CN115960206A - Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof - Google Patents
Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of medical immunology and oncology, and discloses thymus-dependent lymphocyte antigen epitope peptides of primary liver cancer-related antigens and application thereof, wherein the antigen epitope peptides are respectively HLA-A0201, HLA-A1101, HLA-A2402, HLA-A3101, HLA-A0206, HLA-A0207, HLA-A3303, HLA-A3001, HLA-A0203, HLA-A1102, HLA-A0301, HLA-A0101 and HLA-A2601 molecule-restricted antigen peptides, and total 110 types of the antigen peptides can be specifically combined with cytotoxic thymus-dependent lymphocytes to stimulate the later to activate, proliferate and differentiate, thereby playing the role of anti-tumor immune effect; the antigen peptides can be used for preparing therapeutic and preventive vaccines of liver cancer, can also be used for preparing detection reagents for detecting specific cytotoxic thymus-dependent lymphocytes of antigens related to the liver cancer, and have potential application values in prevention, treatment and diagnosis of the liver cancer.
Description
The invention relates to a division of a thymus-dependent lymphocyte epitope peptide with the patent name of 'primary liver cancer-associated antigen and application thereof', the application number of which is '201911286067.5' and the application date of which is '2019-12-13'.
Technical Field
The invention belongs to the field of medical immunology and oncology, and particularly relates to thymus-dependent lymphocyte epitope peptides of three primary liver cancer-related antigens and application thereof.
Background
The primary liver cancer is the lethal cause of 4 th common malignant tumor and 3 rd tumor in China, and seriously threatens the life and health of people in China. The pathological type of the primary liver cancer is hepatocellular carcinoma (HCC) which accounts for 85 to 90 percent; there are also a few intrahepatic cholangiocarcinoma (ICC) and HCC-ICC mixed types, which have large differences in pathogenesis, biological behavior, molecular characteristics, clinical manifestations, histopathological morphology, treatment methods, prognosis, etc.
In China, the high risk group of HCC mainly has Hepatitis B Virus (HBV) and/or Hepatitis C Virus (HCV) infection, long-term alcoholism (alcoholic liver disease), non-alcoholic steatohepatitis, food polluted by aflatoxin, cirrhosis caused by various reasons and people with family history of liver cancer, and meanwhile, the risk of men over 40 years old is high. Recent studies suggest that diabetes, obesity, smoking, and the like are also risk factors for HCC and are of interest.
AFP, GPC3 and GP73 are common tumor-associated antigens which are over-expressed in liver cancer, and are widely researched in the aspects of diagnosis, treatment, course monitoring and the like of liver cancer. A positive serum alpha-fetoprotein (AFP) means that the AFP is more than or equal to 400 mu g, chronic or active hepatitis, liver cirrhosis, testicular or ovarian embryonic derived tumors, pregnancy and the like are excluded, and liver cancer is highly suspected. For patients with low AFP elevation, dynamic observation and analysis of liver function change should be performed. Approximately 30% of liver cancer patients have normal AFP levels and should be tested for alpha-fetoprotein heteroplasmons. GPC3 is one of heparan sulfate glycoproteins, and is highly expressed in primary liver cancer tissues, while other normal tissues are not expressed or are low expressed. GPC3 can promote tumor cell proliferation and differentiation, and can promote formation, growth and metastasis of hepatocarcinoma by combining with extracellular matrix, protease and growth factor. GP73 is a Golgi type II transmembrane protein, which is expressed poorly in hepatocytes under physiological conditions. The serum GP73 level of a patient with primary liver cancer is detected to show that the expression of the GP73 is obviously high, and the expression of the GP73 is negatively related to the liver function and the disease condition of the patient, and is one effective index of the damage severity degree of liver cells. The three liver cancer-related tumor antigens are selected for research.
Cytotoxic T Cells (CTL), which are core cells mediating adaptive immune response, play a crucial role in anti-infection, anti-tumor, and hypersensitivity reactions and the development of autoimmune diseases, and T Cell Receptors (TCR) on their cell membranes are capable of specifically recognizing and binding to complexes of MHC class I molecules and antigenic peptides on the surface of antigen presenting cells, i.e., MHC/antigenic peptide complex molecules. CTL epitopes are antigenic peptides bound to MHC class I molecules, which are linear fragments or spatial conformational structures of antigenic molecules that can be specifically recognized by the TCR, and are the basic antigenic units that elicit an immune response, playing a key role in CTL activation.
The MHC system refers to the Major Histocompatibility Complex (MHC), which is a group of closely linked genes in the vertebrate genome that encode molecules expressing MHC class I and class II proteins. HLA (human leukocyte antigen) is the human MHC system, the most complex gene group in humans, and is highly polymorphic in the human population. HLA plays an important role in immune processes such as antigen recognition, antigen presentation and the like, and is capable of influencing immune response of human bodyThe main factors. For human liver cancer, HLA class I molecules are primarily responsible for the presentation of endogenous liver cancer associated antigens to CD8 + CTLs, activated CTLs, apoptosis tumor cells expressing tumor-associated antigens by secreting perforin, granzyme, and the like. Thus, dynamic monitoring of hepatoma associated antigen specific CD8 + The number and the function of the T cells can accurately reflect the specific immune function state of the liver cancer patient aiming at the liver cancer related antigen. Because different people have different HLA molecular types, the processing, treatment and presentation capacities on different antigens of liver cancer are different, and thus different intensities of specific T cell immune response reactions of the antigens related to the liver cancer are caused. According to different HLA molecular types of liver cancer patients, antigen peptides of the presented liver cancer related antigen are selected, and the specificity of the liver cancer related antigen CD8 is dynamically monitored + The number and the reactivity of the T cells have great significance for monitoring the disease process of liver cancer patients, making diagnosis and treatment schemes, observing curative effect, judging prognosis outcome and the like, and are important technical means for realizing accurate medical treatment of liver cancer. Meanwhile, by using the antigen peptides of the liver cancer related antigens combined by the HLA-A molecules with high affinity,base:Sub>A polypeptide vaccine orbase:Sub>A gene vaccine can be prepared to prevent and treat liver cancer.
However, the existing definite primary liver cancer related antigen T cell epitopes presented by various HLA molecules and capable of stimulating organisms to cause T cell response are still very few, so that the development of specific T cell detection on liver cancer patients carrying different HLA alleles is limited, the action research of liver cancer specific T cells in liver cancer occurrence and development is also limited, and personalized detection and accurate immunotherapy based on the individual difference of HLA genes and the difference of presented liver cancer related antigen peptides are further limited.
Disclosure of Invention
The invention solves the technical problems in the prior art, and provides thymus dependent lymphocyte antigen epitope peptide of liver cancer related antigen and application thereof.
In order to solve the problems, the technical scheme of the invention is as follows: the amino acid sequence of the thymus-dependent lymphocyte epitope peptide of the liver cancer related antigen is any one of the epitope peptide sequences shown as follows:
as can be seen from the above tables, the above sequences are antigenic peptide sequences of alpha-fetoprotein (AFP), glypican-3 (GPC 3) and Golgi transmembrane glycoprotein 73 (GP 73), respectively, and they are bound to HLA-A0201, HLA-A1101, HLA-A2402, HLA-A3101, HLA-A0206, HLA-A0207, HLA-A3303, HLA-A3001, HLA-A0203, HLA-A1102, HLA-A0301, HLA-A0101 and HLA-A2601 molecules with high affinity, respectively, to form "HLA/antigenic peptide" complex molecules on the surface of antigen presenting cells, and to CD8 specific to antigenic peptides + The T cells are combined in a cloning way to stimulate the activation, proliferation and differentiation of the T cells, and play a role in resisting the liver cancer immune effect.
The thymus-dependent lymphocyte antigen epitope peptide sequence of the primary liver cancer related antigen can be used for preparing a liver cancer polypeptide vaccine or a gene vaccine: preparing a polypeptide vaccine: one or more polypeptides are artificially synthesized according to the polypeptide sequence of the invention, and are mixed with an adjuvant to prepare a soluble preparation, or a biological nano material is loaded to prepare a nano polypeptide vaccine which is injected into a liver cancer patient to stimulate the activation and proliferation of liver cancer-related antigen specific T cells of the patient and enhance the activity of tumor killing cells, so that the liver cancer polypeptide vaccine is prepared. Preparing a gene vaccine: according to the polypeptide sequence, a recombinant DNA gene segment, a recombinant plasmid or a recombinant virus vector of one or more polypeptides is constructed and injected into a liver cancer patient, so that the recombinant gene expresses one or more polypeptides in vivo, the activation and proliferation of liver cancer-related antigen-specific T cells of the patient are stimulated, the tumor killing activity of the tumor killing cells is enhanced, and the liver cancer gene vaccine is prepared.
The thymus dependent lymphocyte antigen epitope peptide sequence of the primary liver cancer related antigen can be used for preparing a detection preparation or a kit for detecting the specific T cells of the liver cancer related antigen: according to the polypeptide sequence, one or more polypeptides are artificially synthesized, and are used as an antigen preparation in an enzyme-linked immunospot method, 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, so that the activation, proliferation and cytokine secretion of the liver cancer-related antigen specific T cells are stimulated, and the synthetic amount of the cytokine is detected through other combined reagents, so that the number and the reactivity of the specific T cells are reflected; the polypeptide can also be used for preparing a complex (peptide-HLA complex) of human leukocyte antigen and polypeptide and a polymer thereof by a gene engineering technology and a protein engineering technology, and further preparing a fluorescein labeled preparation, and detecting the number of the liver cancer related antigen specific T cells in the patient peripheral blood mononuclear cell population by using a flow cytometry analysis method. The related kit is a liver cancer related antigen specific T cell detection kit formed by assembling the preparation and other commonly used reagents in different detection methods.
The thymus-dependent lymphocyte epitope peptide sequence of the primary liver cancer related antigen can be used for preparing a medicament for treating liver cancer: the polypeptide vaccine or gene vaccine based on the polypeptide sequence of the invention is combined with other immunotherapy preparations or chemotherapy preparations to prepare clinical drugs for treating liver cancer.
The invention virtually predicts the specific epitope peptide sequences of 13 HLA-A molecule restricted liver cancer related antigens by six online epitope prediction databases to obtainbase:Sub>A group of liver cancer related epitope peptide sequences which can be respectively combined with HLA-A0201, HLA-A1101, HLA-A2402, HLA-A3101, HLA-A0206, HLA-A0207, HLA-A3303, HLA-A3001, HLA-A0203, HLA-A1102, HLA-A0301, HLA-A0101 and HLA-A2601 molecules with high affinity, and then verifies the immunogenicity through an ELISPOT functional experiment, thereby providing specific antigenic peptides for preparing liver cancer treatment and preventive vaccines, developing liver cancer related antigen specific T cell detection reagents and the like.
1. Selecting an amino acid sequence of alpha-fetoprotein (AFP), glypican-3 (GPC 3) and Golgi transmembrane glycoprotein 73 (GP 73) as a targeting sequence;
2. the prediction results are selected to obtain six commonly used epitope prediction databases which are accepted by researchers and have higher accuracy: SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC and EPIJEN predict the 13 HLA-A molecule restricted liver cancer related epitope peptide sequences;
3. and according to a certain prediction standard, performing integrated analysis on the prediction results of the six online epitope prediction websites to obtain candidate antigen peptide sequences with relatively consistent prediction results of the six websites.
4. The immunogenicity of the liver cancer related antigen peptide is verified through IFN-gamma ELISPOT cell functional experiments.
The invention is an antigen peptide sequence which can be respectively combined with HLA-A0201, HLA-A1101, HLA-A2402, HLA-A3101, HLA-A0206, HLA-A0207, HLA-A3303, HLA-A3001, HLA-A0203, HLA-A1102, HLA-A0301, HLA-A0101 and HLA-A2601 molecules with high affinity and has immunogenicity in alpha-fetoprotein (AFP), glypican-3 (GPC 3) and Golgi transmembrane glycoprotein 73 (GP 73); also relates to liver cancer polypeptide vaccine and gene vaccine based on the antigen peptide, and reagent and method for detecting liver cancer related antigen specific T cell based on the antigen peptide.
Compared with the prior art, the invention has the advantages that,
HLA-A0201, HLA-A1101, HLA-A2402, HLA-A3101, HLA-A0206, HLA-A0207, HLA-A3303, HLA-A3001, HLA-A0203, HLA-A1102, HLA-A0301, HLA-A0101 and HLA-A2601 molecule-restricted primary liver cancer-associated antigen-specific epitope peptides obtained through online virtual prediction and functional experiment verification have not been reported previously. These HLA-A molecules have not been reported to have limited antigenic peptides related to liver cancer. Therefore, the new epitope peptide sequences provide key antigen components, namely the epitope peptide sequences, required for developing polypeptide vaccines and gene vaccines aiming at liver cancer treatment and prevention, designing reagents and methods for detecting liver cancer related antigen specific T cells and the like; meanwhile, the antigen epitope peptides also provide key antigen components for individual detection and precise medical treatment of liver cancer patients with specific HLA-A alleles.
Description of the drawings:
FIG. 1 shows epitope peptides of liver cancer associated antigen T cells restricted by HLA-A0201 molecules;
a) Identifying detection holes, negative control holes and positive control hole spot diagrams of HLA-A0201 molecule restricted liver cancer associated antigen T cell epitope peptide by an IFN-gamma ELISPOT method;
b) A statistical map of the number of spots in the detection wells and the negative control wells;
c) Dot ratio (P/N) statistical plots for test wells versus negative control wells.
FIG. 2 shows epitope peptides of HLA-A1101 molecule-restricted liver cancer associated antigen T cells;
a) Identifying a detection hole, a negative control hole and a positive control hole dot diagram of the HLA-A1101 molecular restricted liver cancer related antigen T cell epitope peptide by an IFN-gamma ELISPOT method;
b) A statistical plot of the number of spots in the detection wells and the negative control wells;
c) Dot ratio (P/N) statistical plots for test wells versus negative control wells.
FIG. 3 shows HLA-A2402 molecule restricted epitope peptide of liver cancer associated antigen T cell;
a) Identifying a detection hole, a negative control hole and a positive control hole dot diagram of HLA-A2402 molecular restricted liver cancer related antigen T cell epitope peptide by an IFN-gamma ELISPOT method;
b) A statistical map of the number of spots in the detection wells and the negative control wells;
c) Dot ratio (P/N) statistical plots for test wells versus negative control wells.
FIG. 4 shows epitope peptides of HLA-A3101 restricted liver cancer associated antigen T-cell;
a) Identifying a detection hole, a negative control hole and a positive control hole dot diagram of HLA-A3101 molecular restricted liver cancer related antigen T cell epitope peptide by an IFN-gamma ELISPOT method;
b) A statistical map of the number of spots in the detection wells and the negative control wells;
c) Dot ratio (P/N) statistical plots for test wells versus negative control wells.
FIG. 5 shows epitope peptides of HLA-A0206 molecule-restricted liver cancer associated antigen T cells;
a) Identifying a detection hole, a negative control hole and a positive control hole dot diagram of the HLA-A0206 molecule restricted liver cancer related antigen T cell epitope peptide by an IFN-gamma ELISPOT method;
b) A statistical map of the number of spots in the detection wells and the negative control wells;
c) Dot ratio (P/N) statistical plots for test wells versus negative control wells.
FIG. 6 shows epitope peptides of HLA-A0207 molecule-restricted liver cancer associated antigen T cells;
a) Identifying detection holes, negative control holes and positive control hole spot diagrams of HLA-A0207 molecule restricted liver cancer associated antigen T cell epitope peptide by an IFN-gamma ELISPOT method;
b) A statistical map of the number of spots in the detection wells and the negative control wells;
c) Dot ratio (P/N) statistical plots for test wells versus negative control wells.
FIG. 7 shows HLA-A3303 restricted epitope peptides of liver cancer associated antigen T cells;
a) Identifying a detection hole, a negative control hole and a positive control hole dot diagram of HLA-A3303 molecule restricted liver cancer related antigen T cell epitope peptide by an IFN-gamma ELISPOT method;
b) A statistical plot of the number of spots in the detection wells and the negative control wells;
c) Dot ratio (P/N) statistical plots for test wells versus negative control wells.
FIG. 8 shows epitope peptides of HLA-A3001 restricted liver cancer associated antigen T cells;
a) Identifying a detection hole, a negative control hole and a positive control hole dot diagram of HLA-A3001 molecular restricted liver cancer related antigen T cell epitope peptide by an IFN-gamma ELISPOT method;
b) A statistical map of the number of spots in the detection wells and the negative control wells;
c) Dot ratio (P/N) statistical plots for test wells versus negative control wells.
FIG. 9 shows epitope peptides of HLA-A0203 molecule-restricted liver cancer associated antigen T cells;
a) Identifying a detection hole, a negative control hole and a positive control hole dot diagram of the HLA-A0203 molecule restricted liver cancer related antigen T cell epitope peptide by an IFN-gamma ELISPOT method;
b) A statistical map of the number of spots in the detection wells and the negative control wells;
c) Dot ratio (P/N) statistical plots for test wells versus negative control wells.
FIG. 10 shows epitope peptides of HLA-A1102 molecule restricted liver cancer associated antigen T cells;
a) Identifying detection holes, negative control holes and positive control hole spot diagrams of HLA-A1102 molecule restricted liver cancer associated antigen T cell epitope peptide by an IFN-gamma ELISPOT method;
b) A statistical plot of the number of spots in the detection wells and the negative control wells;
c) Dot ratio (P/N) statistical plots for test wells versus negative control wells.
FIG. 11 shows epitope peptides of HLA-A0301 restricted hepatocarcinoma associated antigen T cells;
a) Identifying a detection hole, a negative control hole and a positive control hole spot diagram of HLA-A0301 molecular restricted liver cancer associated antigen T cell epitope peptide by an IFN-gamma ELISPOT method;
b) A statistical plot of the number of spots in the detection wells and the negative control wells;
c) Dot ratio (P/N) statistical plots for test wells versus negative control wells.
FIG. 12 shows epitope peptides of HLA-A0101 restricted liver cancer associated antigen T-cell;
a) Identifying detection holes, negative control holes and positive control hole spot diagrams of HLA-A0101 molecule restricted liver cancer associated antigen T cell epitope peptide by IFN-gamma ELISPOT method;
b) A statistical map of the number of spots in the detection wells and the negative control wells;
c) Dot ratio (P/N) statistical plots for test wells versus negative control wells.
FIG. 13 shows epitope peptides of HLA-A2601 restricted liver cancer associated antigen T cells;
a) Identifying a detection hole, a negative control hole and a positive control hole dot diagram of HLA-A2601 molecular restrictive liver cancer related antigen T cell epitope peptide by an IFN-gamma ELISPOT method;
b) A statistical map of the number of spots in the detection wells and the negative control wells;
c) Dot ratio (P/N) statistical plots for test wells versus negative control wells.
FIG. 14 is a technical experimental scheme for verifying the immunogenicity of the epitope peptide to be identified in example 1.
Detailed Description
Example 1: the invention relates to a liver cancer related antigen T cell epitope restricted by HLA-A0201, HLA-A1101, HLA-A2402, HLA-A3101, HLA-A0206, HLA-A0207, HLA-A3303, HLA-A3001, HLA-A0203, HLA-A1102, HLA-A0301, HLA-A0101 and HLA-A2601 molecules, the sequence of which is screened and identified by the following steps:
1 on-line virtual prediction of dominant T cell epitope peptides of three liver cancer related antigens limited by 13 HLA-A molecules
Selecting three liver cancer related antigen proteins: alpha-fetoprotein (AFP), glypican-3 (GPC 3) and Golgi transmembrane glycoprotein 73 (GP 73), the amino acid sequences of which are obtained by searching a UniProt global protein resource database, and the standard sequence which is researched most is selected; virtually predicting T cell epitope peptides aiming at each liver cancer related antigen protein, which are limited by HLA-A0201, HLA-A1101, HLA-A2402, HLA-A3101, HLA-A0206, HLA-A0207, HLA-A3303, HLA-A3001, HLA-A0203, HLA-A1102, HLA-A0301, HLA-A0101 and HLA-A2601 molecules, by six commonly used epitope peptide prediction databases such as SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC and EPIJEN, wherein the SVMHC database comprises two prediction algorithms of MHCPEP model and SYFEPITHI model; the IEDB database contains three methods ANN, SMM and ARB, of which ANN and SMM methods of better statistical significance were selected in this experiment. The epitope peptide prediction database website is shown in table 1.
The antigen binding grooves of HLA class I molecules are closed at two ends, and the length of the received antigen peptide is 8-11 amino acid residues, wherein 9 and 10 amino acids are the most common, so that polypeptides with the lengths of 9 and 10 amino acids are mainly selected as a research object in the experiment. Respectively inputting the amino acid sequence of each liver cancer related antigen protein intobase:Sub>A corresponding amino acid sequence input box ofbase:Sub>A prediction database website, respectively selecting the length of epitope peptide to be 9 and 10 amino acids, then selectingbase:Sub>A specific HLA-A molecule, and carrying out online virtual prediction on the T cell epitope peptide of the liver cancer related antigen.
Table 1 epitope peptide prediction database website
Aiming at each HLA-A molecule and each liver cancer related antigen protein, the polypeptides predicted by different databases are respectively arranged according to the scores from high to low, and epitope peptides which at least meet the scoring standards of more than two prediction methods are selected as candidate epitope peptides. For each HLA-A molecule, aiming at each liver cancer related antigen protein, 1-5 polypeptides with the highest score (highest affinity) are selected from candidate epitope peptides as epitope peptides to be identified. And (3) aiming at the 13 HLA-A molecules, screening and predicting 160 dominant T cell epitope peptides to be identified in total.
2 separating peripheral blood PBMC of liver cancer patient
Taking fresh anticoagulated whole blood stored at room temperature, and properly diluting the anticoagulated whole blood with sterile PBS; adding 1 time of original blood volume of human lymphocyte separation liquid (Dake is biology, shenzhen) into a 15mL centrifuge tube; slowly spreading the diluted blood on the separation solution, centrifuging at room temperature for 20min, and at 2500rpm; sucking a mononuclear cell (PBMCs) layer, and centrifugally washing for 2 times; resuspending with serum-free medium (Dake is biological, shenzhen), counting cells, adjusting cell concentration to 2 × 10 6 The volume is/mL for standby.
3 identifying immunogenicity of liver cancer associated antigen T cell epitope peptide by IFN-gamma ELISPOT method
The technical route is shown in fig. 14:
firstly, screening out liver cancer patients who have positive reaction to the mixed peptide group: mixing epitope peptides to be identified, which are limited by each HLA-A molecule and aim at three liver cancer related proteins, into one group, wherein 13 groups are formed, and each group comprises 8-9 epitope peptides; taking ELISPOT plate (Dake is biology, shenzhen) pre-coated with anti-human IFN-gamma antibody, activating for 8min by serum-free medium (200 μ L/well), and adding PBMC suspension (100 μ L/well) of liver cancer patients into each well; then adding mixed peptides limited by each HLA-A molecule into the detection holes, wherein the single polypeptide in the mixed peptides is 15 mu g/mL, adding PHA (2.5 mu g/mL) into the positive control holes, and adding DMSO polypeptide dissolving solution with the same concentration as the detection holes into the negative control holes; placed at 37 ℃ and 5% CO 2 Incubating in an incubator for 24h; lysing and washing the cells according to the human IFN- γ ELISPOT kit instructions; adding biotin-labeled anti-human IFN-gamma antibody working solution (100 mu L/well) into each well, and incubating for 1h at 37 ℃; adding enzyme-linked avidin working solution (100 mu L/well) after washing the plate, and continuously incubating for 1h at 37 ℃; after washing the plate, adding the AEC color developing solution (100 mu L/well) prepared in situ, and developing for 20min at room temperature in a dark place; washing the plate with deionized water for 4-5 times to stop color development; and placing the ELISPOT plate in a dark place, airing, and then conveying to Shenzhendake as a biotechnology company for automatically scanning and counting spots. If the number of the negative pore spots is 0-5, judging that the CTL reaction is positive if the number of the detection pore spots is not less than 6; and if the number of the spots in the negative control hole is not less than 6, judging that the CTL reaction is positive if the number of the spots in the negative control hole is not less than 2 times that in the detection hole.
Identification of immunogenicity of individual epitope peptides: collecting peripheral blood PBMC of hepatocarcinoma patient with CTL positive reaction to the mixed peptide, adding into the ELISPOT plate, supplementing single hepatocarcinoma-associated antigen polypeptide (15 μ g/mL) in positive mixed peptide into each detection well, setting positive control well and negative control well as above, setting at 37 deg.C, and 5% CO 2 Incubate for 24h in incubator, and perform ELISPOT detection as above. A CTL reaction positive hole indicates that the epitope peptide to be identified in the hole has immunogenicity.
Determination of HLA-A restriction: and (3) carrying out HLA-A allele typing on each liver cancer patient, and preliminarily determining which HLA-A molecules are restricted and presented by combining the virtual affinity of the epitope peptide. Selecting homozygote infected persons of the 13 HLA-A alleles from liver cancer patients, taking PBMCs of the homozygote infected persons, taking the PBMCs of the homozygote infected persons and all epitope peptides which are verified to have immunogenicity and are limited by the HLA-A molecules, carrying out ELISPOT detection again, and further determining the HLA-A molecule limitation of each epitope peptide.
4HLA-A allelic typing
Selecting a liver cancer patient with CTL positive reaction in an epitope peptide identification experiment, taking 200 mu L of anticoagulation blood, and extracting genomic DNA by using a human whole blood genomic DNA extraction kit (Tiangen organism, beijing); and (3) carrying out PCR by adopting HLA-A site specific primers A1 and A3, amplifying DNA sequences of exon 2, intron 2, exon 3 and partial introns l and 3 of the site A, wherein the size ofbase:Sub>A product is 985bp. The amplification conditions were: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 15s; annealing at 62 ℃ for 15s; extension for 90s at 72 ℃;35 cycles; extension at 72 ℃ for 5min. The amplified products were size-characterized by 1% agarose gel electrophoresis and sent to Shanghai Sangni Biotech for purification and bidirectional sequencing. PCR reagents were purchased from Biotech, nanjing Novozam.
TABLE 2 HLA-A site-specific PCR amplification primers
Splicing sequencing results of the exon 2 and the exon 3 intobase:Sub>A complete HLA-A overlapping sequence (Contig) by Seqman software ofbase:Sub>A Lasergene program, carefully checking whether bases subjected to bidirectional sequencing are completely consistent, finding out bases of heterozygote and replacing the bases with merged bases, wherein 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, and K represents G and T, and finally determining the sequence fragment of the amplified HLA-A allele. And (3) comparing the spliced HLA-A base sequence with exon 2 and exon 3 sequences of all HLA-A alleles inbase:Sub>A database by utilizingbase:Sub>A Nucleotide BLAST tool untilbase:Sub>A completely matched gene combination is obtained, thereby determining the HLA-A alleles.
150 patients with positive CTL response to the liver cancer mixed peptide were selected from 500 liver cancer patients by IFN-gamma ELISPOT method. PBMCs of the patients are collected again, immunogenicity of individual epitope peptides is verified by an ELISPOT method, and then combined analysis is performed in combination with HLA-A alleles of the patients and virtual predicted affinities of the epitope peptides to the alleles.
The results show that: there are 9 liver cancer-associated antigenic peptides (P1-P9) that stimulated CTL positive responses in PBMCs from HLA-base:Sub>A x 02;
there are 15 liver cancer-associated antigenic peptides (P10-P24) that stimulated CTL positive responses in PBMCs from HLA-base:Sub>A x 11;
there are 10 liver cancer-associated antigenic peptides (P25-P34) that stimulated CTL positive responses in PBMCs from HLA-base:Sub>A 24;
there are 9 liver cancer-associated antigenic peptides (P35-P43) that stimulated CTL positive responses in PBMCs from HLA-base:Sub>A 31;
there are 9 liver cancer-associated antigenic peptides (P4, P44-P51) that stimulated CTL positive responses in PBMCs from HLA-base:Sub>A 02;
there are 9 liver cancer-associated antigenic peptides (P1, P4, P7-P8, P52-56) that stimulated CTL positive in PBMCs from HLA-A02;
there were 9 liver cancer-associated antigenic peptides (P41, P57-64) that stimulated CTL-positive in PBMCs of HLA-A33 positive patients (FIG. 7);
there are 10 liver cancer-associated antigenic peptides (P10, P65-P73) that stimulated CTL positive responses in PBMCs from HLA-base:Sub>A 30;
there are 8 liver cancer-associated antigenic peptides (P2, P74-P80) that stimulated CTL positive responses in PBMCs from HLA-base:Sub>A x 02 positive patients (fig. 9);
there are 10 liver cancer-associated antigenic peptides (P10-P12, P15, P41, P81-P85) that stimulate CTL positive responses in PBMCs from HLA-base:Sub>A 11;
12 liver cancer-associated antigenic peptides (P10, P16, P68, P86-94) stimulated CTL positive responses in PBMCs of HLA-A03-positive patients (FIG. 11);
there are 9 liver cancer-associated antigenic peptides (P95-P103) that stimulated CTL positive responses in PBMCs from HLA-base:Sub>A 01 positive patients (fig. 12);
there were 8 liver cancer-associated antigenic peptides (P96, P104-P110) that stimulated CTL-positive in PBMCs from HLA-A26.
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 any combination or equivalent changes made on the basis of the above-mentioned embodiments are also within the scope of the present invention.
Attached:
the amino acid sequences of three liver cancer related antigens used for predicting the epitope are as follows:
AFP (P02771): human body protein
GPC3 (P51654): human body protein
GP73 (Q8 NBJ4/B3KNK 9): human body protein
Claims (6)
1. The thymus-dependent lymphocyte epitope peptide of the primary liver cancer related antigen is characterized in that the amino acid sequence of the epitope peptide is any one of epitope peptide sequences limited by HLA-A molecules: RLQDVLQF, GFNYWIASSR, LVLAALVACI.
2. An epitope peptide obtained by removing or replacing single amino acid by using the thymus-dependent lymphocyte epitope peptide of the primary liver cancer-associated antigen in claim 1.
3. The thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-associated antigen as claimed in claim 1 and the use of the antigen epitope peptide as claimed in claim 2 in the preparation of liver cancer polypeptide vaccine or gene vaccine.
4. The use of the thymus-dependent lymphocyte epitope peptide of primary liver cancer-associated antigen of claim 1 and the use of the epitope peptide of claim 2 in the preparation of a detection preparation or kit for detecting liver cancer-associated antigen-specific T cells.
5. The use of claim 4, wherein the detection reagent is an ELISA reagent, an intracellular cytokine fluorescent staining reagent, an ELISA reagent, a human leukocyte antigen multimer fluorescent staining or a flow cytometry reagent.
6. The use of the thymus-dependent lymphocyte epitope peptide of liver cancer-associated antigen as defined in claim 1 and the epitope peptide of liver cancer-associated antigen as defined in claim 2 in the preparation of a medicament for treating liver cancer.
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| CN115785254A (en) | 2023-03-14 |
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| CN116496383A (en) | 2023-07-28 |
| CN110964093B (en) | 2023-05-23 |
| CN115785252A (en) | 2023-03-14 |
| CN115960205A (en) | 2023-04-14 |
| CN110964093A (en) | 2020-04-07 |
| CN115785253A (en) | 2023-03-14 |
| CN116496382A (en) | 2023-07-28 |
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