Construction Of PcDNA3.1(+)/hTSHR And Its Expression In COS-7 Cells

OBJECTIVE: To construct the recombinant eukaryotic expression vector pcDNA3.1(+)hTSHR and then express it in COS-7 cells.METHODS: The full length cDNA sequence of hTSHR was obtained via the plasmid vector pBluescript SK(-)/hTSHR cutted with EcoR I and Xba I, and then was 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 expressed on the membrane of COS-7 cells was detected by RT-PCR and immunocytochemical staining. RESULTS: Obtained full-length sequence of hTSHR was identitical with that released in Genbank. Restriction enzyme digestion, PCR amplifying and DNA sequencing confirmed that the eukaryotic expression vector pcDNA3.1/hTSHR was constructed correctly. The expression of human TSHR on COS-7 cells was proved by RT-PCR and immunocytochemical staining. CONCLUTION: The eukaryotic expression plasmid pcDNA3.1/hTSHR was successfully contructed, which will contribute to further studies on the TSHR function and to the establishment of a good animal model for Graves' disease.