Recombination plasmid related with EB virus specificity TCR gene and method for constructing anti-EBV specific CTL

A recombinant plasmid, EB virus technology, applied in genetic engineering, plant genetic improvement, recombinant DNA technology and other directions, can solve the problems of difficult to popularize, insufficient dose and effect, time-consuming and cumbersome operation, etc., and achieves multi-operability and versatility. Effect

Active Publication Date: 2008-05-21
JINAN UNIVERSITY
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Problems solved by technology

However, in the past, relying solely on the expansion of specific cytotoxic T cells from donors, the source was often limited or the dose-effectiveness was insufficient; foreign countries have used the introduction of suicide genes or genetically modified dendritic cells as tumor vaccines for post-HSCT tumor immunotherapy, Some results have been achieved, but the operation is time-consuming and cumbersome, making it difficult to promote

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0040] 1 Construct the TCR Vα-pIRES-TCR Vβ recombinant plasmid associated with the Epstein-Barr virus-specific cytotoxic T cell clone;

[0041] First, normal human T cells (HLA-A02 restricted) were induced by the nonapeptide fragment GLCTLVAML of the Epstein-Barr virus-associated antigen BMLF and cultured in vitro to obtain Epstein-Barr virus-specific cytotoxic T cell clones. Bovine serum, 20U / mL recombinant human IL-2, 100U / mL penicillin-streptomycin in IMDM medium, 37°C, 5% (v / v) CO 2 In a saturated humidity incubator, conventional in vitro culture and expansion; then collect Epstein-Barr virus-specific cytotoxic T cell clones 1×10 7 Routinely perform RNA extraction and cDNA synthesis (operate according to the instructions of the corresponding kit), and then perform PCR amplification using TCRVα and TCRVβ subfamily primers to analyze the predominantly expressed TCR Vα in EB virus-specific cytotoxic T cell clones and TCR Vβ subfamilies, with specific upstream primers corresp...

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Abstract

The invention discloses a recombinant plasmid relevant to the EB virus characteristic TCR gene and a method for reverting normal T cell to obtain the anti-EBV characteristic CTL. The recombinant plasmid consists of TCRV Alpha 15 gene, pIRES vector and TCR V Beta 1gene; the TCRV Alpha 15-p-IRES-TCR V V Alpha 1gene recombinant plasmid relevant to the clone of EB virus characteristic cell toxin T cell of the invention is delivered to the peripheral blood T cell to obtain a large number of high-efficiency cell toxin T cells of the antigen relevant to the characteristic EV virus; in this way, the cell of EBV<+> can be killed on purpose and the safety property is high; therefore, the invention has comparatively good prospects for curing diseases relevant to the EB virus.

Description

technical field [0001] The invention belongs to the technical field of anti-virus and anti-tumor immunity in hematological tumors, in particular to a TCRVα15-pIRES-TCRVβ1 recombination related to EBV (Epstein Barr virus) specific TCR (T cell receptor, T cell receptor) gene A plasmid, and a method for transducing normal T cells to obtain anti-EBV specific CTL (cytotoxic T cells, cytotoxic T cells). Background technique [0002] Epstein Barr virus (EBV) is closely related to malignant tumors of lymphoid tissue. anti-EBV + Epstein-Barr virus-specific cytotoxic T cells (cytotoxic T cells, CTL) of tumor cells exert their anti-tumor or anti-viral properties, and can effectively eliminate EBV + Tumor cells have good application prospects for the treatment of Epstein-Barr virus-related tumors. However, simply expanding the specific cytotoxic T cells of the donor often has limited sources or insufficient dose-effectiveness; foreign countries have used the introduction of suicide g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/12
Inventor 李扬秋吴秀丽朱康儿杨力建陈少华
Owner JINAN UNIVERSITY
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