Thymus dependent lymphocyte antigen epitope peptide of hepatitis B virus and application of thymus dependent lymphocyte antigen epitope peptide

Abstract

The invention discloses thymus dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen and application, and belongs to the field of medical immunology and infectious pathology. The invention discloses thymus dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen. The amino acid sequence of the epitope peptide comprises any one or more of SYVNVNMGL, SYVNVNMGL, SYVNVNMGL, SYVNVNMGL, SYVNVNMGL LATWVGSNL (Lattice, Attention, an RRMETTVNA (radio frequency identification tag); mAARVCCQL is used as a template; the amino acid sequence is an amino acid sequence obtained by removing or replacing a single amino acid of the amino acid sequence. The epitope peptide disclosed by the invention can be specifically combined with cytotoxic thymus dependent lymphocytes to stimulate the cytotoxic thymus dependent lymphocytes to activate, proliferate and differentiate, so that the immune effect of resisting the hepatitis B virus is exerted; the antigen peptides can be used for preparing therapeutic and prophylactic 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 have potential application value in prevention, treatment and diagnosis of hepatitis B.

Description

Technical Field

[0001] The invention belongs to the field of medical immunology and infectious diseases, and in particular relates to a thymus-dependent lymphocyte antigen epitope peptide of a hepatitis B virus antigen and an application thereof. Background Art

[0002] Hepatitis B virus (HBV) infection is one of the most common infectious diseases, which seriously endangers human health. Although HBV vaccines have been promoted for more than 30 years, the infection is still spreading in the world. About 10% of the world's population is infected with HBV, of which more than 257 million people suffer from chronic lifelong infection. At present, there are about 70 million people infected with HBV in my country, about 20-30 million patients with chronic hepatitis B, and about 77% of liver cirrhosis and 84% of hepatocellular carcinoma are caused by chronic hepatitis B. Antiviral treatment is currently the most fundamental treatment for hepatitis B, which is very effective for most patients, mainly including nucleoside (acid) analogs and interferon treatment. However, because HBV forms covalently closed circular DNA (cccDNA) and integrated sequences in the infected cell nucleus and exists for a long time, the current standard treatment can only inhibit viral replication, but cannot completely eliminate HBV. HBsAg often cannot turn negative, and HBV is prone to reactivation after drug withdrawal. Chronic transformation of hepatitis B and recurrence after drug withdrawal are serious problems we are facing now.

[0003] The HBV genome is composed of incomplete double-stranded circular DNA. The long one is the negative strand, containing 3020-3320 bases, and the short one is the positive strand, containing 1700-2800 bases. It includes four open reading frames (ORFs), which are called C, X, P and S coding regions. The C region is composed of pre-C and C genes, which are regulated by different start codons and end at the same stop codon. The pre-C and C genes jointly encode the pre-C protein, which is then cut and processed to form the e antigen (HBeAg). The C gene encodes the capsid protein of the virus, namely HBcAg. The S region is composed of the S gene, PreS1 and PreS2 genes. The translation is initiated by 3 different start codons and terminated at the same stop codon. It encodes the surface antigen (HBsAg) protein, PreS1 protein and PreS2 protein respectively. The P region gene encodes the polymerase protein (HBpol) of HBV. The gene in region X encodes X protein (HBx), which contains 154 amino acids and is the smallest open reading frame.

[0004] Cytotoxic T lymphocytes (CTLs) are core cells that mediate adaptive immune responses and play a vital role in anti-infection, anti-tumor, hypersensitivity reactions and autoimmune diseases. The T cell receptors (TCRs) on their cell membranes can specifically recognize and bind to the complex of MHC class I molecules and antigenic peptides on the surface of antigen-presenting cells, i.e., MHC / antigen peptide complex molecules. CTL epitopes refer to antigenic peptides bound to MHC class I molecules. They are linear fragments or spatial conformational structures in antigen molecules that can be specifically recognized by TCRs. They are the basic antigenic units that cause immune responses and play a key role in the CTL activation process.

[0005] The MHC system refers to the major histocompatibility complex (MHC), which is a group of tightly linked genes in the vertebrate genome that encode and express MHC class I and class II protein molecules. HLA (human leukocyte antigen) is the human MHC system, the most complex gene group in the human body, and is highly polymorphic in the human population. HLA plays an important role in the body's immune processes such as antigen recognition and antigen presentation, and is the main factor affecting the human immune response. HLA class I molecules are mainly responsible for presenting endogenous HBV antigens to CD8 + CTL, activated CTLs secrete perforin and granzyme to cause apoptosis of virus-infected hepatocytes, and secrete specific cytokines to inhibit HBV replication. + The number and function of T cells can accurately reflect the specific immune function status of hepatitis B infected people against HBV. Due to the different HLA molecular types of different people, their processing, handling and presentation capabilities of different HBV antigens are also different, thus causing different degrees of HBV antigen-specific immune response. According to the different HLA molecular types of hepatitis B patients, the HBV-specific antigen peptides presented by them are selected, and the CD8 specific HBV antigen peptides are dynamically monitored. + The number and function of T cells are of great significance for the disease progression monitoring, diagnosis and treatment plan formulation, efficacy observation and prognosis judgment of HBV infected patients, and are an important technical means to achieve precision medical treatment of hepatitis B. At the same time, using these HBV-specific antigen peptides with high affinity binding to HLA-C molecules, peptide vaccines or gene vaccines can also be prepared to prevent and treat HBV infection.

[0006] However, there are still few HBV antigen peptides that have been identified to be presented by various HLA molecules and can stimulate the body to induce T cell responses. This limits the specific T cell detection of hepatitis B patients carrying different HLA alleles, the role of HBV-specific T cells in the occurrence and development of hepatitis B, and personalized detection and precision immunotherapy based on individual differences in HLA genes and the differences in the HBV antigen peptides they present. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides a thymus-dependent lymphocyte antigen epitope peptide of a hepatitis B virus antigen and its application.

[0008] The technical solution of the present invention is as follows:

[0009] The first aspect of the present invention relates to a thymus-dependent lymphocyte antigen epitope peptide of a hepatitis B virus antigen, wherein the amino acid sequence of the antigen epitope peptide is any one or more of the following sequences:

[0010] SYVNVNMGL;

[0011] LATWVGSNL;

[0012] RRMETTVNA;

[0013] MAARVCCQL;

[0014] The amino acid sequence above is an amino acid sequence after a single amino acid is removed or replaced.

[0015] The second aspect of the present invention relates to the use of the above-mentioned thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen in the preparation of hepatitis B polypeptide vaccine or gene vaccine.

[0016] The third aspect of the present invention relates to the nucleic acid molecule of the thymus-dependent lymphocyte antigen epitope peptide of the above-mentioned hepatitis B virus antigen.

[0017] The fourth aspect of the present invention relates to a nucleic acid molecule vector.

[0018] The fifth aspect of the present invention relates to a vaccine for treating or preventing hepatitis B virus, comprising the above-mentioned nucleic acid molecule or the above-mentioned vector.

[0019] The sixth aspect of the present invention relates to the use of the above-mentioned thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen in the preparation of a drug for treating and preventing hepatitis B.

[0020] The seventh aspect of the present invention relates to the use of the above-mentioned thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen in the preparation of a kit for detecting hepatitis B virus antigen-specific T cells.

[0021] Optionally, the kit includes: ELISA reagents, intracellular cytokine fluorescence staining reagents, ELISA reagents, human leukocyte antigen polymer fluorescence staining or flow cytometry reagents.

[0022] The eighth aspect of the present invention relates to a monoclonal antibody capable of specifically binding to the above-mentioned epitope peptide.

[0023] The ninth aspect of the present invention relates to a drug for treating hepatitis B, comprising the above-mentioned monoclonal antibody.

[0024] More specifically, the present invention provides a novel thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen, the amino acid sequence of which is any one of the epitope peptide sequences shown below:

[0025]

[0026] As can be seen from the above table, the above sequences are the antigen peptide sequences of hepatitis B virus polymerase protein (HBeAg) and X protein (HBx), which bind to HLA-C0401, C1402, C0303, C0102, C0801, C0302, C0702 and other molecules with high affinity, form MHC / antigen peptide complex molecules on the surface of antigen presenting cells, and bind to antigen peptide-specific CD8 + T cell clones bind to stimulate their activation, proliferation and differentiation, and exert immune effect against hepatitis B virus.

[0027] The thymus-dependent lymphocyte antigen epitope peptide sequence of the hepatitis B virus antigen can be used to prepare a hepatitis B polypeptide vaccine or a gene vaccine: Preparation of polypeptide vaccine: one or more antigen peptides are artificially synthesized according to the antigen epitope peptide sequence of the present invention, mixed with an adjuvant to form a soluble preparation, or loaded with biological nanomaterials to form a nano polypeptide vaccine, which is injected into a hepatitis B virus infected person or a hepatitis B patient to stimulate the activation and proliferation of the patient's hepatitis B virus antigen-specific T cells, and enhance their tumor killing cell activity, thereby preparing a hepatitis B polypeptide vaccine. Preparation of gene vaccine: according to the polypeptide sequence of the present invention, a recombinant DNA gene fragment, a recombinant plasmid or a recombinant virus vector of one or more polypeptides is constructed, injected into a hepatitis B virus infected person or a hepatitis B patient, so that the recombinant gene expresses one or more polypeptides in vivo, stimulates the activation and proliferation of hepatitis B virus antigen-specific T cells, and enhances their activity of killing virus-infected cells, thereby preparing a hepatitis B gene vaccine.

[0028] The thymus-dependent lymphocyte antigen epitope peptide sequence of the hepatitis B virus antigen can be used to prepare a detection preparation or kit for detecting hepatitis B virus antigen-specific T cells: according to the antigen epitope peptide sequence of the present invention, one or more antigen peptides are artificially synthesized, and in the enzyme-linked immunospot method, intracellular cytokine fluorescence staining method and enzyme-linked immunosorbent assay, as an antigen preparation, mixed with the patient's peripheral blood mononuclear cells (PBMC) to stimulate the activation, proliferation and secretion of cytokines by hepatitis B virus antigen-specific T cells, and then the synthesis amount of the cytokine is detected by other combined reagents, thereby reflecting the number and reactivity of specific T cells; these polypeptides can also be used to prepare human leukocyte antigen and polypeptide complexes (peptide-HLA complex) and polymers thereof through genetic engineering technology and protein engineering technology, and then further prepared into a fluorescein-labeled preparation, and the number of hepatitis B virus antigen-specific T cells in the patient's peripheral blood PBMC cell population is detected by flow cytometry. The relevant kit refers to a hepatitis B virus antigen-specific T cell detection kit assembled from the above-mentioned preparation and other reagents commonly used in different detection methods.

[0029] The thymus-dependent lymphocyte antigen epitope peptide sequence of the hepatitis B virus antigen can be used to prepare drugs for treating hepatitis B: the above-mentioned polypeptide vaccine or gene vaccine based on the antigen epitope peptide sequence of the present invention is combined with other immunotherapy preparations or chemotherapeutic preparations to prepare clinical drugs for treating hepatitis B.

[0030] The present invention utilizes six online epitope prediction databases to virtually predict HBV specific antigen epitope peptide sequences restricted by HLA-C molecules, and obtains a group of HBV specific antigen epitope peptide sequences that can respectively bind to HLA-C0102, C0602, C0702, C0801, C0304, C0302, C0303, C0401, C1402, C1202, C1502, C1403, C1203, and C0701 molecules with high affinity, and then verifies the immunogenicity thereof through intracellular cell staining experiments, thereby providing specific antigen peptides for preparing therapeutic and preventive vaccines for HBV infection and developing HBV antigen-specific T cell detection reagents and the like.

[0031] 1. Select the amino acid sequences of hepatitis B virus polymerase protein (HBeAg) and X protein (HBx) as targeting sequences;

[0032] 2. Select six epitope prediction databases with high accuracy and widely recognized by researchers: SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC and EPIJEN to predict the HBV specific antigen epitope peptide sequences restricted by the above 14 HLA-C molecules;

[0033] 3. According to certain prediction criteria, the prediction results of six online epitope prediction websites were integrated and analyzed to obtain candidate antigen peptide sequences with relatively consistent prediction results from the six websites.

[0034] 4. The immunogenicity of HBV antigen epitope peptide was verified through IFN-γICS cell functional experiment.

[0035] 5. The peptide competition binding experiment of HLA-C molecules was carried out by using the HMy2.CIR cell line expressing specific HLA-C molecules to analyze the binding force between positive epitope peptides and specific HLA-C molecules.

[0036] 6. The immunogenicity of the positive epitope peptide in stimulating specific T cell activation and proliferation in vivo was verified by inoculating C57BL / 6J mice.

[0037] The present invention relates to antigenic peptide sequences in hepatitis B virus polymerase protein (HBeAg) and X protein (HBx) that can respectively bind to HLA-C0401, C1402, C0303, C0102, C0801, C0302, and C0702 molecules with high affinity and have immunogenicity; it also relates to hepatitis B polypeptide vaccines, gene vaccines, and methods for treating and preventing hepatitis B based on the above antigenic peptides, as well as reagents and methods for detecting hepatitis B virus antigen-specific T cells based on the above antigenic peptides.

[0038] Compared with the prior art, the advantages of the present invention are as follows:

[0039] The HBV-specific antigen epitope peptides restricted by HLA-C0401, C1402, C0303, C0102, C0801, C0302, and C0702 molecules obtained through online virtual prediction and functional experimental verification have not been reported before. These HLA-C molecules have not been reported to have restricted HBV antigen peptides before. Therefore, these new antigen epitope peptide sequences will provide the key antigen components, namely, antigen epitope peptide sequences, required for the development of therapeutic and preventive peptide vaccines and gene vaccines for hepatitis B, and the design of reagents and methods for detecting hepatitis B virus antigen-specific T cells; at the same time, these antigen epitope peptides also provide key antigen components for personalized testing and precision medicine for hepatitis B patients with these specific HLA-C alleles. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 PBMCs from hepatitis B patients were inoculated into 96-well plates and incubated with candidate epitope peptides corresponding to the HLA-C genotype of the patients for 6 hours. Then, IFN-γICS was used to detect CD3 + / CD8 + The proportion of IFN-γ secreting cells in the T cell population. (A) The CD3+ / CD8 + Flow cytometry scatter plot of IFN-γ secretion in T cell population after stimulation with specific epitope peptides; (B) CD3 + / CD8 + Statistical graph of the proportion of cells in a T cell population that secrete IFN-γ after being stimulated by specific epitope peptides. No. 1: epitope peptide No. 1 (SEQ ID NO.1); No. 2: epitope peptide No. 2 (SEQ ID NO.2); No. 3: epitope peptide No. 3 (SEQ ID NO.3); No. 4: epitope peptide No. 4 (SEQ ID NO.4).

[0041] Figure 2 Peptide competition binding assay to analyze the binding affinity of four hepatitis B T cell epitopes to specific HLA-C molecules. No. 1: Epitope peptide No. 1 (SEQ ID NO.1); No. 2: Epitope peptide No. 2 (SEQ ID NO.2); No. 3: Epitope peptide No. 3 (SEQ ID NO.3); No. 4: Epitope peptide No. 4 (SEQ ID NO.4). The blue solid line and red solid line show the cell fluorescence peaks at 5μM and 15μM concentrations of the tested peptide, respectively, while the gray filled line represents the maximum fluorescence intensity of the cell when there is only GFP-reference peptide. The dark blue solid line in the NC graph represents the negative control background fluorescence of the cells in the peptide-free well.

[0042] Figure 3 It provides a technical roadmap for epitope peptide prediction and screening, immunogenicity verification, and peptide competition binding experiments.

[0043] Figure 4 The epitope peptide induced specific CD8 + T cell response

[0044] The mixed polypeptide vaccine was prepared with four positive epitope peptides and used in combination with adjuvant poly(I:C) to immunize C57BL / 6J mice on days 0, 7, and 21, respectively. Seven days after the last immunization, the mouse spleen cells were taken and restimulated with PBS and each positive epitope peptide for 10 h, and then BFA / Monesion (to block cytokine release) was added to continue culturing for 6 h. Then, IFN-γ intracellular staining and flow cytometry analysis were performed to detect IFN-γ. + / CD3 + / CD8 + The frequency of T cells. Figure 4 A is a representative flow cytometry analysis; B is the IFN-γ + / CD3 + / CD8 + T cell frequency statistics. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] This embodiment discloses HLA-C0401, C1402, C0303, C0102, C0801, C0302, C0702 molecule restricted HBV specific antigen peptides, the sequences of which are screened and identified by the following steps:

[0047] 1. Online virtual prediction of dominant T cell epitope peptides of 2 HBV antigens restricted by 14 HLA-C molecules

[0048] Two HBV proteins were selected: hepatitis B virus polymerase protein (HBeAg, sequence is SEQ ID NO.7) and X protein (HBx, sequence is SEQ ID NO.8), and their amino acid sequences were obtained by searching the UniProt global protein resource database, and the HBV C type HBV protein sequence with the most infection in China was selected; then, six commonly used epitope peptide prediction databases such as SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC, and EPIJEN were used to virtually predict the T cell epitope peptides for each of the above HBV proteins restricted by HLA-C0102, C0602, C0702, C0801, C0304, C0302, C0303, C0401, C1402, C1202, C1502, C1403, C1203, and C0701 molecules, among which the SVMHC database includes the MHCPEP model and the SYFPEITHI The IEDB database includes three methods: ANN, SMM and ARB. In this example, the ANN and SMM methods with better statistical significance are selected. The websites of these epitope peptide prediction databases are shown in Table 1.

[0049] The antigen binding groove of HLA class I molecules is closed at both ends, and the length of the antigen peptide accepted is 8-11 amino acid residues, among which 9 and 10 amino acids are the most common. Therefore, in this embodiment, polypeptides with a length of 9 and 10 amino acids are selected as research objects. The amino acid sequence of each HBV protein is input into the corresponding amino acid sequence input box of the prediction database website, and then the length of the epitope peptide is selected as 9 and 10 amino acids, respectively, and then a specific HLA-C molecule is selected to perform online virtual prediction of the T cell epitope peptide of the HBV antigen.

[0050] Table 1 Epitope peptide prediction database website

[0051]

[0052] For each HLA-C molecule and each HBV protein, the 9 peptides and 10 peptides predicted by different databases were ranked from high to low according to the scores, and then the epitope peptides that met the scoring criteria of at least two prediction methods were selected as candidate epitope peptides. For each HLA-C molecule and each HBV protein, 1-4 peptides with the highest scores (highest affinity) were selected from the candidate epitope peptides as epitope peptides to be identified, and a total of 51 dominant T cell epitope peptides to be identified were screened and predicted.

[0053] 2. Isolation of PBMC from peripheral blood of patients with chronic HBV infection

[0054] Take fresh anticoagulated whole blood stored at room temperature and dilute it appropriately with sterile PBS; add 1 times the volume of original blood human lymphocyte separation solution (Dakoway Biotechnology, Shenzhen) into a 15 mL centrifuge tube; slowly spread the diluted blood on the separation solution and centrifuge at room temperature for 20 min, 2500 rpm; aspirate the mononuclear cell (PBMCs) layer and wash it twice by centrifugation; resuspend it with serum-free culture medium (Dakoway Biotechnology, Shenzhen) and count the cells.

[0055] 3. Identification of the immunogenicity of HBV antigen T cell epitope peptides by intracellular cytokine staining

[0056] Technical route such as Figure 3 As shown:

[0057] 200 μL of peripheral blood was taken from fresh anticoagulated blood samples of hepatitis B patients for HLA-C allele typing, and the remaining blood samples were used to separate and prepare PBMCs. The cells were seeded in 96-well U-shaped cell culture plates, and 5×10 5 cells, 200 μL / well; according to the HLA-C typing results, the predicted candidate epitope peptides that bind to the HLA-C molecule were added to a 96-well plate (2 μg / well, final concentration 80 μg / mL) and co-cultured with PBMCs. At the same time, PHA control wells (PHA final concentration 10 μg / mL) and peptide-free wells were set up, and 10% FBS-RPMI 1640 culture medium was added to 200 μL / well, and the cells were placed in a cell culture incubator for co-incubation for 6 h. Brefeldin ASolution / Monensin Solution (1000×) blocking agent was added simultaneously for co-culture, and then IFN-γ intracellular fluorescence staining was performed, and flow cytometry analysis and CD3 + / CD8 + IFN-γ in T cell population + Compared with the wells without peptide, the frequency of cells expressing IFN-γ + / CD8 +The candidate epitope peptides with a T cell frequency increased by more than 2 times were set as positive epitope peptides, that is, the epitope peptides could stimulate the memory CD8 + T cells become activated and secrete cytokines.

[0058] Through the IFN-γICS experiment, PBMCs from 100 HBV-infected patients were collected and co-cultured with related peptides from 51 candidate epitope peptides. Among them, 4 candidate epitope peptides (sequences were SYVNVNMGL; LATWVGSNL; RRMETTVNA; MAARVCCQL;) induced strong CD8 + T cell response, IFN-γ expression in PBMCs of HBV-infected patients + / CD8 + A 10-fold increase in T cell frequency or IFN-γ in five HBV-infected patients + / CD8 + The T cell frequency increased by more than 3 times, so the four candidate epitope peptides were determined to be positive epitopes and dominant epitope peptides with strong immunogenicity ( Figure 1 ).

[0059] 4. HLA-C allele typing

[0060] Chronic HBV infection patients who showed CTL positive reaction in epitope peptide identification experiment were selected, and 200 μL anticoagulated blood was taken. Genomic DNA was extracted using human whole blood genomic DNA extraction kit (Tiangen Biotechnology, Beijing); PCR was performed using HLA-C locus specific primers CF and CR to amplify the DNA sequence of exon 2, intron 2, exon 3 and part of introns 1 and 3 of the C locus, and the product size was 985 bp. The amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 62℃ annealing for 15 s; 72℃ extension for 90 s; 35 cycles; 72℃ extension for 5 min. The amplified product was identified by 1% agarose gel electrophoresis and sent to Shanghai Shenggong Biotechnology Co., Ltd. for purification and bidirectional sequencing. PCR reagents were purchased from Nanjing Novozyme Biotechnology Co., Ltd., see Table 2.

[0061] Table 2 HLA-C locus-specific PCR amplification primers

[0062]

[0063] The sequencing results of exon 2 and exon 3 were assembled into a complete HLA-C overlapping sequence (Contig) by using the Seqman software of the Lasergene program. The bases of the bidirectional sequencing were carefully checked to see if they were complete and consistent. The bases of the heterozygotes were found and replaced with annexed bases, such as M for A and C, R for A and G, W for A and T, S for C and G, Y for C and T, and K for G and T. Finally, the sequence fragments of the amplified HLA-C alleles were determined. The Nucleotide BLAST tool was used to compare the assembled HLA-C base sequence with the exon 2 and exon 3 sequences of all HLA-C alleles in the database until a completely matched gene combination was obtained, thereby determining the HLA-C alleles.

[0064] 5. Peptide competition binding experiment of HLA-C molecules

[0065] The 51 candidate epitopes, including the four positive epitope peptides No. 1, No. 2, No. 3, and No. 4 screened in the above steps and verified by the peptide-PBMCs costimulation experiment, were assigned to the relevant epitope peptide queues of each HLA-C molecule according to the predicted HLA-C restriction and the HLA-C allele type of the patient with positive T cell response, and used as the peptides to be tested to conduct peptide competition binding experiments with the fluorescent reference peptides of the HLA-C molecule.

[0066] The sequences of fluorescent reference peptides C0401, C0801, C0702, and C1402 are the same as those of W26, so peptide competition binding experiments were not performed. Finally, these four positive peptides all caused the cell fluorescence peak to shift to the left ( Figure 2 ), indicating that these positive epitope peptides can compete with the fluorescent reference peptides for binding to the relevant HLA-C molecules on the HMy2.CIR cell membrane. These four positive epitope peptides all showed high or medium affinity binding to the corresponding HLA-C molecules (Table 3).

[0067] Table 3 Peptide competition binding assay analysis of the binding affinity of 4 HBV positive T cell epitopes to 8 dominant HLA-C molecules

[0068]

[0069] 6. Immunization of C57BL / 6J mice with epitope peptides to verify their immunogenicity in inducing specific T cell responses in vivo

[0070] The four positive epitope peptides were made into mixed peptides and then mixed with the adjuvant poly I:C. C57BL / 6J mice were randomly divided into 2 groups, 3 mice in each group. The experimental group was vaccinated with peptide cocktail / poly(I:C) (epitope peptide: 10μg / single peptide, poly(I:C): 100μg / mouse) on day 0, day 7, and day 21, respectively; the adjuvant group was also vaccinated with poly(I:C) (100μg / mouse) on day 0, day 7, and day 21, respectively. Four subcutaneous injections were made (neck back, tail base, bilateral groins).

[0071] On the 28th day, mice were killed and spleen cell suspension was routinely prepared. The cell concentration was adjusted to 1×10 7 / mL, inoculated into 48-well cell culture plates (0.5mL / well), each mouse spleen cell inoculation well was divided into 5 groups, and PBS (negative control) and each positive epitope peptide (20μg / mL / peptide) were added respectively; after culturing for 10 hours, BFA and Monensin were added to each well and incubated for another 6 hours; then the cells in each well were collected, blocked with FcR Blocking Reagent mouse for 15 minutes, and then co-stained with FITC-anti-mouse CD3 and PE-anti-mouse CD8a for 30 minutes; after washing, each tube was treated with a membrane permeabilization agent, and finally APC-anti-mouse IFN-γ monoclonal antibody was added for intracellular staining for 30 minutes; after washing, CD3 was analyzed by flow cytometry + / CD8 + / IFN-γ + T cell frequency, observation of specific CD8 induced by epitope peptide + The strength of the T cell response.

[0072] The results showed that the four positive epitope peptides in this example (peptide cocktail / polyI:C group) all induced strong epitope peptide-specific CD8 + T cell response. Compared with the adjuvant group, the IFN-γ induced by these four epitope peptides + / CD8 + The T cell frequency was 2-7 times that of the adjuvant group.

[0073] The above results show that the epitope peptides of this example can induce specific immune responses in mice and T cells are effectively activated. Therefore, the epitope peptides of this example can be used in the preparation of hepatitis B vaccine. + The activation of T cells reflects that the above-mentioned epitope peptides may also have a certain preventive or therapeutic effect on tumors (such as those caused by hepatitis B).

[0074] It should also be noted that the epitope peptides of this embodiment can also be combined with other pharmaceutically acceptable excipients in practical applications, such as hepatitis B drugs or vaccines, to further optimize the effects of the product, such as some excipients used to maintain the stability of the drug or vaccine and to achieve the delivery of specific targeting functions. These excipients can be directly obtained by those skilled in the art from the prior art and will not be described in detail in this application.

[0075] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0076] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

[0077] Attachment:

[0078] The amino acid sequences of two hepatitis B virus antigens used to predict antigenic epitopes are as follows:

[0079] SEQ ID NO.7

[0080] HBeAg(Q913A8):

[0081] Molecular type of sequence: PRT

[0082] Scientifically named biological genus and species: Human hepatitis B virus

[0083]

[0084] SEQ ID NO.8

[0085] HBx(Q913A9):

[0086] Molecular type of sequence: PRT

[0087] Scientifically named biological genus and species: Human hepatitis B virus

Claims

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 or more of the following sequences: SYVNVNMGL; LATWVGSNL; RRMETTVNA; MAARVCCQL; The amino acid sequence above is an amino acid sequence after a single amino acid is removed or replaced.

2. Use of the thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen according to claim 1 in the preparation of hepatitis B polypeptide vaccine or gene vaccine.

3. A nucleic acid molecule encoding the thymus-dependent lymphocyte antigen epitope peptide of the hepatitis B virus antigen according to claim 1. A vector comprising the nucleic acid molecule according to claim 3.

5. A vaccine for treating or preventing hepatitis B virus, characterized in that: Containing the nucleic acid molecule according to claim 3 or the vector according to claim 4.

6. Use of the thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen according to claim 1 in the preparation of a drug for treating and preventing hepatitis B.

7. Use of the thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen according to claim 1 in preparing a kit for detecting hepatitis B virus antigen-specific T cells.

8. The use of the thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus according to claim 1 in a kit for preparing hepatitis B virus antigen-specific T cells according to claim 7, characterized in that: The kit comprises: an enzyme-linked immunosorbent assay reagent, an intracellular cytokine fluorescent staining reagent, an enzyme-linked immunosorbent assay reagent, and a human leukocyte antigen polymer fluorescent staining or flow cytometric analysis reagent.

9. A monoclonal antibody, characterized in that Capable of specifically binding to the epitope peptide according to claim 1.

10. A drug for treating hepatitis B, characterized in that: Comprising the monoclonal antibody according to claim 9.

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