CN110964093B - 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 PDF

Info

Publication number
CN110964093B
CN110964093B CN201911286067.5A CN201911286067A CN110964093B CN 110964093 B CN110964093 B CN 110964093B CN 201911286067 A CN201911286067 A CN 201911286067A CN 110964093 B CN110964093 B CN 110964093B
Authority
CN
China
Prior art keywords
hla
liver cancer
leu
glu
gln
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201911286067.5A
Other languages
Chinese (zh)
Other versions
CN110964093A (en
Inventor
沈传来
金萧萧
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Dahu Biotechnology Co ltd
Southeast University
Original Assignee
Nanjing Dahu Biotechnology Co ltd
Southeast University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to CN202210907711.1A priority Critical patent/CN115960206A/en
Priority to CN202210905733.4A priority patent/CN115785254A/en
Priority to CN202210905726.4A priority patent/CN115785253A/en
Priority to CN201911286067.5A priority patent/CN110964093B/en
Application filed by Nanjing Dahu Biotechnology Co ltd, Southeast University filed Critical Nanjing Dahu Biotechnology Co ltd
Priority to CN202210912052.0A priority patent/CN116496382A/en
Priority to CN202210912056.9A priority patent/CN116496383A/en
Priority to CN202210905704.8A priority patent/CN115925876A/en
Priority to CN202210905721.1A priority patent/CN115785252A/en
Priority to CN202210907742.7A priority patent/CN115960207A/en
Priority to CN202210907701.8A priority patent/CN115960205A/en
Publication of CN110964093A publication Critical patent/CN110964093A/en
Application granted granted Critical
Publication of CN110964093B publication Critical patent/CN110964093B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001111—Immunoglobulin superfamily
    • A61K39/001114—CD74, Ii, MHC class II invariant chain or MHC class II gamma chain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Oncology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Zoology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Hospice & Palliative Care (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)

Abstract

The invention belongs to the field of medical immunology and oncology, and discloses a thymus-dependent lymphocyte epitope peptide of primary liver cancer related antigens and application thereof, wherein the epitope peptide is respectively limited antigen peptides of 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, 110 of which can be specifically combined with cytotoxic thymus-dependent lymphocytes to stimulate the activation, proliferation and differentiation of the later, thereby playing an anti-tumor immune effect; the antigen peptide can be used for preparing therapeutic and preventive vaccines of liver cancer, can also be used for preparing detection reagents for detecting liver cancer related antigen specific cytotoxic type thymus dependent lymphocytes, and has potential application value in prevention, treatment and diagnosis of liver cancer.

Description

Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer related antigen and application thereof
Technical Field
The invention belongs to the fields 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
Primary liver cancer is the cause of death of the 4 th common malignant tumor and the 3 rd tumor in China, and seriously threatens the life and health of people in China. The pathological type of primary liver cancer is mainly hepatocellular carcinoma (hepatocellular carcinoma, HCC) accounting for 85% -90%; there are also a few intrahepatic cholangiocarcinoma (intrahepatic cholangiocarcinoma, ICC) and HCC-ICC blends, etc., which differ significantly in pathogenesis, biological behavior, molecular characteristics, clinical manifestations, histopathological morphology, treatment methods, prognosis, etc.
In China, the high risk group of HCC mainly comprises the group of hepatitis B virus (hepatitis B virus, HBV) and/or hepatitis C virus (hepatitis C virus, HCV) infection, long-term alcoholism (alcoholic liver disease), nonalcoholic steatohepatitis, food polluted by aflatoxin, liver cirrhosis caused by various reasons and family history of liver cancer, and at the same time, men over 40 years of age have a great risk. Recent studies suggest that diabetes, obesity, smoking, and the like are also risk factors for HCC, and are of great concern.
AFP, GPC3, GP73 are several common tumor-associated antigens that are overexpressed in liver cancer, and have been widely studied in diagnosis, treatment, disease course monitoring, and the like of liver cancer. Serum alpha-fetoprotein (AFP) positive means that the AFP is more than or equal to 400 mug, and chronic or active hepatitis, liver cirrhosis, testicle or ovary embryo-derived tumor, pregnancy and the like are eliminated, and liver cancer is highly suspected. For those with low AFP elevation, dynamic observation should be performed and analyzed in comparison with liver function changes. About 30% of liver cancer patients have normal AFP levels and alpha fetoprotein heterosomes should be detected. GPC3 is one of heparan sulfate glycoproteins, highly expressed in primary liver cancer tissue, while other normal tissues are substantially not expressed or are expressed in low levels. GPC3 can promote proliferation and differentiation of tumor cells, and promote formation, growth and metastasis of liver cancer tumor by combining extracellular matrix, protease, growth factor, etc. GP73 is a golgi type II transmembrane protein, which is expressed poorly in hepatocytes under physiological conditions. The serum GP73 level of the primary liver cancer patient is detected to be obviously high in expression, and the expression of the GP73 is inversely related to the liver function and the illness state of the patient, so that the serum GP73 level is one of effective indexes of the severity degree of the hepatic cell destruction. The patent selects the three liver cancer related tumor antigens for research.
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. For human liver cancer, HLA class I molecules are mainly responsible for the presentation of endogenous liver cancer-associated antigens to CD8 + CTLs, activated CTLs, apoptosis tumor cells expressing tumor-associated antigens by secretion of perforin, granzyme, and the like. Thus, liver cancer-associated antigen-specific CD8 was dynamically monitored + The number and the function of the T cells can accurately reflect the specific immune function state of a liver cancer patient aiming at liver cancer related antigens. Because of different HLA molecular types of different people, the processing, treatment and presenting capacities of different antigens of liver cancer are different, so that the specific T cell immune response reaction of the liver cancer related antigens with different intensities is caused. According to different HLA molecular types of liver cancer patients, selecting antigen peptide of presented liver cancer related antigen, and dynamically monitoring the specificity of the liver cancer related antigen to CD8 + The number and the reactivity 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 liver cancer patients, and are important technical means for realizing accurate medical treatment of liver cancer. Meanwhile, the antigen peptide of liver cancer related antigen combined by the HLA-A molecules with high affinity can be used for preparing polypeptide vaccine or gene vaccine for preventing and treating liver cancer.
However, currently, the number of primary liver cancer related antigen T cell epitopes which are clearly presented by various HLA molecules and can stimulate organisms to cause T cell response is still very small, so that the development of specific T cell detection on liver cancer patients carrying different HLA alleles is limited, the research on the action of liver cancer specific T cells in the development of liver cancer is also limited, and the personalized detection and accurate immunotherapy based on individual differences of HLA genes and the presented liver cancer related 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 liver cancer related antigen and the 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 antigen epitope peptide of the liver cancer-related antigen is any one of epitope peptide sequences shown as the following:
Figure SMS_1
/>
Figure SMS_2
/>
Figure SMS_3
/>
Figure SMS_4
as can be seen from the above table, the above sequences are the antigen peptide sequences of Alpha Fetoprotein (AFP), glypican-3 (GPC 3) and Golgi transmembrane glycoprotein 73 (GP 73), respectively, and bind with high affinity 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, respectively, to form "HLA/antigen peptide" complex molecules on the surface of antigen presenting cells, and bind with antigen peptideCD8 with different specificity + The T cell clone is combined to stimulate the activation, proliferation and differentiation of the T cell clone and play the role of anti-liver cancer immune effect.
The thymus-dependent lymphocyte antigen epitope peptide sequence of the primary liver cancer related antigen can be used for preparing liver cancer polypeptide vaccine or gene vaccine: preparation of polypeptide vaccine: the polypeptide sequence of the invention is synthesized into one or more polypeptides artificially, and the one or more polypeptides are mixed with an adjuvant to prepare a soluble preparation, or the soluble preparation is loaded by biological nano materials to prepare a nano polypeptide vaccine, and the nano polypeptide vaccine is injected into a liver cancer patient to excite the activation and proliferation of liver cancer related antigen specific T cells of the patient and enhance the activity of tumor killing cells of the patient, so that the liver cancer polypeptide vaccine is prepared. Gene vaccine preparation: according to the polypeptide sequence, a recombinant DNA gene fragment, recombinant plasmid or recombinant viral vector of one or more polypeptides is constructed, and is injected into a liver cancer patient, so that the recombinant gene expresses one or more polypeptides in the body, activates and proliferates liver cancer related antigen specific T cells of the patient, and enhances the activity of tumor killing cells of the patient, thereby preparing the liver cancer gene vaccine.
The thymic 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 liver cancer related antigen specific T cells: according to the polypeptide sequence, one or more polypeptides are synthesized artificially, and are 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 liver cancer related antigen-specific T cells, and then the synthesis amount of the cytokines is detected by 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 using a genetic engineering technology and a protein engineering technology, and further preparing a fluorescein-labeled preparation, and detecting the number of liver cancer-related antigen-specific T cells in a peripheral blood mononuclear cell population of a patient by using a flow cytometry method. The related kit is a liver cancer related antigen specific T cell detection kit assembled by the preparation and other reagents commonly used in different detection methods.
The thymic dependent lymphocyte antigen epitope peptide sequence of the primary liver cancer related antigen can be used for preparing medicines for treating liver cancer: the polypeptide vaccine or gene vaccine based on the polypeptide sequence of the invention is combined with other immunotherapeutic preparations or chemotherapeutic preparations to prepare the clinical medicine for treating liver cancer.
The invention virtually predicts the liver cancer related antigen specific antigen epitope peptide sequences of 13 HLA-A molecules limited by six online epitope prediction databases to obtain a group of liver cancer related antigen epitope peptide sequences which can be 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, HLA-A2601 molecules in high affinity, and then verifies the immunogenicity thereof through ELISPOT functional experiments, thereby providing specific antigen peptides for preparing liver cancer therapeutic and preventive vaccines, developing liver cancer related antigen specific T cell detection reagents and the like.
1. Selecting Alpha Fetoprotein (AFP), phosphatidylinositol proteoglycan-3 (GPC 3) and Golgi transmembrane glycoprotein 73 (GP 73) 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 the above 13 HLA-A molecule restricted liver cancer related epitope peptide sequences;
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 the liver cancer related antigen peptide is verified by IFN-gamma ELISPOT cell functional experiments.
The invention is an antigenic peptide sequence which can be 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 respectively in Alpha Fetoprotein (AFP), phosphatidylinositol proteoglycan-3 (GPC 3) and golgi transmembrane glycoprotein 73 (GP 73) in high affinity and has immunogenicity; also relates to a liver cancer polypeptide vaccine and gene vaccine based on the antigen peptide, and a reagent and a method for detecting liver cancer related antigen-specific T cells based on the antigen peptide.
The advantages of the present invention over the prior art are as follows,
the primary liver cancer related antigen-specific epitope peptides obtained through online virtual prediction and functional experiment verification are not reported before, namely 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-A 2601. These HLA-A molecules have not previously been reported to have a restricted liver cancer associated antigenic peptide. Therefore, the new epitope peptide sequences provide key antigen components, namely epitope peptide sequences, required for developing therapeutic and preventive polypeptide vaccines and gene vaccines for liver cancer, designing reagents and methods for detecting liver cancer related antigen-specific T cells and the like; meanwhile, the epitope peptides also provide key antigen components for individual detection and accurate medical treatment of liver cancer patients aiming at the specific HLA-A alleles.
Description of the drawings:
FIG. 1 shows HLA-A0201 molecule limited liver cancer related antigen T cell epitope peptide;
a) Identifying detection holes, negative control holes and positive control Kong Ban dot patterns of HLA-A0201 molecule restrictive liver cancer related antigen T cell epitope peptide by using an IFN-gamma ELISPOT method;
b) Counting the spot numbers of the detection holes and the negative control holes;
c) Statistical plot of spot number ratio (P/N) of test wells to negative control wells.
FIG. 2 shows HLA-A1101 molecule limited liver cancer associated antigen T cell epitope peptide;
a) Identifying detection holes, negative control holes and positive control Kong Ban dot patterns of HLA-A1101 molecule restrictive liver cancer related antigen T cell epitope peptide by using an IFN-gamma ELISPOT method;
b) Counting the spot numbers of the detection holes and the negative control holes;
c) Statistical plot of spot number ratio (P/N) of test wells to negative control wells.
FIG. 3 shows HLA-A2402 molecule-restricted liver cancer associated antigen T cell epitope peptide;
a) Identifying detection holes, negative control holes and positive control Kong Ban dot patterns of HLA-A2402 molecule restrictive liver cancer related antigen T cell epitope peptide by using an IFN-gamma ELISPOT method;
b) Counting the spot numbers of the detection holes and the negative control holes;
c) Statistical plot of spot number ratio (P/N) of test wells to negative control wells.
FIG. 4 shows HLA-A3101 molecule-restricted liver cancer associated antigen T cell epitope peptides;
a) Identifying detection holes, negative control holes and positive control Kong Ban dot patterns of HLA-A3101 molecule restrictive liver cancer related antigen T cell epitope peptide by using an IFN-gamma ELISPOT method;
b) Counting the spot numbers of the detection holes and the negative control holes;
c) Statistical plot of spot number ratio (P/N) of test wells to negative control wells.
FIG. 5 shows HLA-A0206 molecule limited liver cancer associated antigen T cell epitope peptide;
a) Identifying detection holes, negative control holes and positive control Kong Ban dot patterns of HLA-A0206 molecule restrictive liver cancer related antigen T cell epitope peptide by using an IFN-gamma ELISPOT method;
b) Counting the spot numbers of the detection holes and the negative control holes;
c) Statistical plot of spot number ratio (P/N) of test wells to negative control wells.
FIG. 6 shows HLA-A0207 molecule limited liver cancer associated antigen T cell epitope peptide;
a) Identifying detection holes, negative control holes and positive control Kong Ban dot patterns of HLA-A0207 molecule restrictive liver cancer related antigen T cell epitope peptide by using an IFN-gamma ELISPOT method;
b) Counting the spot numbers of the detection holes and the negative control holes;
c) Statistical plot of spot number ratio (P/N) of test wells to negative control wells.
FIG. 7 shows HLA-A3303 molecule-restricted liver cancer associated antigen T cell epitope peptides;
a) Identifying detection holes, negative control holes and positive control Kong Ban dot patterns of HLA-A3303 molecule restrictive liver cancer related antigen T cell epitope peptide by using an IFN-gamma ELISPOT method;
b) Counting the spot numbers of the detection holes and the negative control holes;
c) Statistical plot of spot number ratio (P/N) of test wells to negative control wells.
FIG. 8 shows HLA-A3001 molecule-restricted liver cancer associated antigen T cell epitope peptides;
a) Identifying detection holes, negative control holes and positive control Kong Ban dot patterns of HLA-A3001 molecule-restricted liver cancer related antigen T cell epitope peptide by using an IFN-gamma ELISPOT method;
b) Counting the spot numbers of the detection holes and the negative control holes;
c) Statistical plot of spot number ratio (P/N) of test wells to negative control wells.
FIG. 9 shows HLA-A0203 molecule limited liver cancer associated antigen T cell epitope peptide;
a) Identifying detection holes, negative control holes and positive control Kong Ban dot patterns of HLA-A0203 molecule restrictive liver cancer related antigen T cell epitope peptide by using an IFN-gamma ELISPOT method;
b) Counting the spot numbers of the detection holes and the negative control holes;
c) Statistical plot of spot number ratio (P/N) of test wells to negative control wells.
FIG. 10 shows HLA-A1102 molecule-restricted liver cancer associated antigen T cell epitope peptides;
a) Identifying detection holes, negative control holes and positive control Kong Ban dot patterns of HLA-A1102 molecule-restricted liver cancer related antigen T cell epitope peptide by using an IFN-gamma ELISPOT method;
b) Counting the spot numbers of the detection holes and the negative control holes;
c) Statistical plot of spot number ratio (P/N) of test wells to negative control wells.
FIG. 11 shows HLA-A0301 molecule restricted liver cancer associated antigen T cell epitope peptide;
a) Identifying detection holes, negative control holes and positive control Kong Ban dot patterns of HLA-A0301 molecule restrictive liver cancer related antigen T cell epitope peptide by using an IFN-gamma ELISPOT method;
b) Counting the spot numbers of the detection holes and the negative control holes;
c) Statistical plot of spot number ratio (P/N) of test wells to negative control wells.
FIG. 12 shows HLA-A0101 molecule-restricted liver cancer associated antigen T cell epitope peptides;
a) Identifying detection holes, negative control holes and positive control Kong Ban dot patterns of HLA-A0101 molecule restrictive liver cancer related antigen T cell epitope peptide by using an IFN-gamma ELISPOT method;
b) Counting the spot numbers of the detection holes and the negative control holes;
c) Statistical plot of spot number ratio (P/N) of test wells to negative control wells.
FIG. 13 shows HLA-A2601 molecule restricted liver cancer associated antigen T cell epitope peptide;
a) Identifying detection holes, negative control holes and positive control Kong Ban dot patterns of HLA-A2601 molecule restrictive liver cancer related antigen T cell epitope peptide by using an IFN-gamma ELISPOT method;
b) Counting the spot numbers of the detection holes and the negative control holes;
c) Statistical plot of spot number ratio (P/N) of test wells to 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 sequences of the liver cancer related antigen T cell epitopes restricted by the 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 are screened and identified by the following steps:
1 on-line virtual prediction of dominant T cell epitope peptide of three liver cancer related antigens limited by 13 HLA-A molecules
Three liver cancer related antigen proteins are selected: alpha Fetoprotein (AFP), phosphatidylinositol proteoglycan-3 (GPC 3) and Golgi transmembrane glycoprotein 73 (GP 73), obtaining the amino acid sequence thereof through searching a UniProt global protein resource database, and selecting the most studied standard sequence; virtual prediction is carried out on T cell epitope peptides 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 and are aiming at each liver cancer related antigen 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 most common, so that polypeptides of 9 and 10 amino acids in length are mainly selected as subjects for this experiment. The amino acid sequences of the liver cancer related antigen proteins are respectively input into the corresponding amino acid sequence input boxes of the forecast database website, the lengths of the epitope peptides are respectively selected to be 9 and 10 amino acids, then specific HLA-A molecules are selected, and the T cell epitope peptides of the liver cancer related antigens are virtually forecast on line.
Table 1 epitope peptide predictive database website
Figure SMS_5
Aiming at each HLA-A molecule and each liver cancer related antigen protein, the polypeptides predicted by different databases are respectively arranged from high score to low score, and then epitope peptides meeting at least two prediction method scoring standards are selected as candidate epitope peptides. For each HLA-A molecule, aiming at each liver cancer related antigen protein, 1-5 polypeptides with highest scores (highest affinity) are selected from candidate epitope peptides to be used as epitope peptides to be identified. For the 13 HLA-A molecules described above, a total screen predicts 160 dominant T cell epitope peptides to be identified.
2 separation of peripheral blood PBMC of liver cancer patient
Taking fresh anticoagulation whole stored at room temperatureBlood, diluted appropriately 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 liver cancer related antigen T cell epitope peptide by IFN-gamma ELISPOT method
The technical route is shown in fig. 14:
first screening liver cancer patients with 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 13 groups, wherein each group comprises 8-9 epitope peptides; taking an ELISPOT plate (Daidae is biological, shenzhen) pre-coated with an anti-human IFN-gamma antibody, activating for 8min by using a serum-free culture medium (200 mu L/well), and adding PBMC suspension (100 mu L/well) of a liver cancer patient into each well; then, adding mixed peptide limited by each HLA-A molecule into a detection hole, wherein the single polypeptide in the mixed peptide is 15 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 24 hours; 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 negative Kong Ban points is 0-5, detecting Kong Ban points is not less than 6, and judging that the CTL reaction is positive; if the number of the negative control hole spots is not less than 6, the number of the detection holes is not less than 2 times of the number of the negative hole spots, and the CTL reaction is judged to be positive.
Identifying immunogenicity of a single epitope peptide: re-collecting peripheral blood PBMC of liver cancer patient with CTL positive reaction to the mixed peptide, adding into the above ELISPOT plate, each timeThe single liver cancer related antigen polypeptide (15 mug/mL) in the positive mixed peptide is added into each detection hole, a positive control hole and a negative control hole are arranged at the same time, the temperature is 37 ℃ and the concentration of CO is 5% 2 Incubators were incubated for 24h 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 liver cancer patient was genotyped with HLA-A alleles, and the virtual affinity of the epitope peptide was bound, initially determined which HLA-A molecules bound and presented. The homozygote infected person of the 13 HLA-A alleles is selected from liver cancer patients, PBMC thereof is taken, and the HLA-A molecular restriction of each epitope peptide is further determined by carrying out ELISPOT detection again with all epitope peptides which are verified to have immunogenicity and are restricted by the HLA-A molecules.
4HLA-A allele typing
Selecting liver cancer 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.
TABLE 2 HLA-A site specific PCR amplification primers
Figure SMS_6
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.
150 patients with CTL positive response to the liver cancer mixed peptide were selected from 500 liver cancer 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 with HLA-A 02:01 positive were stimulated to exhibit CTL positive responses by 9 liver cancer-associated antigenic peptides (P1-P9) (FIG. 1);
there are 15 liver cancer associated antigenic peptides (P10-P24) that stimulated CTL positive responses in PBMCs of HLA-A 11:01 positive patients (fig. 2);
PBMC from patients with HLA-A 24:02 positive were stimulated to exhibit CTL positive responses by 10 liver cancer associated antigenic peptides (P25-P34) (FIG. 3);
PBMC from HLA-A 31:01 positive patients were stimulated to exhibit CTL-positive responses by 9 liver cancer-associated antigenic peptides (P35-P43) (FIG. 4);
PBMC from patients with HLA-A 02:06 positive were stimulated to exhibit CTL positive responses by 9 liver cancer associated antigenic peptides (P4, P44-P51) (FIG. 5);
there were 9 liver cancer associated antigenic peptides (P1, P4, P7-P8, P52-56) that stimulated CTL positive response in PBMCs of HLA-A 02:07 positive patients (fig. 6);
there were 9 liver cancer associated antigenic peptides (P41, P57-64) that stimulated CTL positive responses in PBMCs of HLA-A x 33:03 positive patients (fig. 7);
there are 10 liver cancer associated antigenic peptides (P10, P65-P73) that stimulated CTL positive responses in PBMCs of HLA-A x 30:01 positive patients (fig. 8);
8 liver cancer associated antigenic peptides (P2, P74-P80) stimulated CTL positive responses in PBMC of HLA-A 02:03 positive patients (FIG. 9);
there are 10 liver cancer associated antigenic peptides (P10-P12, P15, P41, P81-P85) that stimulated CTL positive response in PBMCs of HLA-A 11:02 positive patients (fig. 10);
there are 12 liver cancer associated antigenic peptides (P10, P16, P68, P86-94) that stimulated CTL positive responses in PBMC from HLA-A 03:01 positive patients (FIG. 11);
PBMC from patients positive for HLA-A 01:01 were stimulated to exhibit CTL-positive responses by 9 liver cancer-associated antigenic peptides (P95-P103) (FIG. 12);
8 liver cancer associated antigenic peptides (P96, P104-P110) stimulated CTL positive responses in PBMC from HLA-A 26:01 positive patients (FIG. 13).
It should be noted that the foregoing is merely a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and any combination or equivalent transformation made on the basis of the foregoing embodiment falls within the scope of the present invention.
The method comprises the following steps:
the amino acid sequences of three liver cancer-associated antigens used to predict epitopes are as follows:
AFP (P02771): human protein
Figure SMS_7
GPC3 (P51654): human protein
Figure SMS_8
/>
Figure SMS_9
GP73 (Q8 NBJ4/B3KNK 9): human protein
Figure SMS_10
。/>
Sequence listing
<110> Nanjing Dajingkou biotechnology Co., ltd
Southeast University
<120> thymus-dependent lymphocyte epitope peptide of primary liver cancer-associated antigen and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 600
<212> PRT
<213> Human protein (Human protein)
<400> 1
Met Lys Trp Val Glu Ser Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr
1 5 10 15
Glu Ser Arg Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu
20 25 30
Asp Ser Tyr Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr
35 40 45
Ile Phe Phe Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser
50 55 60
Lys Met Val Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp
65 70 75 80
Glu Gln Ser Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu
85 90 95
Glu Leu Cys His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp
100 105 110
Cys Cys Ser Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His
115 120 125
Lys Lys Pro Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro Glu Pro
130 135 140
Val Thr Ser Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn
145 150 155 160
Lys Phe Ile Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro
165 170 175
Thr Ile Leu Leu Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys
180 185 190
Cys Lys Ala Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr
195 200 205
Val Thr Lys Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys
210 215 220
Ala Val Met Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val
225 230 235 240
Thr Lys Leu Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln
245 250 255
Lys Leu Val Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly
260 265 270
Asp Val Leu Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile
275 280 285
Cys Ser Gln Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys
290 295 300
Leu Thr Thr Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp
305 310 315 320
Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp
325 330 335
Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala
340 345 350
Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser
355 360 365
Val Ile Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys
370 375 380
Phe Gln Thr Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu
385 390 395 400
Leu Gln Lys Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys
405 410 415
Gly Leu Phe Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Ala Phe Leu
420 425 430
Val Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met
435 440 445
Ala Ile Thr Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu
450 455 460
Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile
465 470 475 480
Ile Gly His Leu Cys Ile Arg His Glu Met Thr Pro Val Asn Pro Gly
485 490 495
Val Gly Gln Cys Cys Thr Ser Ser Tyr Ala Asn Arg Arg Pro Cys Phe
500 505 510
Ser Ser Leu Val Val Asp Glu Thr Tyr Val Pro Pro Ala Phe Ser Asp
515 520 525
Asp Lys Phe Ile Phe His Lys Asp Leu Cys Gln Ala Gln Gly Val Ala
530 535 540
Leu Gln Thr Met Lys Gln Glu Phe Leu Ile Asn Leu Val Lys Gln Lys
545 550 555 560
Pro Gln Ile Thr Glu Glu Gln Leu Glu Ala Val Ile Ala Asp Phe Ser
565 570 575
Gly Leu Leu Glu Lys Cys Cys Gln Gly Gln Glu Gln Glu Val Cys Phe
580 585 590
Ala Glu Glu Gly Gln Lys Leu Ile
595 600
<210> 2
<211> 580
<212> PRT
<213> Human protein (Human protein)
<400> 2
Met Ala Gly Thr Val Arg Thr Ala Cys Leu Val Val Ala Met Leu Leu
1 5 10 15
Ser Leu Asp Phe Pro Gly Gln Ala Gln Pro Pro Pro Pro Pro Pro Asp
20 25 30
Ala Thr Cys His Gln Val Arg Ser Phe Phe Gln Arg Leu Gln Pro Gly
35 40 45
Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu Gln Val
50 55 60
Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Glu Lys
65 70 75 80
Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln Ser Ala
85 90 95
Ser Met Glu Leu Lys Phe Leu Ile Ile Gln Asn Ala Ala Val Phe Gln
100 105 110
Glu Ala Phe Glu Ile Val Val Arg His Ala Lys Asn Tyr Thr Asn Ala
115 120 125
Met Phe Lys Asn Asn Tyr Pro Ser Leu Thr Pro Gln Ala Phe Glu Phe
130 135 140
Val Gly Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu Gly Ser Asp
145 150 155 160
Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu Phe Pro
165 170 175
Val Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser Ala Leu
180 185 190
Asp Ile Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys Val Phe
195 200 205
Gly Asn Phe Pro Lys Leu Ile Met Thr Gln Val Ser Lys Ser Leu Gln
210 215 220
Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu Val Ile
225 230 235 240
Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp Cys Gly Arg Met Leu
245 250 255
Thr Arg Met Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met Val Lys
260 265 270
Pro Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met Ala Gly
275 280 285
Val Val Glu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu
290 295 300
Glu Leu Val Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn Val Leu
305 310 315 320
Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile Gln Tyr Val Gln Lys
325 330 335
Asn Ala Gly Lys Leu Thr Thr Thr Ile Gly Lys Leu Cys Ala His Ser
340 345 350
Gln Gln Arg Gln Tyr Arg Ser Ala Tyr Tyr Pro Glu Asp Leu Phe Ile
355 360 365
Asp Lys Lys Val Leu Lys Val Ala His Val Glu His Glu Glu Thr Leu
370 375 380
Ser Ser Arg Arg Arg Glu Leu Ile Gln Lys Leu Lys Ser Phe Ile Ser
385 390 395 400
Phe Tyr Ser Ala Leu Pro Gly Tyr Ile Cys Ser His Ser Pro Val Ala
405 410 415
Glu Asn Asp Thr Leu Cys Trp Asn Gly Gln Glu Leu Val Glu Arg Tyr
420 425 430
Ser Gln Lys Ala Ala Arg Asn Gly Met Lys Asn Gln Phe Asn Leu His
435 440 445
Glu Leu Lys Met Lys Gly Pro Glu Pro Val Val Ser Gln Ile Ile Asp
450 455 460
Lys Leu Lys His Ile Asn Gln Leu Leu Arg Thr Met Ser Met Pro Lys
465 470 475 480
Gly Arg Val Leu Asp Lys Asn Leu Asp Glu Glu Gly Phe Glu Ser Gly
485 490 495
Asp Cys Gly Asp Asp Glu Asp Glu Cys Ile Gly Gly Ser Gly Asp Gly
500 505 510
Met Ile Lys Val Lys Asn Gln Leu Arg Phe Leu Ala Glu Leu Ala Tyr
515 520 525
Asp Leu Asp Val Asp Asp Ala Pro Gly Asn Ser Gln Gln Ala Thr Pro
530 535 540
Lys Asp Asn Glu Ile Ser Thr Phe His Asn Leu Gly Asn Val His Ser
545 550 555 560
Pro Leu Lys Leu Leu Thr Ser Met Ala Ile Ser Val Val Cys Phe Phe
565 570 575
Phe Leu Val His
580
<210> 3
<211> 401
<212> PRT
<213> Human protein (Human protein)
<400> 3
Met Met Gly Leu Gly Asn Gly Arg Arg Ser Met Lys Ser Pro Pro Leu
1 5 10 15
Val Leu Ala Ala Leu Val Ala Cys Ile Ile Val Leu Gly Phe Asn Tyr
20 25 30
Trp Ile Ala Ser Ser Arg Ser Val Asp Leu Gln Thr Arg Ile Met Glu
35 40 45
Leu Glu Gly Arg Val Arg Arg Ala Ala Ala Glu Arg Gly Ala Val Glu
50 55 60
Leu Lys Lys Asn Glu Phe Gln Gly Glu Leu Glu Lys Gln Arg Glu Gln
65 70 75 80
Leu Asp Lys Ile Gln Ser Ser His Asn Phe Gln Leu Glu Ser Val Asn
85 90 95
Lys Leu Tyr Gln Asp Glu Lys Ala Val Leu Val Asn Asn Ile Thr Thr
100 105 110
Gly Glu Arg Leu Ile Arg Val Leu Gln Asp Gln Leu Lys Thr Leu Gln
115 120 125
Arg Asn Tyr Gly Arg Leu Gln Gln Asp Val Leu Gln Phe Gln Lys Asn
130 135 140
Gln Thr Asn Leu Glu Arg Lys Phe Ser Tyr Asp Leu Ser Gln Cys Ile
145 150 155 160
Asn Gln Met Lys Glu Val Lys Glu Gln Cys Glu Glu Arg Ile Glu Glu
165 170 175
Val Thr Lys Lys Gly Asn Glu Ala Val Ala Ser Arg Asp Leu Ser Glu
180 185 190
Asn Asn Asp Gln Arg Gln Gln Leu Gln Ala Leu Ser Glu Pro Gln Pro
195 200 205
Arg Leu Gln Ala Ala Gly Leu Pro His Thr Glu Val Pro Gln Gly Lys
210 215 220
Gly Asn Val Leu Gly Asn Ser Lys Ser Gln Thr Pro Ala Pro Ser Ser
225 230 235 240
Glu Val Val Leu Asp Ser Lys Arg Gln Val Glu Lys Glu Glu Thr Asn
245 250 255
Glu Ile Gln Val Val Asn Glu Glu Pro Gln Arg Asp Arg Leu Pro Gln
260 265 270
Glu Pro Gly Arg Glu Gln Val Val Glu Asp Arg Pro Val Gly Gly Arg
275 280 285
Gly Phe Gly Gly Ala Gly Glu Leu Gly Gln Thr Pro Gln Val Gln Ala
290 295 300
Ala Leu Ser Val Ser Gln Glu Asn Pro Glu Met Glu Gly Pro Glu Arg
305 310 315 320
Asp Gln Leu Val Ile Pro Asp Gly Gln Glu Glu Glu Gln Glu Ala Ala
325 330 335
Gly Glu Gly Arg Asn Gln Gln Lys Leu Arg Gly Glu Asp Asp Tyr Asn
340 345 350
Met Asp Glu Asn Glu Ala Glu Ser Glu Thr Asp Lys Gln Ala Ala Leu
355 360 365
Ala Gly Asn Asp Arg Asn Ile Asp Val Phe Asn Val Glu Asp Gln Lys
370 375 380
Arg Asp Thr Ile Asn Leu Leu Asp Gln Arg Glu Lys Arg Asn His Thr
385 390 395 400
Leu

Claims (5)

1. The thymus-dependent lymphocyte antigen epitope peptide of the primary liver cancer related antigen is characterized in that the amino acid sequence of the antigen epitope peptide is one of HLA-A molecule restricted epitope peptide sequences: YQDEKAVLV.
2. The use of the thymic dependent lymphocyte epitope peptide of a primary liver cancer associated antigen according to claim 1 in the preparation of a liver cancer polypeptide vaccine or a genetic vaccine.
3. The use of the thymic dependent lymphocyte epitope peptide of a primary liver cancer associated antigen according to claim 1 in the preparation of a detection preparation or a kit for detecting liver cancer associated antigen specific T cells.
4. The use according to claim 3, 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.
5. The use of the thymic dependent lymphocyte epitope peptide according to claim 1, as a primary liver cancer associated antigen, in the preparation of a medicament for treating liver cancer.
CN201911286067.5A 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer related antigen and application thereof Active CN110964093B (en)

Priority Applications (10)

Application Number Priority Date Filing Date Title
CN202210905726.4A CN115785253A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN201911286067.5A CN110964093B (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer related antigen and application thereof
CN202210905721.1A CN115785252A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210912052.0A CN116496382A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer related antigen and application thereof
CN202210905733.4A CN115785254A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210905704.8A CN115925876A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210907711.1A CN115960206A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210907742.7A CN115960207A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210907701.8A CN115960205A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210912056.9A CN116496383A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer related antigen and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911286067.5A CN110964093B (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer related antigen and application thereof

Related Child Applications (9)

Application Number Title Priority Date Filing Date
CN202210905733.4A Division CN115785254A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210905704.8A Division CN115925876A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210905726.4A Division CN115785253A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210905721.1A Division CN115785252A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210907742.7A Division CN115960207A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210912052.0A Division CN116496382A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer related antigen and application thereof
CN202210912056.9A Division CN116496383A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer related antigen and application thereof
CN202210907711.1A Division CN115960206A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210907701.8A Division CN115960205A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof

Publications (2)

Publication Number Publication Date
CN110964093A CN110964093A (en) 2020-04-07
CN110964093B true CN110964093B (en) 2023-05-23

Family

ID=70034249

Family Applications (10)

Application Number Title Priority Date Filing Date
CN202210907742.7A Pending CN115960207A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210905726.4A Pending CN115785253A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210912056.9A Pending CN116496383A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer related antigen and application thereof
CN202210907711.1A Pending CN115960206A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN201911286067.5A Active CN110964093B (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer related antigen and application thereof
CN202210912052.0A Pending CN116496382A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer related antigen and application thereof
CN202210905721.1A Pending CN115785252A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210905733.4A Pending CN115785254A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210907701.8A Pending CN115960205A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210905704.8A Pending CN115925876A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof

Family Applications Before (4)

Application Number Title Priority Date Filing Date
CN202210907742.7A Pending CN115960207A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210905726.4A Pending CN115785253A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210912056.9A Pending CN116496383A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer related antigen and application thereof
CN202210907711.1A Pending CN115960206A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof

Family Applications After (5)

Application Number Title Priority Date Filing Date
CN202210912052.0A Pending CN116496382A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer related antigen and application thereof
CN202210905721.1A Pending CN115785252A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210905733.4A Pending CN115785254A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210907701.8A Pending CN115960205A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
CN202210905704.8A Pending CN115925876A (en) 2019-12-13 2019-12-13 Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof

Country Status (1)

Country Link
CN (10) CN115960207A (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022022696A1 (en) * 2020-07-30 2022-02-03 香雪生命科学技术（广东）有限公司 High-affinity tcr for recognizing afp
CN113956342B (en) * 2021-12-22 2022-03-04 北京大学人民医院 Tumor neogenesis antigen polypeptide and application thereof
CN115785206B (en) * 2022-06-10 2024-03-12 河北博海生物工程开发有限公司 Lung cancer specific molecular target 07 and uses thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2021500852A (en) * 2017-08-18 2021-01-14 グリットストーン オンコロジー インコーポレイテッド Antigen-binding protein that targets shared antigens

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
原发性肝癌高表达抗原GPC-3的HLA-A2限制性细胞毒性T淋巴细胞表位预测;雷俊华，等;《解放军医药杂志》;20130828;第25卷(第08期);第26-28页 *
肝癌相关肿瘤抗原HLA-A2限制性CTL表位的预测;董海龙，等;《第四军医大学学报》;20031231;第24卷(第6期);第492-494页 *

Also Published As

Publication number Publication date
CN115960207A (en) 2023-04-14
CN115785254A (en) 2023-03-14
CN115925876A (en) 2023-04-07
CN115960206A (en) 2023-04-14
CN116496383A (en) 2023-07-28
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

Similar Documents

Publication Publication Date Title
CN110964093B (en) Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer related antigen and application thereof
CN109081867B (en) Cancer specific TCR and assay techniques and uses thereof
CN111116719B (en) Thymus dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen and its application
CN110627895B (en) Lung cancer specific TCR and analysis technology and application thereof
CN105524984A (en) Method and equipment for neoantigen epitope prediction
CN110172080B (en) Thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen and application thereof
CN114377112A (en) Immune inducer
CN113956342B (en) Tumor neogenesis antigen polypeptide and application thereof
CN116970058B (en) Tumor neoantigen polypeptide aiming at TP53 gene R249S mutation and application thereof
WO2003030725A2 (en) Pancreatic cancer diagnosis and therapies
CN112940105B (en) HLA-A11 restrictive hepatitis B virus HBc 141-151 T cell receptor of epitope peptide and application thereof
WO2011038689A1 (en) Novel human cytokine vstm1-v2 and use thereof
CN113416240B (en) Universal antigen peptide library and kit for inducing tumor specific immune response
CN111434674B (en) Polypeptide composition and use thereof in cancer immunotherapy
CN106119195A (en) A kind of for inducing the test kit and abductive approach producing the DC cell identifying HPV
CN111647069B (en) Improved TCR and application thereof
CN116948004B (en) Tumor new antigen polypeptide aiming at CTNNB1 gene H36P mutation and application thereof
Malone et al. Performance of neoantigen prediction for the design of TG4050, a patient specific neoantigen cancer vaccine
CN114591404A (en) Hepatitis B virus antigen peptide suitable for leukocyte antigen haplotype as HLA-A2 individual and application thereof
CN114675035A (en) Antigen-specific thymus-dependent lymphocyte universality detection technical scheme suitable for extensive population in east Asia region
CN1331885C (en) Tumor antigen protein and tumor antigen peptide
CN117964720A (en) HLA-A 02:01 restrictive SARS-CoV-2T cell epitope peptide and its application
CN117999350A (en) Identification method of T cell epitope sequence and application thereof
CN116589557A (en) Tumor neoantigen specific TCR and application thereof
WO2010042852A2 (en) T-cell-based identification of tissue antigens

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant