Construction of pcDNA3.1-myc/PINK1 and its expression in COS-7 cells
- VernacularTitle:帕金森病PINK1蛋白真核表达载体pcDNA3.1-myc/PINK1的构建及其在COS-7细胞中的表达
- Author:
Yuhu ZHANG
;
Yuecun LIU
;
Shaohua LI
;
Lijuan WANG
- Publication Type:Journal Article
- Keywords:
Parkinson disease/GE;
Gene expression;
Genetic vectors;
Transfection;
Cells,cultured
- From:
Journal of Chinese Physician
2009;11(12):1598-1600
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct eukaryotic expression vector of PINK1 (pcDNA3. 1-myc/PINK1) and verify PINK1 expression in pcDNA3.1-myc/PINK1 transfected COS-7 cells. Methods The cDNA fragment encoding human PINK1 gene was amplified by PCR method from human cDNA library. After nucleotide sequencing, this cDNA fragment was inserted into an eukaryotic expression vector pcDNA3. 1-myc-his( - )B using gene cloning and DNA recombination. The recombinant was then transferred into COS-7 cells by liposome. The expression of the target molecule was assayed by western blotting. Results The sequence of DNA fragment amplified by PCR was consistent with that published in Genbank, and digestion of the recombinant plasmid with EcoR Iand BamH Iliberated DNA fragments with expected size. PINK1 was expressed and synthesized in transfected cells after 48h culture. Conclusion An eukaryotic expression plasmid containing human PINK1 gene was successfully constructed, and it can express out objective protein, which has laid a concrete foundation for future study on PINK1.
