Gene cloning and expression of human TSHR ectodomain in eukaryotic cells / 西安交通大学学报(医学版)

ABSTRACT

Objective To clone and construct the plasmid containing human thyroid stimulating hormone receptor(TSHR) gene ectodomain,and then identify the immunoreactivity of the purified recombinant protein.Methods TSHR total RNA was extracted from human thyroid,and cDNA was obtained with RT-PCR technique.Human TSHR ectodomain gene(hETSHR) was cloned into pcDNA3.1(+) vector.The recombined construct was transfected into CHO cells by Lipofectin 2000.The transcript mRNA was detected by RT-PCR,and protein immunoreactivity was assayed by TSHR antibody with immunocytochemistry staining and Western blot.Results DNA sequencing results showed that the recombinant of human thyrotropin receptor ectodomain had confirmed sequence reported in GenBank.The fusion protein had the immunoreactivity.Conclusion Human thyroid stimulating hormone receptor was successfully cloned in eukaryotic expression vector and the constructor was expressed in eukaryotic cells very well.