人促甲状腺素受体膜外区基因克隆及真核表达

Gene cloning and expression of human TSHR ectodomain in eukaryotic cells

目的克隆人促甲状腺激素受体胞外段基因,构建重组真核表达质粒,获得具有免疫学活性的纯化重组蛋白。方法应用RT-PCR技术从人甲状腺组织得到TSHR膜外区cDNA,插入真核表达载体pcDNA3.1(+),转染CHO细胞,RT-PCR鉴定基因转录,用免疫细胞化学染色法和Western Blot鉴定表达蛋白。结果经测序证实筛选得到的重组体中存在序列正确的TSHR膜外区基因,表达蛋白具有免疫活性。结论成功克隆了人促甲状腺素受体膜外区基因和构建了真核表达载体,并将其在真核细胞中成功表达。

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.