Construction and Expression of Human pEGFP-GEF-H1 Vector
10.3969/j.issn.1008-7125.2014.09.007
- VernacularTitle:人pEGFP-GEF-H1载体的构建及其表达
- Author:
Weidong XU
;
Yuanxia QIAN
;
Jun CAO
- Publication Type:Journal Article
- Keywords:
Colonic Neoplasms;
Guanine Nucleotide Exchange Factors;
Rho Protein;
Vector Construction;
Transfection
- From:
Chinese Journal of Gastroenterology
2014;(9):540-543
- CountryChina
- Language:Chinese
-
Abstract:
Background:Studies have shown that GEF-H1 can activate Rho protein, and is closely associated with tumorigenesis,tumor progression,infiltration,migration. Aims:To construct human pEGFP-GEF-H1 vector and stably transfect into colon cancer HCT116 cells,and determine the expression of GEF-H1. Methods:The gene sequence was designed according to the Online primers synthesis software for constructing recombinant plasmid pEGFP-GEF-H1. HCT116 cells were transfected with plasmid pEGFP-GEF-H1,empty plasmid,respectively,and HCT116 cells without transfection were served as blank control group. Expression of green fluorescence protein was observed under fluorescence microscope. mRNA and protein expressions of GEF-H1 were determined by RT-PCR and Western blotting,respectively. Results:Human pEGFP-GEF-H1 vector was successfully constructed and identified by sequence analysis. After transfection with pEGFP-GEF-H1,green fluorescence could be observed in HCT116 cells,and the mRNA and protein expressions of GEF-H1 were higher than those in empty plasmid group and blank control group. Conclusions:Recombinant plasmid pEGFP-GEF-H1 is successfully constructed in vitro,and transfection into HCT116 cells leads to high expression of GEF-H1, thereby can provide experimental materials needed for further study on correlation between colon cancer and GEF-H1.
