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Congenital heart disease ventricular septal defect cell model constructed by transduced SV40LT antigen gene and cell bank thereof

A technology for congenital heart disease and ventricular septal defect, applied in the field of medical cardiovascular disease research, can solve problems such as inability to carry out research projects

Inactive Publication Date: 2015-03-18
翁炳焕
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] However, there has been no literature report on the construction of an immortal cell model and its cell bank that can be used to study the pathogenesis of congenital heart disease ventricular septal defect from the cellular level in vitro, and it is impossible to carry out related research projects

Method used

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  • Congenital heart disease ventricular septal defect cell model constructed by transduced SV40LT antigen gene and cell bank thereof

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Embodiment Construction

[0015] 1. Extraction of SV40 large T antigen DNA: ① SV40 DNA digestion: buy SV40 freeze-dried powder or SV40 plasmid containing large T antigen gene from the market, and dissolve it in an appropriate amount of H 2 O or TE buffer, add 2uL10× enzyme digestion buffer and 18uL H 2 O, add restriction enzyme 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 (or add 0.5mol / L EDTA ) Terminate the reaction to prepare for electrophoresis. ②SV40 DNA electrophoresis: Take electrophoresis grade agarose and use electrophoresis buffer to make a 10% agarose gel, pour it on the sealed gel filling platform, insert the sample comb, and remove the sealing tape from the gel making platform after the gel has solidified , Pull out the comb and put it into the electrophoresis tank with enough electrophoresis buffer. The buffer is about 1mm above the surface of the gel. Prepare a DNA sample with an appropriate amount of 10...

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Abstract

The invention relates to construction of an SV40LT antigen gene transduced congenital heart disease ventricular septal defect cell model and a cell bank of the SV40LT antigen gene transduced congenital heart disease ventricular septal defect cell model applied to the field of medical research. A construction method is mainly characterized by comprising the following steps: according to a conventional route, constructing an SV40LTag-pcDNA3.1(-) recombinant by using T4DNA ligase, BamHI, pcDNA3.1(-)DNA and SV40LTag DNA; and purifying the recombinant by virtue of competent escherichia coli, then transferring the recombinant to congenital heart disease ventricular septal defect cells which are subjected to collagenase II digestive in-vitro subculture and logarithmic growth to ensure that the recombinant is integrated with DNA, performing extended culture, screening the cells containing positive recombinants for cloning by virtue of G418, and screening the cells of which the cellular morphology, growth curves, chromosome karyotypes, carcinogenicity tests in nude mice, transfection cell SV40 large T gene detection, mRNA product determination and DNA sequence determination results can meet the characteristics of immortalized cells and are the same as or similar to that of primary cells as an SV40LT transduced congenital heart disease ventricular septal defect in-vitro study cell model so as to lay a foundation for studying pathogenesis of the cells in vitro from cell levels for a long time.

Description

Technical field [0001] The present invention relates to transduction of SV40LT antigen gene (monkey kidney virus 40 large T antigen gene) to construct a cell model of congenital heart disease ventricular septal defect and its cell bank, which is mainly used in the field of medical cardiovascular disease research, and is used for congenital heart disease ventricular septal defect. Research provides cell models and preserves its scientific research resources. Background technique [0002] Congenital heart disease, ventricular septal defect, is one of the birth defects with the highest incidence and has become a serious social problem. In my country, 112,000 to 160 thousand new cases of congenital heart disease occur every year, which will cause economic losses of 10.9 billion to 15.6 billion. The cost of treatment of congenital heart disease is as high as 12 billion yuan per year, causing a serious psychological burden and spiritual burden to the society and family. Suffering has s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C40B50/06C40B40/02
Inventor 翁炳焕黄荷凤徐晨明李晓杨红卫陈雁
Owner 翁炳焕
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