a peptide library; (2) Input the sequence information of BLA with gene number AY357253, BLB2*021:01 with gene number ADO14476 and each peptide in the peptide library into AlphaFold 3 in sequence to obtain the predicted structure of the complex of each peptide in the peptide library with MHC class II molecules. (3) Comprehensively evaluate whether the pLDDT confidence value of the peptide and MHC class II molecules is >80 and the molecular interface interaction characteristics to screen out candidate epitope peptides with binding stability and structural reliability. (4) One of the candidate epitope peptides was linked to the N-terminus of the β chain of an MHC class II molecule via a flexible linker, and the recombinant plasmid BLB2*21 was synthesized. (5) Design a primer set for point mutation, use the primer set to perform PCR amplification on the remaining candidate epitope peptides, ligate the amplification product to the recombinant plasmid BLB2*21, perform homologous recombination, transform into competent cells, culture, sequence, and obtain inclusion bodies. (6) The inclusion bodies were successively refolded and purified to obtain the complex of the chicken Marek's virus MHC class II molecule restriction epitope peptide; The amino acid sequence of the flexible linker is shown in SEQ ID No. 17; All candidate epitope peptides are 15-peptides.

[0011] This invention also provides the application of the chicken Marek's virus MHC class II restricted epitope peptide in the preparation of products against chicken Marek's virus.

[0012] Preferably, the anti-Marek's virus product includes an anti-Marek's virus vaccine or a T-cell immune activator.

[0013] More preferably, the anti-Marek's virus vaccine is a vector vaccine, an epitope vaccine, or an mRNA vaccine.

[0014] The present invention also provides a chicken Marek's virus vaccine, comprising the chicken Marek's virus MHC class II molecule restriction epitope peptide.

[0015] This invention also provides the application of the chicken Marek's virus MHC class II restriction epitope peptide in the preparation of reagents for detecting the immune status of chicken Marek's virus.

[0016] This invention also provides a method for constructing recombinant plasmid BLB2*21, comprising the following steps: The 15-peptide of Marek's virus was linked to the N-terminus of the β chain of an MHC class II molecule using a flexible linker to obtain the recombinant plasmid BLB2*21. The amino acid sequence of the flexible linker is shown in SEQ ID No. 17.

[0017] The beneficial effects of this invention compared to the prior art are as follows: This invention utilizes AF3 (AlphaFold 3) to predict and construct the three-dimensional structure of complexes between chicken MHC class II molecules and candidate antigen peptides of Marek's virus. The complex binding energy and molecular interface interaction parameters are used to score the structure of candidate antigen peptides, screening for candidate epitopes with high binding potential. This invention, by using the above method to screen for restriction epitopes of the chicken Marek's virus MHC class II molecule BLB2*21, avoids the biases of traditional bioinformatics predictions and ensures the binding activity of epitopes through precise structural analysis and crystallization verification. Simultaneously, the clearly defined method for constructing prokaryotic expression plasmids for MHC class II molecules provides a stable tool for subsequent research. The four identified epitopes possess clear binding specificity and application value, providing a material and theoretical basis for the development of chicken Marek's virus epitope vaccines, the preparation of immunoassay reagents, and disease-resistant breeding, which is of great significance for the efficient prevention and control of chicken Marek's disease. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of a method for screening chicken MHC class II Marek's virus restriction epitope peptides based on AF3 structure prediction. Figure 2 The structure of the complex formed by the 19 peptides of Marek's virus predicted by AF3 and BLB2*21; Figure 3 pBLB2*21 inclusion body and BLA inclusion body with 10 core motifs; Figure 4 The results of molecular sieve purification of peptides that are weakly bound to BLB2*21; Figure 5 Results of molecular sieve gel chromatography and ion column purification of BLB2*21 strongly bound peptides (AQVLEIAAARSVNCS and VLEIAAARSVNCSAG); Figure 6Results of molecular sieve gel chromatography and ion column purification of BLB2*21 strongly bound peptides (TVLNASEQNAGIYIR and LSECRFASNAQVLEI); Figure 7 The image shows the appearance of the protein crystal of pBLB2*21 (peptide TVLNASEQNAGIYIR); Figure 8 The overall structure of pBLB2*21 (peptide is TVLNASEQNAGIYIR) is shown. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] This invention provides a chicken Marek's virus MHC class II restricted epitope peptide, the amino acid sequence of which is shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4.

[0026] In this invention, the amino acid sequence of the restricted epitope peptide of the chicken Marek's virus MHC class II molecule is: AQVLEIAAARSVNCS (SEQ ID No. 1), VLEIAAARSVNCSAG (SEQ ID No. 2), TVLNASEQNAGIYIR (SEQ ID No. 3) or LSECRFASNAQVLEI (SEQ ID No. 4).

[0027] The present invention also provides a gene encoding the restriction epitope peptide of the chicken Marek's virus MHC class II molecule, the nucleotide sequence of which is shown in SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 or SEQ ID No. 8.

[0028] In this invention, the nucleotide sequence of the gene is: GCACAGGTTCTGGAAATCGCGGCGGCACGTAGCGTTAACTGCTCC (SEQ ID No. 5), GTTCTGGAAATCGCGGCGGCACGTTCTGTTAACTGCTCTGCGGGC (SEQ ID No. 6), ACCGTTCTGAACGCGAGCGAACAGAACGCGGGTATCTACATCCGT (SEQ ID No. 7), CTGTCTGAATGCCGTTTCGCATCTAACGCGCAGGTTCTGGAAATC (SEQ ID No. 8).

[0029] The present invention also provides primer sets for amplifying the aforementioned genes, including primer set 1 for amplifying SEQ ID No. 5, primer set 2 for amplifying SEQ ID No. 6, primer set 3 for amplifying SEQ ID No. 7, or primer set 4 for amplifying SEQ ID No. 8; The primer set 1 includes an upstream primer 1 with a nucleotide sequence as shown in SEQ ID No. 9 and a downstream primer 1 with a nucleotide sequence as shown in SEQ ID No. 10; The primer set 2 includes an upstream primer 2 with a nucleotide sequence as shown in SEQ ID No. 11 and a downstream primer 2 with a nucleotide sequence as shown in SEQ ID No. 12; The primer set 3 includes an upstream primer 3 with a nucleotide sequence as shown in SEQ ID No. 13 and a downstream primer 3 with a nucleotide sequence as shown in SEQ ID No. 14; The primer set 4 includes an upstream primer 4 with a nucleotide sequence as shown in SEQ ID No. 15 and a downstream primer 4 with a nucleotide sequence as shown in SEQ ID No. 16.

[0030] In this invention, the nucleotide sequence of upstream primer 1 is AGTTCACGCTACGAGCTGCCGCGATCTCCAGTACCTGAGCCATATGTATATCTCCTTCT (SEQ ID No. 9), and the nucleotide sequence of downstream primer 1 is AGCTCGTAGCGTGAACTGCTCCTCTGGCGGTGGCAGCCTT (SEQ ID No. 10); the nucleotide sequence of upstream primer 2 is ACGTAGCGTCAACTGCTCTGCTGGTTCTGGCGGTGGCAGCCTT (SEQ ID No. 11), and the nucleotide sequence of downstream primer 2 is AGCAGTTGACGCTACGTGCCGCAGCGATCTCCAGGACCATATGTATATCTCCTTCTT (SEQ ID No. 12); the nucleotide sequence of upstream primer 3 is GAACAGAACGCAGGCATCTACATCCGCTCTGGCGGTGGCAGCCTT (SEQ ID No. 10). No. 13), the nucleotide sequence of the downstream primer 3 is ATGCCTGCGTTCTGTTCGGACGCGTTCAGAACAGTCATATGTATATCTCCTT (SEQ ID No. 14); the nucleotide sequence of the upstream primer 4 is TTTCGCTTCTAACGCTCAGGTTCTGGAGATCTCTGGCGGTGGCAGCCTT (SEQ ID No. 15); the nucleotide sequence of the downstream primer 4 is GAGCGTTAGAAGCGAAACGACACTCAGACAGCATATGTATATCTCCTT (SEQ ID No. 16).

[0031] The present invention also provides a method for screening complexes of restricted epitope peptides of chicken Marek's virus MHC class II molecules, comprising the following steps: (1) The gE protein of Marek's virus was truncated in 15 amino acid lengths using a step-by-step method to form a peptide library; (2) Input the sequence information of BLA with gene number AY357253, BLB2*021:01 with gene number ADO14476 and each peptide in the peptide library into AlphaFold 3 in sequence to obtain the predicted structure of the complex of each peptide in the peptide library with MHC class II molecules. (3) Comprehensively evaluate whether the pLDDT confidence value of the peptide and MHC class II molecules is >80 and the molecular interface interaction characteristics to screen out candidate epitope peptides with binding stability and structural reliability. (4) One of the candidate epitope peptides was linked to the N-terminus of the β chain of an MHC class II molecule via a flexible linker, and the recombinant plasmid BLB2*21 was synthesized. (5) Design a primer set for point mutation, use the primer set to perform PCR amplification on the remaining candidate epitope peptides, ligate the amplification product to the recombinant plasmid BLB2*21, perform homologous recombination, transform into competent cells, culture, sequence, and obtain inclusion bodies. (6) The inclusion bodies were successively refolded and purified to obtain the complex of the chicken Marek's virus MHC class II molecule restriction epitope peptide; The amino acid sequence of the flexible linker is shown in SEQ ID No. 17; All candidate epitope peptides are 15-peptides.

[0032] In this invention, in step (4), the candidate epitope peptide is preferably SFTIYEPCIFHPKEP.

[0033] In this invention, in step (5), the PCR amplification reaction program is as follows: 98℃ pre-denaturation for 1 min; 98℃ denaturation for 15 s, 58℃ annealing for 30 s, 72℃ extension for 4 min, this step is repeated 30 times; 72℃ extension for 5 min; storage at 8℃; the PCR amplification reaction system consists of: 25 μL 2×Phanta Max Master Mix, 1 μL upstream primer, 1 μL downstream primer, 1 μL DNA template, 22 μL ddH2O; the culture temperature is preferably 35~40℃, more preferably 36~38℃, and even more preferably 37℃; the culture time is preferably 8~12 h, more preferably 9~11 h, and even more preferably 10 h.

[0034] In this invention, in step (6), the refolding process is preferably performed using a refolding solution, which consists of 300 ml glycerol, 50 ml TRIS8.0, 1 ml EDTA, 0.5516 g oxidized glutathione and 0.9904 g reduced glutathione; the purification method is preferably chromatography, and the purification process is preferably performed by first performing molecular sieve gel chromatography and then anion exchange chromatography.

[0035] This invention also provides the application of the chicken Marek's virus MHC class II restricted epitope peptide in the preparation of products against chicken Marek's virus.

[0036] In this invention, the anti-Marek's virus product preferably includes an anti-Marek's virus vaccine or a T-cell immune activator; the anti-Marek's virus vaccine is preferably a vector vaccine, an epitope vaccine, or an mRNA vaccine.

[0037] The present invention also provides a chicken Marek's virus vaccine, comprising the chicken Marek's virus MHC class II molecule restriction epitope peptide.

[0038] This invention also provides the application of the chicken Marek's virus MHC class II restriction epitope peptide in the preparation of reagents for detecting the immune status of chicken Marek's virus.

[0039] This invention also provides a method for constructing recombinant plasmid BLB2*21, comprising the following steps: The 15-peptide of Marek's virus was linked to the N-terminus of the β chain of an MHC class II molecule using a flexible linker to obtain the recombinant plasmid BLB2*21. The amino acid sequence of the flexible linker is shown in SEQ ID No. 17.

[0040] In this invention, the amino acid sequence of the 15-peptide of Marek's virus is AQVLEIAAARSVNCS (SEQ ID No. 1), VLEIAAARSVNCSAG (SEQ ID No. 2), TVLNASEQNAGIYIR (SEQ ID No. 3), LSECRFASNAQVLEI (SEQ ID No. 4).

[0041] Example 1: AF3 Prediction Screening for Marek's Virus Peptides 1.1 Construction of MDV viral peptide library using the step-by-step method According to the article by Professor Kaufman of the University of Edinburgh (Samer H, Michael G, Stefan S, et al. The dominantly expressed class II molecule from a resistant MHC haplotype presents only a few Marek's disease virus peptides by using an unprecedented binding motif.[J].PLoS biology,2021,19(4):e3001057-e3001057.DOI:10.1371 / JOURNAL.PBIO.3001057.), it is known that the gE protein of Marek's virus presents a large number of antigenic peptides. Based on this, we used the stepwise method to extract the amino acid sequence of the gE protein of Marek's virus into peptides of 15 amino acid lengths to form a peptide library.

[0042] 1.2 AF3 Prediction Structure The sequence information of each peptide from BLA (GenBank: AY357253), BLB2*021:01 (GenBank: ADO14476), and the peptide library was sequentially input into AF3 to obtain the predicted structure of the complex of each peptide in the peptide library with MHC class II molecules. Based on the results of AF3, the pLDDT value scoring system for MHC class II molecule binding peptides, i.e., pLDDT value (>80), and molecular interface interaction characteristics (number of hydrogen bonds ≥5, interface area ≥800Å) were used. 2 A comprehensive evaluation was conducted using peptides with binding free energy ≤ -10 kcal / mol to obtain candidate epitope peptides, core motifs, and their 15 peptides related to BLB2*21, exhibiting high binding stability and structural reliability, as shown in Table 1. The predicted three-dimensional structure of the AF3 complex is shown below. Figure 2 As shown.

[0043] Table 1. Viral peptides, core motifs, and their 15-peptide amino acid sequences predicted by AF to bind to BLB2*21.

[0044] Example 2: Construction of expression vector and prokaryotic expression 2.1 Construction of BLB2*21 plasmid One of the Marek's virus 15 peptides (SFTIYEPCIFHPKEP) with a high AF3 prediction score was linked to the nitrogen terminus of the β chain of an MHC-II molecule (BLB2*021:01(GenBank:ADO14476)) via a linker (SGGGSLVPRGSGGGGS(SEQ ID No.17)), and the sequence was given to Nanjing GenScript Biotech Co., Ltd. to synthesize plasmid BLB2*21.

[0045] 2.2 Primer Design The sequence of the BLB2*21 recombinant plasmid synthesized by Nanjing GenScript Biotech Co., Ltd. is as follows: >BLB2*021:01-SFTIYEPCIFHPKEP SFTIYEPCIFHPKEPSSGGGSLVPRGSGGGGSTRPSAFFFYGKIGECHYLNGTERVRFLDRQIYNRQQFAHFDSDVGKFVADTPLGERQAEYWNSNAELLENLMNEVDRVCRHNYGI LESFTVQRSVEPKVRVSALQSGSLPETDRLACYVTGFYPPEIEVKWFLNGREETERVVSTDVMQNGDWTYQVLVVLETVPRRGDSYVCRVEHASLRQPISQAWEPPADAGRSK (SEQ ID No.45) Based on the BLB2*021:01-SFTIYEPCIFHPKEP recombinant plasmid synthesized by GenScript, primers for the remaining 9 viral 15 peptides with high AF3 scores were designed using the online single-point mutation tool on the Novizan website (https: / / crm.vazyme.com / cetool / singlepoint.html). The nucleotide sequences of the 15 peptides are shown in Table 2, and the primer sequences are shown in Table 3.

[0046] Table 2. Nucleotide sequences of BLB2*21 viral peptides

[0047] Table 3 Primer sequences of BLB2*21 viral peptide

[0048] Table 4 PCR reaction system

[0049] Table 5 PCR reaction procedure

[0050] 2.3 Glue Recycling and Homologous Recombination The target gene was gel-recovered using an Axygen gel recovery kit, and the gel-recovered product was then subjected to homologous recombination.

[0051] Table 6 Homologous recombination reaction system

[0052] After mixing the above substances, heat in a 37°C metal bath for 30 minutes, then in ice water for 5 minutes.

[0053] 2.4 Transformation Thaw competent BL21-DE3 cells (purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.) in an ice box for 5 minutes. In a clean bench, add 10 μL of homologous recombination plasmid to 50 μL of competent cells, mix thoroughly by pipetting, incubate on ice for 30 minutes, heat shock at 42°C for 45 seconds, and incubate on ice for 5 minutes. Do not shake the centrifuge tubes during this process. Add 1 ml of antibiotic-free LB medium (purchased from Beijing Solarbio Science & Technology Co., Ltd.) to each tube, mix well, and incubate at 37°C and 200 rpm on a shaker. After 1 hour, centrifuge the shaken bacterial culture, discard 800 μL of the supernatant, and streak 200 μL on an ampicillin-resistant plate (purchased from Beijing Solarbio Science & Technology Co., Ltd.). Incubate overnight at 37°C with the plate inverted. Pick single colonies and place them in ampicillin-resistant LB medium. Shake at 170 rpm for 10 hours. Take 200 μL for sequencing. Inoculate the successfully sequenced bacterial culture into a 1 L Erlenmeyer flask for expansion culture. Add IPTG after 2 hours. After 500 minutes, collect the bacterial culture and sonicate to disrupt it, and extract inclusion bodies.

[0054] 2.5 Inclusion bodies Centrifuge the sonicated bacterial culture, discard the supernatant, scrape off the bacterial skin, and centrifuge three times at 4°C, 6500 rpm, 10 min with 25 mL of washing buffer (as shown in Table 7). Resuspend the culture in 25 mL of resuspension buffer (as shown in Table 8) with 25 μL of DTT, and add 30 μL of the resuspension buffer to 10 μL of protein loading buffer (purchased from Beijing Solarbio Science & Technology Co., Ltd.) for water bath denaturation. Perform SDS-PAGE verification. Centrifuge the remaining liquid at 4°C, 6500 rpm, 10 min, discard the supernatant, weigh the net weight of the inclusion bodies, and calculate the amount of denaturing buffer required to dissolve the inclusion bodies (as shown in Table 9). Adjust the final concentration to 30 mg / mL and stir overnight with a magnetic stirrer. Store at -20°C. The verification results are as follows: Figure 3 As shown.

[0055] Table 7 Washing Liquid System

[0056] Table 8 Resuspension System

[0057] Table 9 Denaturing Liquid System

[0058] Example 3 Protein refolding and purification 3.1 Molecular refolding of the BLB2*21 complex The inclusion bodies α and β chains of the BLB2*21 complex molecule were added to the refolding solution in the same proportion and refolded for 7 days.

[0059] Table 10 Refolding Liquid System

[0060] 3.2 BLB2*21 Molecular Concentration Pour the refolded liquid into a concentration vessel and concentrate it to approximately 20 ml at 4°C. Then add 60 ml of molecular sieve replacement solution and continue concentrating until it reaches 15 ml. Transfer the concentrated liquid to a 15 ml centrifuge tube and centrifuge at 7000 rpm for 30 min at 4°C. Pour the centrifuged liquid into a 10 kDa ultrafiltration tube and centrifuge again to approximately 1 ml. Centrifuge once more, collect the supernatant, filter it through a 0.22 μm filter, and store it for later use.

[0061] 3.3 Purification of BLB2*21 molecules 3.3.1 Molecular sieve gel chromatography Turn on the purification instrument, place the steel ball into the molecular sieve, connect the molecular sieve chromatography column, name the file, set the parameters: flow rate 1 ml / min, pressure 0.6 MPa, sample peak collection 1 mL, peak collection start UV value 20 mAU, inject the sample into the loading loop and load the target protein so that the peak position is approximately 80 ml, collect the target protein, and perform SDS-PAGE verification. Results are as follows: Figure 4 , Figure 5 A, 5B and Figure 6 As shown in A and 6B.

[0062] The results showed that 4 out of 10 viral peptides were strongly bound.

[0063] 3.3.2 Anion Exchange Chromatography The collected target protein from the previous step was concentrated to approximately 1 ml using a 10 kDa ultrafiltration tube and set aside. The purification instrument was turned on, and pumps A and B were inserted into solutions A and B, respectively. Both solutions A and B contained Tris-HCl pH 8.0 and NaCl, but solution A contained 0.5844 g of NaCl, while solution B contained 58.44 g of NaCl. An anion exchange chromatography column was installed, and the parameters were set as follows: flow rate 2 mL / min, alarm pressure 0.3 MPa, sample peak collection 1 mL, peak collection start UV value 10 mAU, 50% B pump for 40 min. The sample was then injected into the loading loop for loading. When the pump was at 50% B, it was switched to 100% B pump for 0 min. After the appearance of the contaminating protein peak, the pump was switched to 100% A pump for 20 min. The target protein appeared at approximately a conductivity of 19. The protein was collected and validated by SDS-PAGE. The results are as follows. Figure 5 C, 5D and Figure 6 As shown in C and 6D.

[0064] The results showed that AQVLEIAAARSVNCS, VLEIAAARSVNCSAG, TVLNASEQNAGIYIR, and LSECRFASNAQVLEI could form relatively stable MHC II-peptide complexes with BLB2*021.

[0065] Example 4: Crystal Structure Verification 4.1 Initial screening of protein crystals The four pBLB2*021 complex proteins obtained by molecular sieve gel chromatography and anion exchange chromatography were concentrated to below 200 μL using a 10 kDa ultrafiltration tube. The proteins were then replaced with molecular sieves 2-3 times, and the protein concentrations were measured. The solutions were diluted to 4 mg / mL and 8 mg / mL. Crystallization conditions were screened using a crystallization kit (purchased from Hamptom). The screening conditions were: Customized-H084-PEGRx 2-39 (10% v / v Polyetnylene glycol 200, 0.1M BIS-TRIS propane pH 9.0, 18% w / v Polyethylene glycol 8000).

[0066] 4.2 Optimization of protein crystallization conditions The precipitant, protein concentration, and temperature were optimized based on the initial screening conditions. Specifically, the protein concentration was set to 2, 4, 6, 8, 10, and 12 mg / mL; the temperature was set to 8℃ and 18℃, or the precipitant concentration was serially diluted. Observations were recorded weekly, and one pBLB2*021 complex (TVLNASEQNAGIYIR) crystal was obtained. The optimal protein concentration was determined to be 18℃ and 6 mg / mL. The ideal conditions for pBL2*021:01 complex crystal growth were: Customized-H084-PEGRx 2-39 (10% v / v Polyetnyleneglycol 200, 0.1M BIS-TRIS propane, pH 9.0, 18% w / v Polyethylene glycol 8000). Under these conditions, the crystal appearance was as follows. Figure 7 As shown in Figure A. Figure 7 B and 7C represent the preliminary crystal appearances of the pBLB2*021 complex (VLEIAAARSVNCSAG) and the pBLB2*021 complex (LSECRFASNAQVLEI), but their structures have not yet been resolved and require further optimization. The pBLB2*021 complex (AQVLEIAAARSVNCS) has not yet been crystallized.

[0067] 4.3 Protein crystal diffraction Crystals with good appearance and growth were selected and subjected to diffraction at the Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences. The diffraction data were analyzed using CCP4 and COOT software to obtain the structure of the pBLB2*021 complex protein (TVLNASEQNAGIYIR), as shown below. Figure 8 As shown.

[0068] As can be seen from the above embodiments, the present invention provides chicken Marek's virus MHC class II molecule restriction epitope peptides, genes, primer sets, screening methods, and applications. The present invention constructs the three-dimensional structure of the complex between chicken MHC class II molecule BLB2*21 and chicken Marek's virus candidate antigen peptides by predicting AF3, and simulates the spatial conformation of the two binding. Based on parameters such as complex binding energy and molecular interface interactions (such as hydrogen bonds and hydrophobic interactions), the candidate antigen peptides are structurally scored, and candidate epitope peptides with high binding potential are screened.

[0069] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Marek's disease virus MHC class II molecule-restricted epitope peptide, characterized in that, The amino acid sequence of the chicken Marek's disease virus MHC class II molecule-restricted epitope peptide is shown as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, or SEQ ID No.

4.

2. A gene encoding the Marek's disease virus of chicken MHC class II molecule restricted epitope peptide according to claim 1, characterized in that, The nucleotide sequence of the gene is shown as SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, or SEQ ID No.

8.

3. A primer set for amplifying the gene of claim 2, characterized in that, The primer set 1 for amplifying SEQ ID No. 5, the primer set 2 for amplifying SEQ ID No. 6, the primer set 3 for amplifying SEQ ID No. 7, or the primer set 4 for amplifying SEQ ID No. 8; The primer set 1 comprises an upstream primer 1 with a nucleotide sequence shown as SEQ ID No. 9 and a downstream primer 1 with a nucleotide sequence shown as SEQ ID No. 10; The primer set 2 comprises an upstream primer 2 with a nucleotide sequence shown as SEQ ID No. 11 and a downstream primer 2 with a nucleotide sequence shown as SEQ ID No. 12; The primer set 3 comprises an upstream primer 3 with a nucleotide sequence shown as SEQ ID No. 13 and a downstream primer 3 with a nucleotide sequence shown as SEQ ID No. 14; The primer set 4 comprises an upstream primer 4 with a nucleotide sequence shown as SEQ ID No. 15 and a downstream primer 4 with a nucleotide sequence shown as SEQ ID No.

16.

4. A method of screening for a complex of the Marek's disease virus MHC class II molecule-restricted epitope peptide according to claim 1, characterized by, The method comprises the following steps: (1) truncating the gE protein of Marek's disease virus by the walking method at a length of 15 amino acids to form a peptide library; (2) inputting the sequence information of BLA with gene number AY357253, BLB2*021:01 with gene number ADO14476, and each peptide in the peptide library into AlphaFold 3 in sequence to obtain the predicted structure of the complex of each peptide in the peptide library and the MHC class II molecule; (3) comprehensively evaluating whether the pLDDT confidence value of the peptide and the MHC class II molecule is >80 and the molecular interface interaction characteristics to screen out candidate epitope peptides with binding stability and structural reliability; (4) connecting one of the candidate epitope peptides to the N-terminus of the β chain of the MHC class II molecule through a flexible linker to synthesize and obtain a recombinant plasmid BLB2*21; (5) designing a primer set for point mutation, using the primer set to perform PCR amplification on the remaining candidate epitope peptides, connecting the amplification products to the recombinant plasmid BLB2*21, homologously recombining, transforming into competent cells, culturing, and sequencing to obtain inclusion bodies; (6) sequentially subjecting the inclusion bodies to renaturation and purification treatment to obtain the complex of the chicken Marek's disease virus MHC class II molecule-restricted epitope peptide; The amino acid sequence of the flexible linker is shown as SEQ ID No. 17; The candidate epitope peptides are all 15 peptides.

5. The chicken Marek's disease virus MHC class II molecule-restricted epitope peptide in the preparation of an anti-chicken Marek's disease virus product according to claim 1.

6. Use according to claim 5, characterized in that, The anti-chicken Marek's disease virus product includes an anti-chicken Marek's disease virus vaccine or a T cell immune activator.

7. Use according to claim 6, characterized in that, The anti-chicken Marek's disease virus vaccine is a vector vaccine, an epitope vaccine or an mRNA vaccine.

8. A vaccine against Marek's virus in chickens, characterized in that, The chicken Marek's disease virus MHC class II molecule-restricted epitope peptide of claim 1.

9. Use of the chicken Marek's disease virus MHC class II molecule-restricted epitope peptide of claim 1 in the preparation of a reagent for detecting the immune status of a chicken Marek's disease virus.

10. A method of constructing a recombinant plasmid BLB2*21, characterized by, The method comprises the following steps: The 15-peptide of the Marek's disease virus is connected to the N-terminal of the MHC class II molecule beta chain through a flexible linker to obtain the recombinant plasmid BLB2*21. The amino acid sequence of the flexible linker is shown in SEQ ID No. 17.

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