Construction and identification of eukaryotic expression vector of rat GPR17 gene.
- Author:
Ka-Na LIN
1
;
San-Hua FANG
;
Bei-Lei CAI
;
Xin-Xin WANG
;
Yun-Bi LU
;
Wei-Ping ZHANG
;
Er-Qing WEI
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Base Sequence; DNA, Complementary; genetics; Escherichia coli; genetics; metabolism; Female; Genetic Vectors; genetics; HEK293 Cells; Humans; Male; Molecular Sequence Data; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; biosynthesis; genetics; Recombinant Proteins; biosynthesis; genetics; Transfection
- From: Journal of Zhejiang University. Medical sciences 2009;38(6):584-590
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct the eukaryotic expression vector of rat GPR17 (rGPR17) cDNA,and to identify its function in HEK293 cells.
METHODSTotal RNA was extracted from rat brain tissue; full-length GPR17 cDNA was prepared by RT-PCR, and cloned into pcDNA3.1(+) plasmid. The recombinant plasmid was converted into E.coli DH5alpha and confirmed by PCR, double enzyme digestion analysis and DNA sequencing. The recombinant plasmid pcDNA3.1(+)-rGPR17 was transiently transfected into HEK293 cells using Lipofectamin 2000. Expression of rGPR17 gene was confirmed by RT-PCR and immunofluorescence staining. The exogenous LTD(4) enhanced intracellular calcium was measured using Fluo-4.
RESULTRT-PCR, double enzyme digestion analysis and sequencing showed that the rGPR17 gene was cloned into recombinant vector, and the recombinant rGPR17 was expressed after transfection in HKE293 cells. LTD(4) increased intracellular calcium release in the transfected HEK293 cells.
CONCLUSIONThe eukaryotic expression vector of rGPR17 cDNA has been constructed; it is functionally expressed in HEK293 cells. This work provides a basis for further research of the GPR17 receptor and its antagonists.
