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Afp158-166 specific tcr gene and its transgenic T cell, in vitro proliferation method and application

A specific, transgenic technology, applied in gene therapy, genetic engineering, plant gene improvement, etc., can solve the problems of efficiency and economical adverse effects of clinical application of cell therapy

Active Publication Date: 2016-01-27
GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current in vitro proliferation system needs to add antigens, co-stimulatory molecules and cytokines to the culture system, or needs to culture autologous dendritic cells, its efficiency and economy have a negative impact on the clinical application of cell therapy

Method used

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  • Afp158-166 specific tcr gene and its transgenic T cell, in vitro proliferation method and application
  • Afp158-166 specific tcr gene and its transgenic T cell, in vitro proliferation method and application
  • Afp158-166 specific tcr gene and its transgenic T cell, in vitro proliferation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1A

[0052] Example 1AFP 158-166 Acquisition of specific TCR genes

[0053] (1) Peripheral blood mononuclear cells of genotype HLA-A0201 were separated by density gradient centrifugation, and CD14 was screened by immunomagnetic bead method + Monocytes culture dendritic cells (DC) with IL-4 and GM-CSF and promote their maturation with TNF-α;

[0054] (2) Mature DC load AFP 158-166 Activate and amplify AFP after antigenic peptide 158-166 antigen-specific CD8 + T lymphocytes;

[0055] (3) Separation of Pro5MHC-AFP by fluorescence activated cell sorting 158-166 Pentamer and CD8 double positive cells;

[0056] (4) extract AFP 158-166 Specific T cell total RNA, reverse transcribed cDNA, PCR amplified AFP 158-166 Specific T cell TCRα / β gene, OverlappingPCR to obtain the full-length AFP connected by 2A sequence 158-166 Specific TCR genes;

[0057] (5) Build AFP 158-166 Specific TCR lentiviral expression plasmid, send the plasmid to Beijing Aoke Dingsheng Company for sequencing i...

Embodiment 2

[0058] Embodiment 2AFP 158-166 Specific TCR transgenic T cells are constructed by the following method:

[0059] with AFP 158-166 Specific TCR gene constructs AFP 158-166 Specific TCR lentiviral vector (Lenti-Pac TM HIVExpressionPackagingKit), infecting polyclonal T cells obtained from peripheral blood of genotype HLA-A0201 by density gradient centrifugation to establish AFP 158-166 Specific TCR transgenic T cells.

Embodiment 3

[0060] Example 3AFP 158-166 Identification of specific TCR transgenic T cells

[0061] (1) Pro5AFP 158-166 -MHC pentamer (FCM) detection of AFP 158-166 Proportion of specific TCR transgenic T cells: Pro5AFP 158-166 - MHC pentamer and CD8 double positive cell ratio was 1.8%, see figure 2 .

[0062] (2) Identification of AFP by enzyme-linked immunospot technique (ELISPOT) 158-166 Function of specific TCR transgenic T cells: AFP 158-166 Specific TCR transgenic T cell group, obvious speckle formation can be seen, and the average spot area is correlated with DC-induced AFP 158-166 There was no statistical difference in the specific T cell group, but the number of spots was significantly increased by nearly 2 times, and there was a statistical difference, see Figure 4 .

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Abstract

The invention discloses an AFP[158-166] specific TCR gene, its transgenic T cell, and an in-vitro proliferation method and a use of the transgenic T cell. The AFP[158-166] specificity TCR gene is represented by SEQ ID NO.1 in a sequence table. The transgenic T cell of the AFP[158-166] specific TCR gene has a specific killing effect on AFP positive liver cancer cells. Artificial antigen presentation cells can present AFP[158-166] epitope peptides through the MHCI type molecule of the cell surface when the artificial antigen presentation cells are secreted by IL-15 to induce the AFP specific immune reaction, and the stimulation of the AFP[158-166] specific TCR gene transgenic T cell by the AFP[158-166] specific TCR gene can improve the proportion of the transgenic T cell, enhances the specificity and enhances the T cell activity and the antitumor capability in an auxiliary manner through the secretion of IL-15.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to AFP 158-166 Specific TCR gene, AFP 158-166 Specific TCR transgenic T cells, AFP 158-166 In vitro proliferation method and AFP of specific TCR transgenic T cells 158-166 Application of specific TCR transgenic T cells in the preparation of drugs for treating liver cancer. Background technique [0002] Tumor-specific T cell adoptive cell therapy (ACT) is the latest progress in tumor immunotherapy. Cellular immunity especially CD8 + T cell-mediated specific MHC-I molecule-restricted cellular immune function plays a decisive role in anti-tumor immunity. T lymphocytes are activated by specific T cell receptor (Tcell receptor, TCR) recognition of tumor cell surface antigen peptide-MHC complex. Activated T cells can directly lyse tumor cells, or inhibit tumor growth and prevent tumor angiogenesis by secreting cytokines, such as interferon and tumor necrosis factor. [0003] Adva...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/867C12N5/10A61K48/00A61K39/00A61P1/16A61P35/00
Inventor 何向辉孙龙昊章志翔刘彤
Owner GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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