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Method for constructing hTERT mediated T lymphocytic model and cell bank thereof

A lymphocyte and cell technology, applied in the field of medical immunology research, can solve problems such as inability to carry out research, achieve the effect of normal differentiation and maintain chromosomes

Inactive Publication Date: 2015-03-18
翁炳焕
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Problems solved by technology

[0011] However, there have been no literature reports on the construction of immortal T cell models and cell banks using hTERT that can be used to study the pathogenesis of T lymphocyte-related diseases from the cellular level in vitro, and it is impossible to carry out research on related projects

Method used

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Embodiment Construction

[0015] 1. Extraction of hTERT: ① Digestion of pClneo-hTERT: hTERT is located between the EcoRI and SalI sites of the plasmid pClneo-hTERT, and the multiple cloning site (MCS) of the pLXSNneo vector contains EcoRI and XhoI restriction sites. Commercially purchased pCIneo-hTERT plasmid, dissolved in an appropriate amount of ultra-clean H 2 In O or TE buffer, add 2uL 10× digestion buffer and 18uL H 2O, add restriction endonuclease EcoR I and Xho I 0.5ul each, incubate at 37°C for 1h, heat at 75°C for 15min, inactivate the enzyme, add 5uL electrophoresis sample buffer (also by adding 0.5mol / L EDTA) The reaction was terminated, and hTERT was amplified according to the conventional PCR method, and the amplified product was collected for electrophoresis. ②hTERT electrophoresis: Take electrophoresis grade agarose and use electrophoresis buffer to make 10% agarose gel, pour it on the gel filling platform that has been sealed, insert a sample comb, and remove the sealing tape from the ...

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Abstract

The invention relates to a method for constructing hTERT mediated T lymphocytic model and a cell bank thereof in the field of medicines. The method is mainly characterized by comprising the following steps: carrying out double enzyme digestion on a plasmid pCIneo-hTERT and a carrier pLXSNneo with incision enzymes EcoR I and Xho I, connecting hTERT and pLXSNneo digested products subjected to PCR amplification and gel electrophoresis separation by virtue of Ligation Mix to construct a pLXSNneo-hTERT recombinant, transforming DH5a competent cells for purifying, amplifying and selecting ampicillin-resistant bacterial colony extraction plasmids, and performing in-vitro subculture on T lymphocytes in logarithmic growth by virtue of lipofection transfection, so that the recombinant is integrated with DNA of cells; performing enlarged culture, performing G418 screening on cells containing positive recombinants, screening cells of which the cellular morphology, growth curve, karyotype, inoculated nude mice test, transfection cell telomerase activity, hTERT mRNA expression, immunohistochemistry, and cell generation cycle and apoptosis rate accord with the characteristics of immortalized cells and which are the same or similar to primary cells to serve as an hTERT mediated in-vitro T lymphocytic research model so as to be cryopreserved in liquid nitrogen. Therefore, a foundation is laid for long-term in-vitro research of pathogenesis of related diseases from the cellular level.

Description

technical field [0001] The invention relates to the construction of a telomerase reverse transcriptase catalytic subunit (hTERT)-mediated T lymphocyte model and its cell bank, which is mainly used in the field of medical immunology research, and is used for developing T lymphocytes or various T lymphocyte-related This research provides cell models and preserves its scientific research resources. Background technique [0002] The formed components in human blood are divided into red blood cells, white blood cells and platelets. The white blood cells are mainly neutrophils, lymphocytes and monocytes. Lymphocytes are divided into T lymphocytes (T cells) and B lymphocytes (B cells). [0003] It is known that T cells are differentiated from lymphoid stem cells in the thymus, and they are the most numerous and most complex type of lymphocytes. According to their functions, they can be divided into three subgroups: helper T cells, suppressor T cells and cytotoxic T cells. There...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C40B50/06C40B40/02
Inventor 翁炳焕徐向荣沈国松李晓严恺黄荷凤
Owner 翁炳焕
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