人促甲状腺激素受体全长表达载体在真核细胞内的稳定表达

Stable expression of pcDNA3.1-hTSHR in CHO cells

目的构建pcDNA3.1/人TSH受体(human thyroid-stimulating hormone receptor,hTSHR)基因真核表达载体(已完成),将该真核表达载体在CHO细胞(中国仓鼠卵巢细胞)中表达,并筛选获得稳定表达细胞株。方法通过脂质体介导,用重组质粒pcDNA3.1-hTSHR转染CHO细胞,以G 418筛选稳定表达细胞株,挑取单克隆,扩大培养并传代;取P5代细胞,抽提基因组DNA及蛋白分别用PCR及Western blot方法鉴定被转染CHO细胞hTSHR的表达情况。结果得到阳性单克隆5个,传代培养。P5代细胞PCR扩增产物为约530 bp特异性条带,大小与预期相符,未见非特异性条带。Western印记法结果表明被转染CHO传代细胞有hTSHR蛋白表达。结论pcDNA3.1-hTSHR真核表达载体可以在CHO细胞中稳定表达,成功构建稳定表达细胞株。为测定自身免疫性甲状腺疾病患者血清TSHR奠定了基础。

Objective To construct the recombinant eukaryotic expression vector with full length cDNA sequence of pcDNA3, 1/human thyroid-stimulating hormone receptor （hTSHR） （accomplished） and to express it in CHO cells （Chinese hamster ovary cells）, then screen the stable expression cell lines. Methods Constructed pcDNA3. 1-hTSHR was transferred into CHO cells by liposome. The stable expression cell lines were screened by using G 418 and were cultured for generations. The fifth generation was selected and DNA and protein expressions of hTSHR in transferred CHO cells were detected by PCR and Western blot. Results Five monoclones with full length sequence of hTSHR gene were obtained. One band of approximately 530 bp in size was amplified by PCR which agreed with anticipation. Western blot confirmed that the expressed recombinant hTSHR protein was present and active in CHO cells. Conclusion The stable hTSHR expression cell lines has been successfully constructed, which would contribute to further studies on the TSHR function in the blood of patients with autoimmune thyroid disease.