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  • Title: [Construction of pcDNA3.1(+)/ hTSHR and its expression in COS-7 cells].
    Author: Zhan SH, Zhang H, Liu C.
    Journal: Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi; 2004 Jul; 20(4):390-3. PubMed ID: 15207077.
    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.
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