Construction of a recombinant plasmid of BC022687 and identification of its expression and localization in CHO cells.
- Author:
Hong-Fei LI
1
;
Ling ZHANG
;
Yu-Qin SHI
;
Zhi-Qiong WANG
;
Gao-Feng JIANG
;
Shi-Zhen SONG
;
Guo-Qing FU
;
Zhi-Bing ZHANG
Author Information
- Publication Type:Journal Article
- MeSH: Animals; CHO Cells; Centrosome; metabolism; Cilia; metabolism; Cricetinae; Cricetulus; DNA, Complementary; Genetic Vectors; Male; Mice; Plasmids; Recombinant Fusion Proteins; genetics; Transfection
- From: National Journal of Andrology 2013;19(5):387-391
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct a mammalian expression plasmid of the BC022687 gene and investigate the expression and localization of the fusion protein in Chinese hamster ovary (CHO) cells.
METHODSThe BC022687 coding sequence was amplified by polymerase chain reaction (PCR) and subcloned into the pEGFP-C1 vector carrying the gene of green fluorescence protein (GFP). After the target region was sequenced, the recombinant plasmid was transfected into CHO cells, and its expression in the CHO cells was determined by Western blot. The localization of GFP-tagged BC022687 in the CHO cells was observed by laser scanning confocal microscopy.
RESULTSBC022687 was successfully cloned into the mammalian expression vector pEGFP-C1, with the restriction fragment length of 950 bp. The expression of the fusion protein was confirmed, with the relative molecular weight of 64 000. The GFP-tagged BC022687 protein was mainly localized in the cytoplasm, and also presented in the centrioles in the transfected CHO cells.
CONCLUSIONThe successful construction of the plasmid expressing BC022687 in CHO cells has laid a foundation for further studies on the role of this protein in ciliogenesis.
