Cloning of transforming growth factor beta 1 gene and construction of recombinant eukaryotic expression plasmid
- VernacularTitle:转化生长因子?1基因克隆及重组真核表达质粒的构建
- Author:
Yisheng WANG
;
Gang WANG
;
Li YIN
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2007;0(42):-
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Gene transfer to study the effect of a exogenous gene on cells is a commonly used technique for molecular biology. Compared with viral vector,pcDNA4.0-TGF-?1 eukaryotic expression plasmid transfection has no inherent toxicity or cytotoxicity,with high safety. OBJECTIVE: To construct and identify eukaryotic expression plasmid carrying recombined TGF-?1 by cloning transforming growth factor (TGF)-?1 gene. DESIGN,TIME AND SETTING: The in vitro cell experiment was performed at the Opening Laboratory of Key Clinical Medical Sciences,Henan Provincial High-learning School from March 2007 to February 2008. MATERIALS: Clean healthy Sprague Dawley rats aged 2 months,of both gender,were used for isolating RNA from cells. METHODS: The TGF-?1 mRNA was extracted from the rat bone marrow cells,and amplified by reverse transcription-polymerase chain reaction (RT-PCR),and cloned into the pGEM-T-vector and identified by PCR. Then TGF-?1 gene was subcloned into pcDNA4.0 plasmid vector. The inserting DNA sequences were analyzed. MAIN OUTCOME MEASURES: Expression of TGF-?1 cDNA,pGEM-T-TGF-?1 and recon pcDNA4.0-TGF-?1. RESULTS: There was an obvious electrophoresis strip of TGF-?1 cDNA by RT-PCR. The obtained pGEM-T-TGF-?1 was confirmed correct with PCR amplification and sequence identification. Recombinant eukaryotic expression plasmid pcDNA4.0-TGF-?1 was obtained and identified by digestion. The inserted sequences were conformity to the designed sequences. CONCLUSION: Rat TGF-?1 gene has been successfully cloned,and recombinant TGF-?1 eukaryotic expression vector has been constructed successfully.