[Construction of pcDNA3.1(+)/ hTSHR and its expression in COS-7 cells]

Abstract

Aim: 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.

Conclusion: 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.