[Construction of pcDNA3.1(+)/ hTSHR and its expression in COS-7 cells].
OBJECTIVE
To construct the recombinant eukaryotic expression vector pcDNA3.1(+)hTSHR and express it in COS-7 cells.


METHODS
The full length cDNA sequence of hTSHR was obtained via the plasmid vector pBluescript SK(-)/hTSHR cut with EcoR I and Xba I, and then subcloned into eukaryotic expression vector pcDNA3.1(+). Constructed pcDNA3.1/hTSHR was identified by restricting enzyme digestion analysis, PCR amplifying and DNA sequencing. The recombinant expression plasmid was transfected into COS-7 cells by Lipofectin method. Human TSHR protein expression on COS-7 cells was detected by RT-PCR and immunocytochemical staining.


RESULTS
Obtained full-length sequence of hTSHR gene was identical with that included in GenBank. Restriction enzyme digestion, PCR amplifying and DNA sequencing confirmed that pcDNA3.1/hTSHR had been constructed successfully. The recombinant plasmid could express hTSHR protein with activity on COS-7 cells.


CONCLUSIONS
The pcDNA3.1/hTSHR has been successfully constructed, which will contribute to further studies on the TSHR function and to the establishment of a good animal model for Graves' disease.
Zhan
Sheng-hua
SH
Department of Pathology, Chongqing University of Medical Sciences, Chongqing 400016,China. cys01028@hotmail.com
Zhang
Hui
H
Liu
Chun
C
D000818:Animals; D001483:Base Sequence; D019556:COS Cells / Q000378:metabolism; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics*; D005822:Genetic Vectors; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011989:Receptors, Thyrotropin / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D011989:Receptors, Thyrotropin; D011993:Recombinant Fusion Proteins