建立人促甲状腺素受体α亚基HEK 293T细胞稳定表达株

Construction of Stable Expression of Human Thyrotropin Receptor α-Subunits on HEK 293T Cells

为研究抗甲状腺多肽的特异性与亲和力,本研究建立了人促甲状腺素受体(TSHR)α亚基(hTSHRA)稳定表达株。构建hTSHRA到慢病毒质粒GV218上形成GV218-hTSHRA,挑取GV218-hTSHRA构建物阳性大肠杆菌克隆,提取DNA测序结果与人TSHRA序列比对,100%吻合。结果显示:GV218-hTSHRA转染到HEK 293T后,Western blot显示,HEK 293T细胞提取蛋白出现52kD的hTSHRA目的条带。GV218-hTSHRA、辅助质粒在HEK 293T细胞中包装后,HEK 293T细胞表达绿色荧光蛋白。收取浓缩的包装病毒,qPCR测定滴度达到了2×108 TU/mL。再将包装病毒转染到HEK 293T细胞中,hTSHRA即表达在HEK 293T细胞上,细胞传代后hTSHRA仍然稳定表达。结果表明获得GV218-hTSHRA包装质粒,并建立了hTSHRA-HEK 293T稳定表达株,为研究抗甲状腺多肽与人TSHRα亚基特异性与亲和力提供了必要的实验材料。

The aim of this study was to establish stable expression of human thyroid stimulating hormone receptor （TSHR） α-subunit （hTSHRA） on human embryonic kidney 293T （HEK 293T）. HEK 293T cell lines with stable expression of hTSHRA could be used for detecting affinity between hTSHRA and potential TSHR blocking peptide. We firstly constructed hTSHRA gene into lentiviral vectors GV218. The sequence comparison indicated that we had constructed GV218 hTSHRAA. Western blot demonstrated the 52 kD aim band of hTHSRA on over-expressed HEK 293T cells. GV218 hTSHRA constructions and pHelper were then co-transfected into HEK 293T cells to form packaging plasmid. The HEK 293T cells that stably expressed hTSHRA could also express green fluorescent protein. The titer of lentiviral packaging vector is 2×10^8 TU/mL with qPCR. The lentiviral packaging vector thereafter was transfected into HEK 293T cells again. The hTSHRA expressed on the HEK 293T cells. Human TSHRA stably expressed on HEK 293T upon continuously passaging. Therefore, we established hTSHRA stable expression on HEN 293T cells by constructing GV218 hTHSR lentiviral packaging vector. It is a useful tool for studying TSHR affinity with anti thyroid peptide.