CN110964093A - 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
CN110964093A
CN110964093A CN201911286067.5A CN201911286067A CN110964093A CN 110964093 A CN110964093 A CN 110964093A CN 201911286067 A CN201911286067 A CN 201911286067A CN 110964093 A CN110964093 A CN 110964093A
Authority
CN
China
Prior art keywords
hla
liver cancer
leu
glu
antigen
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.)
Granted
Application number
CN201911286067.5A
Other languages
Chinese (zh)
Other versions
CN110964093B (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 CN202210912052.0A priority Critical patent/CN116496382A/en
Priority to CN202210905721.1A priority patent/CN115785252A/en
Priority to CN202210912056.9A priority patent/CN116496383A/en
Priority to CN202210905726.4A priority patent/CN115785253A/en
Application filed by Nanjing Dahu Biotechnology Co ltd, Southeast University filed Critical Nanjing Dahu Biotechnology Co ltd
Priority to CN202210907711.1A priority patent/CN115960206A/en
Priority to CN202210905704.8A priority patent/CN115925876A/en
Priority to CN202210907701.8A priority patent/CN115960205A/en
Priority to CN201911286067.5A priority patent/CN110964093B/en
Priority to CN202210905733.4A priority patent/CN115785254A/en
Priority to CN202210907742.7A priority patent/CN115960207A/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 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

Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related antigen and application thereof
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, disease course monitoring and the like of liver cancer. The positive serum alpha-fetoprotein (AFP) means that AFP is more than or equal to 400 mu g, chronic or active hepatitis, liver cirrhosis, testis or ovary embryonic-derived tumors, pregnancy and the like are excluded, and liver cancer is highly suspected. For patients with low AFP elevation, dynamic observation should be performed and analyzed in comparison with liver function changes. 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 substantially expressed or are low expressed. GPC3 can promote tumor cell proliferation and differentiation, and has effects of promoting formation, growth and metastasis of hepatocarcinoma by combining extracellular matrix, protease and growth factor. GP73 is a golgi type II transmembrane protein, which is rarely expressed physiologically in hepatocytes. The serum GP73 level of a patient with primary liver cancer is detected to show that the expression of the serum GP73 is remarkably high, and the expression of GP73 is negatively related to the liver function and the disease condition of the patient, and is one effective index of the severity of hepatocyte damage. 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 leucocyte 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 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 primarily responsible for 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 human HLA molecular types are different, the processing, treatment and presentation abilities of different antigens of liver cancer are different, thereby causing liver cancer with different strengthsAssociated antigen-specific T cell immune response. According to different HLA molecular types of liver cancer patients, antigen peptides of presented liver cancer related antigens are selected, and the specificity of the liver cancer related antigens, namely 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, a polypeptide vaccine or 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 the occurrence and development of liver cancer is also limited, and the individual difference of HLA genes and the individual difference of presented liver cancer related antigen peptides and the precise immunotherapy are further limited.
Disclosure of Invention
The invention solves the technical problems in the prior art and provides thymus-dependent lymphocyte 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:
Figure BDA0002318008280000021
Figure BDA0002318008280000031
Figure BDA0002318008280000041
Figure BDA0002318008280000051
as can be seen from the above tables, the above sequences are antigenic peptide sequences of alpha-fetoprotein (AFP), glypican-3 (GPC3) and Golgi transmembrane glycoprotein 73(GP73), which are respectively bound 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, form "HLA/antigenic peptide" complex molecules on the surface of antigen presenting cells, and are bound with antigenic peptide-specific CD8+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 liver cancer related antigen specific T cells: 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 of the human leukocyte antigen and the polypeptide (peptide-HLA complex) and a polymer thereof by a genetic 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 peripheral blood mononuclear cell population of the patient by a flow cytometry. 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 obtain 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 (GPC3) and Golgi transmembrane glycoprotein 73(GP73) 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 liver cancer related epitope peptide sequence restricted by the 13 HLA-A molecules;
3. according to a certain prediction standard, the prediction results of the six online epitope prediction websites are subjected to integration analysis, and candidate antigen peptide sequences with more consistent prediction results of the six websites are obtained.
4. The immunogenicity of the liver cancer related antigen peptide is verified through IFN-gamma ELISPOT cell functional experiments.
The invention is an antigenic 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 (GPC3) 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 a detection hole, a negative control hole and a positive control hole dot diagram of the HLA-A0201 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 number ratio (P/N) statistical plots of test wells and 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 number ratio (P/N) statistical plots of test wells and negative control wells.
FIG. 3 shows epitope peptides of HLA-A2402 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 HLA-A2402 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 number ratio (P/N) statistical plots of test wells and negative control wells.
FIG. 4 shows epitope peptides of HLA-A3101 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 HLA-A3101 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 number ratio (P/N) statistical plots of test wells and negative control wells.
FIG. 5 shows epitope peptides of liver cancer associated antigen T cells restricted by HLA-A0206 molecules;
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 plot of the number of spots in the detection wells and the negative control wells;
C) dot number ratio (P/N) statistical plots of test wells and negative control wells.
FIG. 6 shows epitope peptides of HLA-A0207 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-A0207 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 number ratio (P/N) statistical plots of test wells and negative control wells.
FIG. 7 shows epitope peptides of HLA-A3303 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 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 number ratio (P/N) statistical plots of test wells and 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 plot of the number of spots in the detection wells and the negative control wells;
C) dot number ratio (P/N) statistical plots of test wells and 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 plot of the number of spots in the detection wells and the negative control wells;
C) dot number ratio (P/N) statistical plots of test wells and negative control wells.
FIG. 10 shows epitope peptides of HLA-A1102 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-A1102 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 number ratio (P/N) statistical plots of test wells and negative control wells.
FIG. 11 shows epitope peptides of HLA-A0301 molecule-restricted liver cancer 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 number ratio (P/N) statistical plots of test wells and negative control wells.
FIG. 12 shows epitope peptides of HLA-A0101 molecule-restricted liver cancer associated antigen T cells;
A) identifying a detection hole, a negative control hole and a positive control hole spot diagram of the epitope peptide of the HLA-A0101 molecule restricted liver cancer related antigen T cell 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 number ratio (P/N) statistical plots of test wells and 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 plot of the number of spots in the detection wells and the negative control wells;
C) dot number ratio (P/N) statistical plots of test wells and 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 restricted by 13 HLA-A molecules
Selecting three liver cancer related antigen proteins: alpha-fetoprotein (AFP), glypican-3 (GPC3) and Golgi transmembrane glycoprotein 73(GP73), and the amino acid sequences are obtained by searching a UniProt global protein resource database, and the standard sequences which are researched most are selected; then, through SYFPEITHI, BIMAS, SVMHC, IEDB, NETMHC, EPIJEN and other six commonly used epitope peptide prediction databases, the T cell epitope peptides aiming at each of the liver cancer related antigen proteins are virtually predicted, 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 the experiment selects the method of ANN and SMM with better statistical significance. 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 into a corresponding amino acid sequence input box of a prediction database website, respectively selecting the length of epitope peptide to be 9 and 10 amino acids, then selecting 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
Figure BDA0002318008280000101
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 meeting at least more than two prediction method score standards 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 human lymphocyte separation fluid (Dake is biological, Shenzhen) with 1 time of the volume of the original blood into a 15mL centrifuge tube; slowly spreading the diluted blood on the separation liquid, and centrifuging at room temperature for 20min at 2500 rpm; 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 × 106The 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 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 one group, wherein 13 groups are formed, and each group comprises 8-9 epitope peptides; taking ELISPOT plate (Dake is biological, Shenzhen) pre-coated with anti-human IFN-gamma antibody, activating with serum-free medium (200 μ L/well) for 8min, and adding PBMC suspension (100 μ L/well) of liver cancer patients into each well; then adding each HLA-A molecule restricted mixed peptide into the detection hole, wherein the single polypeptide in the mixed peptide is 15 mug/mL, adding PHA (2.5 mug/mL) into the positive control hole, and adding DMSO polypeptide solution with the same concentration as the detection hole into the negative control hole; standing at 37 deg.C for 5% CO2Incubating in an incubator for 24 h; 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 ℃; enzyme linked is added after washing the plateAvidin working solution (100. mu.L/well), and incubation was continued at 37 ℃ for 1 h; 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, and conveying the ELISPOT plate to Shenzhenjn Dake as an automated scanning counting spot of the biotechnology company after airing. 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 negative control hole spots is not less than 6, judging that the CTL reaction is positive if the number of the negative control hole spots is not less than 2 times that of the detection holes.
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 hole, setting positive control hole and negative control hole at 37 deg.C and 5% CO2Incubate 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 allelic gene typing on each liver cancer patient, and preliminarily determining which HLA-A molecules limit and present the epitope peptide 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, and carrying out ELISPOT detection again to further determine 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); the DNA sequences of exon 2, intron 2, exon 3 and partial introns l and 3 of the A site were amplified by PCR using HLA-A site specific primers A1 and A3, and the product size was 985 bp. The amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15 s; annealing at 62 ℃ for 15 s; extension at 72 ℃ for 90 s; 35 cycles; extension at 72 ℃ for 5 min. 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
Figure BDA0002318008280000121
Splicing sequencing results of the exon 2 and the exon 3 into a complete HLA-A overlapping sequence (Contig) by Seqman software of 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 in a database by utilizing a Nucleotide BLAST tool until 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: 9 liver cancer-associated antigenic peptides (P1-P9) stimulated CTL positive responses in PBMCs of HLA-A02: 01 positive patients (FIG. 1);
15 liver cancer-associated antigenic peptides (P10-P24) stimulated CTL positive responses in PBMCs of HLA-A11: 01 positive patients (FIG. 2);
there are 10 liver cancer-associated antigenic peptides (P25-P34) that stimulate CTL positive responses in PBMCs of HLA-A24: 02 positive patients (FIG. 3);
9 liver cancer-associated antigenic peptides (P35-P43) stimulated CTL positive responses in PBMCs of HLA-A31: 01 positive patients (FIG. 4);
9 liver cancer-associated antigenic peptides (P4, P44-P51) stimulated the PBMC of HLA-A02: 06 positive patient to present CTL positive reaction (FIG. 5);
9 liver cancer-associated antigenic peptides (P1, P4, P7-P8, P52-56) stimulated positive CTL response in PBMC of HLA-A02: 07 positive patients (FIG. 6);
9 liver cancer-associated antigenic peptides (P41, P57-64) stimulated CTL positive responses in PBMCs of HLA-A33: 03 positive patients (FIG. 7);
there are 10 liver cancer-associated antigenic peptides (P10, P65-P73) that stimulate the PBMCs of HLA-A30: 01 positive patients to present CTL positive responses (FIG. 8);
8 liver cancer-associated antigenic peptides (P2, P74-P80) stimulated CTL positive responses in PBMCs of HLA-A02: 03 positive patients (FIG. 9);
10 liver cancer-associated antigenic peptides (P10-P12, P15, P41, P81-P85) can stimulate PBMCs of HLA-A11: 02 positive patients to show CTL positive reaction (figure 10);
12 liver cancer-associated antigenic peptides (P10, P16, P68, P86-94) stimulated CTL positive responses in PBMCs of HLA-A03: 01 positive patients (FIG. 11);
9 liver cancer-associated antigenic peptides (P95-P103) stimulated CTL positive responses in PBMCs of HLA-A01: 01 positive patients (FIG. 12);
there are 8 liver cancer-associated antigenic peptides (P96, P104-P110) that stimulated CTL positive in PBMCs from HLA-A26: 01 positive patients (FIG. 13).
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
Figure BDA0002318008280000141
GPC3 (P51654): human body protein
Figure BDA0002318008280000142
Figure BDA0002318008280000151
GP73(Q8NBJ4/B3KNK 9): human body protein
Figure BDA0002318008280000152
。
Sequence listing
<110> Nanjing Dahu Biotechnology Co., Ltd
Southeast university
<120> thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer-related 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 IleLeu 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 AsnGln 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 ThrSer 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 LysLys 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 (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 the following sequences:
FLYAPTILL、KLSQKFTKV、SLVVDETYV、ELFDSLFPV、FLQALNLGI、VMQGCMAGV、YQDEKAVLV、FQLESVNKL、ALSEPQPRL；
KVNFTEIQK、AVSVILRVAK、ITVTKLSQK、SQQDTLSNK、VSVILRVAK、TTDHLKFSK、KLIMTQVSK、VINTTDHLK、KNYTNAMFK、LQSASMELK、STIHDSIQY、SSEVVLDSK、DVFNVEDQK、QQDVLQFQK、LSQCINQMK；
LYAPTILLW、IYEIARRHPF、SYANRRPCF、IFLIFLLNF、NYPSLTPQAF、FYSALPGYI、VFGNFPKLI、NYGRLQQDVL、LYQDEKAVL、RLQQDVLQF；
AVMKNFGTR、IIIGHLCIR、AATVTKELR、MIKVKNQLR、KLCAHSQQR、VSKSLQVTR、KTLQRNYGR、GFNYWIASSR、KGNEAVASR；
AVIADFSGL、YAPTILLWAA、KQEFLINLV、SVVCFFFLV、LTPQAFEFV、IVLGFNYWI、LVLAALVACI、PQVQAALSV；
QLPAFLEEL、KLVLDVAHV、FLIIQNAAV、FVGEFFTDV、VLAALVACI；
LASFVHEYSR、MNKFIYEIAR、PTILLWAAR、HQVRSFFQR、LFIDKKVLK、FNYWIASSR、DTINLLDQR、EVKEQCEER；
QTKAATVTK、ITRKMAATA、YSRRHPQLA、LLRTMSMPK、KVKNQLRFL、KSFISFYSA、RSMKSPPLV、KGNVLGNSK、KQREQLDKI；
FLIFLLNFT、LLNQHACAV、KLTTTIGKL、SLQVTRIFL、TLSSRRREL、ALVACIIVL、VLQDQLKTL；
NAFLVAYTK、AKNYTNAMFK、GSDLQVCLPK、QLLRTMSMPK、LQQDVLQFQK；
ILLWAARYDK、KVNFTEIQK、AITVTKLSQK、KLKSFISFY、CLRGARRDLK、GLGNGRRSMK、VLDSKRQVEK、LIRVLQDQLK、RVRRAAAER；
FSSLVVDETY、FLASFVHEY、KGEEELQKY、YTNAMFKNNY、LTRMWYCSY、EIDKYWREY、ETDKQAALA、QLESVNKLY、QTNLERKFSY；
ETFMNKFIY、EIARRHPFLY、ELIQKLKSF、ELVNGMYRIY、EIVVRHAKNY、VPQGKGNV、QTPQVQAAL。
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 primary liver cancer associated antigen thymus-dependent lymphocyte antigen epitope peptide of claim 1 and the use of the antigen epitope peptide of 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.
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
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
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
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
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
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
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
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
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

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

Family

ID=70034249

Family Applications (10)

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
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
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
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
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
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
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
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
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
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

Family Applications Before (3)

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
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
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

Family Applications After (6)

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
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
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
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
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
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

Country Status (1)

Country Link
CN (10) CN116496382A (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113956342A (en) * 2021-12-22 2022-01-21 北京大学人民医院 Tumor neogenesis antigen polypeptide and application thereof
CN114057864A (en) * 2020-07-30 2022-02-18 香雪生命科学技术（广东）有限公司 High-affinity TCR for identifying AFP
CN114675035A (en) * 2022-03-22 2022-06-28 南京大户生物科技有限公司 Antigen-specific thymus-dependent lymphocyte universality detection technical scheme suitable for extensive population in east Asia region
CN115785206A (en) * 2022-06-10 2023-03-14 河北博海生物工程开发有限公司 Lung cancer specific molecular target 07 and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210147550A1 (en) * 2017-08-18 2021-05-20 Joshua Michael Francis Antigen-binding proteins targeting shared antigens

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210147550A1 (en) * 2017-08-18 2021-05-20 Joshua Michael Francis Antigen-binding proteins targeting shared antigens

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
董海龙，等: "肝癌相关肿瘤抗原HLA-A2限制性CTL表位的预测", 《第四军医大学学报》 *
雷俊华，等: "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", 《解放军医药杂志》 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114057864A (en) * 2020-07-30 2022-02-18 香雪生命科学技术（广东）有限公司 High-affinity TCR for identifying AFP
CN114057864B (en) * 2020-07-30 2023-12-15 香雪生命科学技术（广东）有限公司 High affinity TCR for identifying AFP
CN113956342A (en) * 2021-12-22 2022-01-21 北京大学人民医院 Tumor neogenesis antigen polypeptide and application thereof
CN113956342B (en) * 2021-12-22 2022-03-04 北京大学人民医院 Tumor neogenesis antigen polypeptide and application thereof
CN114675035A (en) * 2022-03-22 2022-06-28 南京大户生物科技有限公司 Antigen-specific thymus-dependent lymphocyte universality detection technical scheme suitable for extensive population in east Asia region
CN115785206A (en) * 2022-06-10 2023-03-14 河北博海生物工程开发有限公司 Lung cancer specific molecular target 07 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

Also Published As

Publication number Publication date
CN115960205A (en) 2023-04-14
CN115785253A (en) 2023-03-14
CN116496383A (en) 2023-07-28
CN115785254A (en) 2023-03-14
CN115960206A (en) 2023-04-14
CN115925876A (en) 2023-04-07
CN110964093B (en) 2023-05-23
CN115785252A (en) 2023-03-14
CN116496382A (en) 2023-07-28
CN115960207A (en) 2023-04-14

Similar Documents

Publication Publication Date Title
CN110964093B (en) Thymus-dependent lymphocyte antigen epitope peptide of primary liver cancer related antigen and application thereof
Greten et al. Immunology and immunotherapy of cholangiocarcinoma
CN111116719B (en) Thymus dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen and its application
CN110172080B (en) Thymus-dependent lymphocyte antigen epitope peptide of hepatitis B virus antigen and application thereof
CN109081867B (en) Cancer specific TCR and assay techniques and uses thereof
CA3131766A1 (en) Cancer biomarkers for durable clinical benefit
CN110627895B (en) Lung cancer specific TCR and analysis technology and application thereof
TW201927809A (en) Method for preparing personalized cancer vaccine
Mota et al. ALK peptide vaccination restores the immunogenicity of ALK-rearranged non-small cell lung cancer
WO2019179040A1 (en) Fusion protein, preparation method therefor and application thereof
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
CN113416240B (en) Universal antigen peptide library and kit for inducing tumor specific immune response
WO2006008633A2 (en) Receptor protein tyrosine phosphatase gamma (ptprg) as marker for dendritic cells cells
WO2011038689A1 (en) Novel human cytokine vstm1-v2 and use thereof
EP3298132A1 (en) Recombinant oncolytic viruses and uses thereof
KR102182555B1 (en) Platform of discovery of cancer therapeutic antigen for activation of T cell-mediated immune response
CN116948004B (en) Tumor new antigen polypeptide aiming at CTNNB1 gene H36P mutation and application thereof
CN109355379A (en) A kind of kit causing deaf gene mutation for detecting autosomal dominant deafness family
CN111434674A (en) Polypeptide compositions and their use in cancer immunotherapy
CN111647069B (en) Improved TCR and application thereof
Pereira et al. Gene expression in IFN-gamma-activated murine macrophages
CN114675035A (en) Antigen-specific thymus-dependent lymphocyte universality detection technical scheme suitable for extensive population in east Asia region
Malone et al. Performance of neoantigen prediction for the design of TG4050, a patient specific neoantigen cancer vaccine

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