CN116355057A - Thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen and application thereof - Google Patents
Thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen and application thereof Download PDFInfo
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Abstract
The invention belongs to the fields of medical immunology and infectious pathology, and particularly relates to a thymus-dependent lymphocyte epitope peptide of 189 hepatitis B virus antigens and application thereof, wherein the epitope peptide is an antigen peptide limited by molecules of HLA-A0201, HLA-A1101, HLA-A2402, HLA-A0207, HLA-A3101, HLA-A0206, HLA-A3303, HLA-A3001, HLA-A0203, HLA-A1102, HLA-A0301, HLA-A2601 and HLA-A0101, can be specifically combined with cytotoxic thymus-dependent lymphocytes, and can stimulate the activation, proliferation and differentiation of the later, thereby playing an immune effect of resisting hepatitis B virus; the antigen peptide can be used for preparing therapeutic and preventive vaccines for hepatitis B virus infection, can also be used for preparing detection reagents for detecting hepatitis B virus specific cytotoxic thymus dependent lymphocytes, and has potential application value in prevention, treatment and diagnosis of hepatitis B.
Description
The scheme is a division of cases with application number 2019112846358 and application date 2019.12.13 aiming at thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen and application thereof.
Technical Field
The invention belongs to the fields of medical immunology and infectious diseases, and particularly relates to a thymus-dependent lymphocyte antigen epitope peptide of 189 hepatitis B virus antigens and application thereof.
Background
Hepatitis b virus (Hepatitis B virus, HBV) infection is one of the most common infectious diseases, severely endangering human health. Statistics of the world health organization, 2015, 7 months, show that 1/3 of the world population is expected to be infected with HBV, about 2.48 hundred million HBV carriers, and later may develop cirrhosis and primary hepatocellular carcinoma. About 78 tens of thousands of HBV infected patients die annually due to disease progression to cirrhosis, liver failure or liver cancer. Epidemiological investigation shows that about 1.2 hundred million people are HBV carriers in China, accounting for half of the total chronic infections. In addition, china is a large country of liver cancer, and accounts for more than 55% of the global liver cancer population.
HBV genome consists of incomplete double-stranded circular DNA, long as negative strand and 3020 to 3320 bases, short as positive strand and 1700 to 2800 bases. Four Open Reading Frames (ORFs), designated C, X, P and S coding regions, respectively, are included. The C region is composed of Pre-C and C genes which are regulated by different initial codons and end at the same stop codon, the Pre-C and C genes jointly code Pre-C protein, and the Pre-C protein is cut to form e antigen (HBeAg). The C gene encodes the viral capsid protein, HBcAg. The S region consists of the S gene, preS1 and PreS2 genes, and is initiated by 3 different start codons, and the translation is terminated at the same stop codon, and the surface antigen (HBsAg) protein, preS1 protein and PreS2 protein are respectively encoded. The P region gene encodes the polymerase protein (HBpol) of HBV. The gene in region X encodes protein X (HBx), which contains 154 amino acids and is the smallest open reading frame.
Cytotoxic T cells (Cytotoxic T lymphocyte, CTL) are core cells mediating adaptive immune responses and play a vital role in the development of anti-infective, anti-neoplastic and hypersensitivity reactions and autoimmune diseases, and their T Cell Receptors (TCR) on their cell membranes are capable of specifically recognizing and binding to complexes of antigen presenting cell surface MHC class I molecules with antigen peptides, i.e. MHC/antigen peptide complex molecules. CTL epitopes refer to antigenic peptides bound to MHC class I molecules, which are linear fragments or spatially conformational structures of the antigenic molecules that can be specifically recognized by TCRs, are the basic antigenic units that elicit an immune response and play a critical role in CTL activation.
The MHC system refers to the major histocompatibility complex (major histocompatibility complex, MHC), a group of closely linked gene groups in the vertebrate genome that encode molecules that express MHC class I and class II proteins. HLA (human leukocyte antigen) is the MHC system of humans, the most complex gene group in humans, and is highly polymorphic in the human population. HLA plays an important role in the immune process of organisms such as antigen recognition, antigen presentation and the like, and is a main factor influencing the immune response of human bodies. HLA class I molecules are mainly responsible for the presentation of endogenous HBV antigens to CD8 + CTL, activated CTLs apoptosis virus-infected hepatocytes by secreting perforin and granzyme, etc., while secreting specific cytokines to inhibit HBV replication. Thus, HBV antigen-specific CD8 was monitored dynamically + The number and the function of the T cells can accurately reflect the specific immune function state of the hepatitis B infected person aiming at HBV. Due to different HLA molecular types of different peopleIts processing, handling and presentation capabilities for different HBV antigens are also different, thus eliciting different degrees of HBV antigen-specific immune response. According to different HLA molecular types of hepatitis B patients, HBV specific antigen peptide presented by the HBV specific antigen peptide is selected, and the specificity of the HBV antigen peptide is dynamically monitored + The number and the function of the T cells are of great significance to the disease process monitoring, diagnosis and treatment scheme formulation, curative effect observation, prognosis and prognosis, and the like of HBV infected persons, and are important technical means for realizing accurate medical treatment of hepatitis B. Meanwhile, polypeptide vaccines or gene vaccines can be prepared by utilizing HBV specific antigen peptides combined with the HLA-A molecules with high affinity, so that HBV infection can be prevented and treated.
However, currently, there are few clear HBV antigen peptides which are presented by various HLA molecules and can stimulate organisms to cause T cell response, so that the development of specific T cell detection on HBV patients carrying different HLA alleles is limited, the research on the action of HBV specific T cells in the development of HBV is also limited, and the personalized detection and accurate immunotherapy based on individual differences of HLA genes and the presented HBV antigen peptide differences are further limited.
Disclosure of Invention
The invention solves the technical problems in the prior art and provides the thymic dependent lymphocyte antigen epitope peptide of the hepatitis B virus antigen and the application thereof.
In order to solve the problems, the technical scheme of the invention is as follows:
the thymus-dependent lymphocyte antigen epitope peptide of the hepatitis B virus antigen has an amino acid sequence of any one of epitope peptide sequences shown as the following:
as can be seen from the above table, the above sequences are the antigen peptide sequences of hepatitis B virus e antigen HBeAg, surface antigen HBsAg, polymerase protein HBpol or X protein (HBx), respectively, and bind with high affinity to HLA-A0201, A1101, A2402, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101, A2601 molecules, respectively, to constitute MHC/antigen peptide complex molecules on the surface of antigen presenting cells, and CD8 specific for antigen peptide + The T cell clone combines to stimulate the activation, proliferation and differentiation of the T cell clone, and plays an immune effect role of resisting hepatitis B virus.
The thymus-dependent lymphocyte antigen epitope peptide sequence of the hepatitis B virus antigen can be used for preparing hepatitis B polypeptide vaccine or gene vaccine: preparation of polypeptide vaccine: the antigen epitope peptide sequence of the invention synthesizes one or more antigen peptides artificially, and is mixed with adjuvant to prepare soluble preparation, or is loaded by biological nanometer material to prepare nanometer polypeptide vaccine, which is injected into hepatitis B virus infected person or hepatitis B patient to excite the specific T cell activation and proliferation of hepatitis B virus antigen of patient and enhance the activity of tumor killing cell, thus preparing hepatitis B polypeptide vaccine. Gene vaccine preparation: according to the polypeptide sequence of the invention, a recombinant DNA gene fragment, recombinant plasmid or recombinant viral vector of one or more polypeptides is constructed, and is injected into the body of a hepatitis B virus infected person or a hepatitis B patient, so that the recombinant gene expresses one or more polypeptides in the body, activates and proliferates the specific T cells of the hepatitis B virus antigen, and enhances the activity of killing virus infected cells, thereby preparing the hepatitis B gene vaccine.
The thymus-dependent lymphocyte antigen epitope peptide sequence of the hepatitis B virus antigen can be used for preparing a detection preparation or a kit for detecting the hepatitis B virus antigen specific T cells: according to the epitope peptide sequence of the invention, one or more antigen peptides are synthesized artificially, and used as antigen preparations in an enzyme-linked immunosorbent assay, an intracellular cytokine fluorescent staining method and an enzyme-linked immunosorbent assay, and are mixed with Peripheral Blood Mononuclear Cells (PBMC) of a patient for culture to stimulate activation, proliferation and secretion of cytokines of hepatitis B virus antigen-specific T cells, and then the synthesis amount of the cytokines is detected by other combined reagents, so that the number and reactivity of the specific T cells are reacted; the polypeptide can also be used for preparing a complex (peptide-HLA complex) of human leukocyte antigen and polypeptide and a polymer thereof by using a genetic engineering technology and a protein engineering technology, and further preparing a fluorescein-labeled preparation, and detecting the number of hepatitis B virus antigen specific T cells in a peripheral blood PBMC cell population of a patient by using a flow cytometry method. The related kit is a hepatitis B virus antigen specific T cell detection kit assembled by the preparation and other reagents commonly used in different detection methods.
The thymus-dependent lymphocyte antigen epitope peptide sequence of the hepatitis B virus antigen can be used for preparing medicines for treating hepatitis B: the polypeptide vaccine or gene vaccine based on the antigen epitope peptide sequence of the invention is combined with other immunotherapeutic preparations or chemotherapeutic preparations to prepare the clinical medicine for treating hepatitis B.
Six HBV specific epitope peptide sequences limited by an online epitope prediction database are utilized to virtually predict the HLA-A molecule, a group of HBV specific epitope peptide sequences which can be combined with the HLA-A0201, A1101, A2402, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101 and A2601 molecules respectively in high affinity are obtained, and then the immunogenicity of the HBV specific epitope peptide sequences is verified through ELISPOT functional experiments, so that specific antigen peptides are provided for preparing therapeutic and prophylactic vaccines of HBV infection, developing HBV antigen specific T cell detection reagents and the like.
1. The method comprises the steps of selecting hepatitis B virus e antigen (HBeAg, including the sequence of HBcAg), surface antigen (HBsAg), polymerase protein (HBpol) and X protein (HBx) amino acid sequences as targeting sequences;
2. selection prediction results six commonly used epitope prediction databases with higher accuracy, acknowledged by researchers, were obtained: SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC and EPIJEN predict HBV specific epitope peptide sequences restricted by the two HLA-A molecules;
3. and carrying out integration analysis on the predicted results of the six online epitope predicted websites according to a certain prediction standard to obtain candidate antigen peptide sequences with more consistent predicted results of the six websites.
4. The immunogenicity of HBV epitope peptides was verified by IFN-gamma ELISPOT cell functional experiments.
The invention relates to an antigen peptide sequence which can be combined with HLA-A0201, A1101, A2402, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101 and A2601 molecules respectively in hepatitis B virus e antigen (HBeAg, including the sequence of HBcAg), surface antigen (HBsAg), polymerase protein (HBpol) and X protein (HBx) with high affinity and immunogenicity; also relates to the hepatitis B polypeptide vaccine, gene vaccine and hepatitis B treatment and prevention method based on the antigen peptide, and the reagent and method for detecting the hepatitis B virus antigen specific T cell based on the antigen peptide.
The advantages of the present invention over the prior art are as follows,
the HLA-A0201, A1101, A2402, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101 and A2601 molecular restriction HBV specific epitope peptide obtained by on-line virtual prediction and functional experiment verification has not been reported before. These HLA-A molecules have not previously been reported to have a limiting HBV antigenic peptide. Therefore, the new epitope peptide sequences provide required key antigen components, namely epitope peptide sequences, for developing therapeutic and prophylactic polypeptide vaccines and gene vaccines for hepatitis B, designing reagents and methods for detecting hepatitis B virus antigen specific T cells and the like; meanwhile, the epitope peptides also provide key antigen components for the individual detection and accurate medical treatment of hepatitis B patients aiming at the specific HLA-A alleles.
Drawings
FIG. 1 shows HLA-A0201 molecule limited HBV antigen T cell epitope peptide; (A) IFN-gamma ELISPOT method for identifying HLA-A0201 molecule restriction HBV antigen T cell epitope peptide detection hole, negative control hole and positive control Kong Ban dot pattern; (B) a statistical plot of spot numbers of the test wells and the negative control wells; (C) A statistical plot of the spot number ratio (P/N) of the test wells to the negative control wells;
FIG. 2 shows HLA-A1101 molecular restricted HBV antigen T cell epitope peptide; (A) IFN-gamma ELISPOT method for identifying HLA-A1101 molecule restriction HBV antigen T cell epitope peptide detection well, negative control well and positive control Kong Ban dot pattern; (B) a statistical plot of spot numbers of the test wells and the negative control wells; (C) A statistical plot of the spot number ratio (P/N) of the test wells to the negative control wells;
FIG. 3 shows HLA-A2402 molecule restricted HBV antigen T cell epitope peptides; (A) IFN-gamma ELISPOT method for identifying HLA-A2402 molecule restricted HBV antigen T cell epitope peptide detection well, negative control well and positive control Kong Ban dot pattern; (B) a statistical plot of spot numbers of the test wells and the negative control wells; (C) A statistical plot of the spot number ratio (P/N) of the test wells to the negative control wells;
FIG. 4 is a schematic diagram of HLA-A3101 molecule-restricted HBV antigen T cell epitope peptide; (A) IFN-gamma ELISPOT method for identifying HLA-A3101 molecule restricted HBV antigen T cell epitope peptide detection well, negative control well and positive control Kong Ban dot pattern; (B) a statistical plot of spot numbers of the test wells and the negative control wells; (C) A statistical plot of the spot number ratio (P/N) of the test wells to the negative control wells;
FIG. 5 shows HLA-A0206 molecule limited HBV antigen T cell epitope peptide; (A) IFN-gamma ELISPOT method for identifying HLA-A0206 molecule restriction HBV antigen T cell epitope peptide detection hole, negative control hole and positive control Kong Ban dot pattern; (B) a statistical plot of spot numbers of the test wells and the negative control wells; (C) A statistical plot of the spot number ratio (P/N) of the test wells to the negative control wells;
FIG. 6 shows HLA-A0207 molecule limited HBV antigen T cell epitope peptide; (A) IFN-gamma ELISPOT method for identifying HLA-A0207 molecule restriction HBV antigen T cell epitope peptide detection hole, negative control hole and positive control Kong Ban dot pattern; (B) a statistical plot of spot numbers of the test wells and the negative control wells; (C) A statistical plot of the spot number ratio (P/N) of the test wells to the negative control wells;
FIG. 7 is a HLA-A3303 molecule-restricted HBV antigen T cell epitope peptide; (A) IFN-gamma ELISPOT method for identifying HLA-A3303 molecule restricted HBV antigen T cell epitope peptide detection well, negative control well and positive control Kong Ban dot pattern; (B) a statistical plot of spot numbers of the test wells and the negative control wells; (C) A statistical plot of the spot number ratio (P/N) of the test wells to the negative control wells;
FIG. 8 shows HLA-A3001 molecule-restricted HBV antigen T cell epitope peptides; (A) IFN-gamma ELISPOT method for identifying HLA-A3001 molecule restricted HBV antigen T cell epitope peptide detection well, negative control well and positive control Kong Ban dot pattern; (B) a statistical plot of spot numbers of the test wells and the negative control wells; (C) A statistical plot of the spot number ratio (P/N) of the test wells to the negative control wells;
FIG. 9 shows HLA-A0203 molecule limited HBV antigen T cell epitope peptide; (A) IFN-gamma ELISPOT method for identifying HLA-A0203 molecule restriction HBV antigen T cell epitope peptide detection hole, negative control hole and positive control Kong Ban dot pattern; (B) a statistical plot of spot numbers of the test wells and the negative control wells; (C) A statistical plot of the spot number ratio (P/N) of the test wells to the negative control wells;
FIG. 10 is a schematic of HLA-A1102 molecule-restricted HBV antigen T cell epitope peptide; (A) IFN-gamma ELISPOT method for identifying HLA-A1102 molecule restricted HBV antigen T cell epitope peptide detection well, negative control well and positive control Kong Ban dot pattern; (B) a statistical plot of spot numbers of the test wells and the negative control wells; (C) A statistical plot of the spot number ratio (P/N) of the test wells to the negative control wells;
FIG. 11 is HLA-A0301 molecule-restricted HBV antigen T cell epitope peptide; (A) The IFN-gamma ELISPOT method identifies the detection wells, negative control wells and positive control Kong Ban dot patterns of HLA-A0301 molecule-restricted HBV antigen T cell epitope peptide; (B) a statistical plot of spot numbers of the test wells and the negative control wells; (C) A statistical plot of the spot number ratio (P/N) of the test wells to the negative control wells;
FIG. 12 shows HLA-A0101 molecule-restricted HBV antigen T cell epitope peptides; (A) IFN-gamma ELISPOT method for identifying HLA-A0101 molecule restriction HBV antigen T cell epitope peptide detection well, negative control well and positive control Kong Ban dot pattern; (B) a statistical plot of spot numbers of the test wells and the negative control wells; (C) A statistical plot of the spot number ratio (P/N) of the test wells to the negative control wells;
FIG. 13 is a schematic diagram of HLA-A2601 molecule restricted HBV antigen T cell epitope peptide; (A) IFN-gamma ELISPOT method for identifying HLA-A2601 molecule restricted HBV antigen T cell epitope peptide detection well, negative control well and positive control Kong Ban dot pattern; (B) a statistical plot of spot numbers of the test wells and the negative control wells; (C) A statistical plot of the spot number ratio (P/N) of the test wells to the negative control wells;
FIG. 14 is a experimental technical scheme for verifying the immunogenicity of epitope peptides to be identified in example 1.
Detailed Description
Example 1:
the HLA-A0201, A1101, A2402, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101 and A2601 molecular restriction HBV specific antigen peptide provided by the invention, and the sequence is screened and identified by the following steps:
1. online virtual prediction of dominant T cell epitope peptides of 13 HLA-A molecule restricted 4 HBV antigens
4 HBV proteins were selected: surface antigen (HBsAg), e antigen (HBeAg, including HBcAg sequence), DNA polymerase (HBpol) and X protein (HBx), and obtaining amino acid sequence by searching UniProt global protein resource database, and selecting HBV C type HBV protein sequence with most Chinese infection; performing virtual prediction on T cell epitope peptides which are limited by HLA-A0201, A1101, A2402, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101 and A2601 molecules and aim at each HBV protein through six commonly used epitope peptide prediction databases such as SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC, EPIJEN, wherein the SVMHC database comprises two prediction algorithms of MHCPEP model and SYFEPITHI model; the IEDB database included three methods, ANN, SMM and ARB, and the experiment selected the ANN and SMM methods with better statistical significance. These epitope peptide predictive database websites are shown in table 1.
The antigen binding groove of HLA class I molecules is closed at both ends and the received antigenic peptide is 8-11 amino acid residues in length, with 9 and 10 amino acids being the most common, so that polypeptides 9 and 10 amino acids in length are selected as subjects for this experiment, respectively. The amino acid sequence of each HBV protein is respectively input into the corresponding amino acid sequence input frame of the forecast database website, the length of the epitope peptide is respectively selected to be 9 and 10 amino acids, then a specific HLA-A molecule is selected, and the T cell epitope peptide of HBV antigen is virtually forecast on line.
Table 1 epitope peptide predictive database website
The predicted 9 peptide and 10 peptide of different databases are respectively arranged according to the scores from high to low for each HLA-A molecule and each HBV protein, and then the epitope peptide which at least meets the scoring standards of more than two prediction methods is selected as the candidate epitope peptide. For each HLA-A molecule, for each HBV protein, the highest scoring (highest affinity) 1-4 polypeptides are selected from the candidate epitope peptides as epitope peptides to be identified.
2. Isolation of peripheral blood PBMC from HBV chronically infected patients
Taking fresh anticoagulated whole blood stored at room temperature, and properly diluting the fresh anticoagulated whole blood with sterile PBS; adding 1 times of human lymphocyte separation liquid (Daidae is biological and Shenzhen) into a 15mL centrifuge tube; slowly spreading diluted blood on the separating liquid, and centrifuging at room temperature for 20min at 2500rpm; sucking up mononuclear cell (PBMCs) layers, and centrifugally washing for 2 times; resuspension with serum-free medium (Daidae organism, shenzhen), cell count, and cell concentration adjustment to 2×10 6 prepare/mL for use.
3. Identification of immunogenicity of HBV antigen T cell epitope peptides by IFN-gamma ELISPOT method
The technical route is shown in fig. 14:
first, HBV chronically infected persons who have positive reaction to the mixed peptide group are screened: mixing each HLA-A molecule limited epitope peptide to be identified for 4 HBV proteins into a group of 8-9 epitope peptides each; ELISPOT plates (Daidae, shenzhen) pre-coated with anti-human IFN-gamma antibodies were activated with serum-free medium (200. Mu.L/well) for 8min, and PBMC suspension (100. Mu.L/well) was added to each well of HBV chronic infected patients; then, adding mixed peptide limited by each HLA-A molecule into a detection hole, wherein the single polypeptide in the mixed peptide is 30 mug/mL, adding PHA (2.5 mug/mL) into a positive control hole, and adding DMSO polypeptide solution with the same concentration as the detection hole into a negative control hole; placing at 37deg.C, 5% CO 2 Incubating the incubator for 20-22h; lysing and washing off cells according to the human IFN-. Gamma.ELISPOT kit instructions; biotin-labeled anti-human IFN-gamma antibody working solution (100. Mu.L/well) was added to each well and incubated at 37℃for 1h; after washing the plates, adding an enzyme-linked avidin working solution (100 mu L/well), and continuing to incubate at 37 ℃ for 1h; after washing the plate, adding the AEC color development liquid (100 mu L/well) which is prepared at present, and developing color for 20min at room temperature in a dark place; washing the plate with deionized water 4-5 times to terminate the color development; the ELISPOT plate is placed in a dark place, dried and then sent to Shenzhen daceae for automatically scanning and counting spots. If the number of the negative Kong Ban points is 0-5, judging that the CTL reaction is positive by the number of the spots of the detection hole and the negative control hole being equal to or larger than 5; if the number of the negative control well spots is not less than 5, the number of the detection well spots is not less than 2 times the number of the negative well spots, and the CTL reaction is judged to be positive.
Identifying immunogenicity of a single epitope peptide: peripheral blood PBMC of HBV chronically infected person with CTL positive reaction to the mixed peptide is collected again, added into the ELISPOT plate, and single HBV polypeptide (30 μg/mL) in the positive mixed peptide is added into each detection hole, and a positive control hole and a negative control hole are arranged at 37 ℃ and 5% CO are arranged 2 Incubators were incubated for 20-22h and ELISPOT assays were performed as above. A CTL reaction positive well indicates that the epitope peptide to be identified in the well is immunogenic.
Determination of HLA-A restriction: each HBV infected person is subjected to HLA-A allele typing, and the virtual affinity of the epitope peptide is combined, and the restriction and presentation of the HLA-A molecule is preliminarily determined. From HBV infected persons, homozygous infected persons of the 13 HLA-A alleles are selected, PBMC thereof are taken, and ELISPOT detection is performed again with all epitope peptides which have been verified to be immunogenic and are limited by the HLA-A molecules, so that the HLA-A molecular limitation of each epitope peptide is further determined.
4. HLA-A allele typing
Selecting HBV chronic infection patients with CTL positive reaction in epitope peptide identification experiment, taking 200 mu L of anticoagulation, and extracting genome DNA by using human whole blood genome DNA extraction kit (Tiangen organism, beijing); PCR was performed using HLA-A site-specific primers A1 and A3, and the DNA sequences of exon 2, intron 2, exon 3, and part of introns l and 3 at the A site were amplified to a size of 985bp. The amplification conditions were: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s; annealing at 62 ℃ for 15s; extending at 72 ℃ for 90s;35 cycles; extending at 72℃for 5min. The amplified products were sized by 1% agarose gel electrophoresis and sent to Shanghai Sanny Biotech for purification and two-way sequencing. PCR reagents were purchased from Nanjinouzan Biotech, inc., see Table 2.
TABLE 2 HLA-A site specific PCR amplification primers
Sequencing results of exon 2 and exon 3 are spliced into a complete HLA-A overlapping sequence (Contig) by Seqman software of a Lasergene program, whether bases sequenced in two directions are complete and consistent is carefully checked, bases of heterozygotes are found and replaced by facultative bases, for example M represents A and C, R represents A and G, W represents A and T, S represents C and G, Y represents C and T, K represents G and T, and sequence fragments of amplified HLA-A alleles are finally determined. The spliced HLA-A base sequences were aligned with the exon 2 and exon 3 sequences of all HLA-A alleles in the database using the nucleotides BLAST tool until a perfectly matched gene combination was obtained, thereby determining the HLA-A alleles.
288 patients who had a CTL positive response to HBV mixed peptide were selected from 612 HBV chronically infected patients by IFN-. Gamma.ELISPOT method. PBMCs from these patients were then re-collected, validated for immunogenicity of individual epitope peptides using ELISPOT method, and then analyzed in combination with HLA-A alleles from the patients and the virtual predicted affinities of epitope peptides for these alleles. The results show that: PBMC from patients positive for HLA-A 02:01 were stimulated to exhibit CTL-positive responses by 26 HBV epitope peptides (P1-P26) (FIG. 1); PBMC from patients positive for HLA-A 11:01 were stimulated to exhibit CTL-positive responses by 23 HBV epitope peptides (P27-P49) (FIG. 2); PBMC from patients with HLA-A 24:02 positive were stimulated to exhibit CTL positive responses by 25 HBV epitope peptides (P50-P74) (FIG. 3); 17 HBV epitope peptides (P36, P54, P75-P89) stimulated CTL-positive response in PBMC of HLA-A 31:01 positive patients (FIG. 4); there are 15 HBV epitope peptides (P9, P24-P26, P54, P90-P99) that stimulate PBMCs of HLA-A 02:06 positive patients to exhibit CTL positive responses (fig. 5); there were 14 HBV epitope peptides (P20, P100-P112) that stimulated CTL positive response by PBMCs from HLA-A 02:07 positive patients (fig. 6); there are 16 HBV epitope peptides (P36, P42, P46, P48, P79, P86, P113-P122) that stimulate PBMCs of HLA-A 33:03 positive patients to exhibit CTL positive responses (fig. 7); there are 16 HBV epitope peptides (P9, P34, P123-P136) that stimulate CTL positive response in PBMCs of HLA-A 30:01 positive patients (fig. 8); PBMC from patients positive for HLA-A.02:03 were stimulated to exhibit CTL-positive responses by 11 HBV epitope peptides (P95, P137-P146) (FIG. 9); PBMC from patients positive for HLA-A 11:02 were stimulated to exhibit CTL-positive responses by 13 HBV epitope peptides (P86, P117, P127, P147-P156) (FIG. 10); there are 13 HBV epitope peptides (P78, P85, P115, P151, P153, P155, P157-P164) that stimulate PBMCs of HLA-A 03:01 positive patients to exhibit CTL positive responses (fig. 11); there are 16 HBV epitope peptides (P47, P50, P115, P158, P164-P175) that stimulate PBMCs of HLA-A 01:01 positive patients to exhibit CTL positive responses (fig. 12); there are 15 HBV epitope peptides (P31, P176-P189) that stimulate CTL positive response in PBMCs from HLA-A 26:01 positive patients (fig. 13). There was a clear difference in immunogenicity between each of the HLA-A molecule restricted HBV epitope peptides.
It should be noted that the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and the equivalents or alternatives made on the basis of the above description are all included in the scope of the present invention.
The method comprises the following steps:
the amino acid sequences of the four hepatitis b virus antigens used to predict the epitope are as follows:
HBsAg(Q913A6)：
molecular type of sequence: PRT (PRT)
Scientifically named biotype: human hepatitis B virus
HBeAg(Q913A8)：
Molecular type of sequence: PRT (PRT)
Scientifically named biotype: human hepatitis B virus
HBpol(Q913A7)：
Molecular type of sequence: PRT (PRT)
Scientifically named biotype: human hepatitis B virus
HBx(Q913A9)：
Molecular type of sequence: PRT (PRT)
Scientifically named biotype: human hepatitis B virus
Claims (6)
1. A thymus-dependent lymphocyte antigen epitope peptide of a hepatitis b virus antigen, characterized in that the amino acid sequence of the antigen epitope peptide is any one of the following sequences:
QAGFFSLTK、TVNAHGNLPK、LKVFVLGGCR。
2. an epitope peptide obtained by removing or replacing a single amino acid using the thymus-dependent lymphocyte epitope peptide of hepatitis B virus antigen according to claim 1.
3. Use of the thymic dependent lymphocyte epitope peptide of hepatitis b virus antigen according to claim 1 or the epitope peptide according to claim 2 in the preparation of hepatitis b polypeptide vaccine or genetic vaccine.
4. Use of the thymic dependent lymphocyte epitope peptide of the hepatitis b virus antigen according to claim 1 or the epitope peptide according to claim 2 in the preparation of a detection preparation or kit for detecting hepatitis b virus antigen specific T cells.
5. The use according to claim 4, wherein the detection reagent is an enzyme-linked immunosorbent assay reagent, an intracellular cytokine fluorescent staining reagent, an enzyme-linked immunosorbent assay reagent, a human leukocyte antigen multimer fluorescent staining or a flow cytometry reagent.
6. Use of the thymic dependent lymphocyte epitope peptide according to claim 1 or the epitope peptide according to claim 2 for preparing a medicament for treating hepatitis b.
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