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T-lymphocytes model and cell bank thereof established by recombination of SV40L and hTERT gens

A technology of lymphocyte and gene recombination, applied in the field of medical immunology research, can solve problems such as inability to carry out research, and achieve the effect of long life

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

[0011] However, so far, there has been no information about simultaneously introducing the simian nephrovirus 40 large T antigen gene (SV40LT) and the catalytic subunit of telomerase reverse transcriptase (hTERT) into T lymphocytes, and using this construction to study T lymphocytes in vitro from the cellular level. Immortal T lymphocyte models and cell banks for the pathogenesis of cells and related diseases have been reported in the literature, and research on related projects cannot be carried out

Method used

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Embodiment Construction

[0015] 1. Extraction of SV40LT and hTERT: (1) Enzyme digestion of SV40LT and hTERT: ① Enzyme digestion of SV40LT: buy SV40 freeze-dried powder or SV40 plasmid containing large T antigen gene from the market, dissolve in appropriate amount of H 2 In O or TE buffer, add 2uL 10× digestion buffer, 18uL H 2O and restriction endonuclease BamH I (1-5U / ugDNA), incubate at 37°C for 1h, heat at 75°C for 15min, inactivate the enzyme, add 5uL electrophoresis loading buffer (also by adding 0.5mol / L EDTA) Stop the reaction and prepare for electrophoresis. ② Digestion of 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. Purchase the pCIneo-hTERT plasmid from the market and dissolve it in an appropriate amount of ultra-clean H 2 In O or TE buffer, add 2uL 10× digestion buffer and 18uL H 2 O, add restriction endonuclease EcoR I and Xho I 0.5ul each, incub...

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Abstract

The invention relates to a T-lymphocytes model and a cell bank thereof established by recombination of SV40L and hTERT gens for medical field. The T-lymphocytes model is characterized in that T4DNA is connected with products passing through BamHI digestion plasmid SV40LTag DNA and vector pc DNA 3.1 (I) DNA to construct an SV40LTag-pcDNA3.1 (I) recombinant; Ligation Mix is connected with products through Eco RI and Xho I double restriction plasmid pCIneo-hTERT and vector pLXSNneo to construct pLXSNneo-hTERT recombinant; the two recombinants are lipofection transfection T-lymphocytes after being purified and identified by competence cell expansion; the cells of positive recombinants screened by G418 are enlarged and cultured to screen cells of which a cellular morphology, a growth curve, a karyotype, a nude mouse carcinogenicity test, a transfection cell telomerase activity, SV40LT and Htert mRNA expression, immunohistochemical, a cell generation cycle and an apoptosis rate conform to the features of immortalized cells andare same or similar to those of primary cells to be used as T-lymphocytes model established by SV40LT and hTERT gene recombination to be stored in liquid nitrogen; the T-lymphocytes model established by recombination of SV40L and hTERT gens lays foundation for in-vitro long-time research of the pathogenesis from cellular level.

Description

technical field [0001] The invention relates to SV40LT (simian kidney virus 40 large T antigen gene) and hTERT (telomerase reverse transcriptase catalytic subunit) gene recombination (SV40LT and hTERT mediated) to construct T lymphocyte model and cell bank, mainly used in medicine In the field of immunology research, it provides cell models and preserves scientific research resources for carrying out various researches on T lymphocytes or T lymphocytes. 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 are the most numerous and most complex type of lymphocytes. According to their functions, they can be divided into three subgroups: helper T c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C40B50/06C40B40/02
Inventor 翁炳焕蔡志坚徐向荣毛愉婵孙亚莉吕时铭
Owner 翁炳焕
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