人促甲状腺激素受体HEK 293T细胞稳定表达株的构建

Stable Expression of Human Thyrotropin Receptor in HEK 293T Cells

目的构建人促甲状腺激素受体(hTSHR)稳定表达株,用于研究抗甲状腺新药。方法 AgeⅠ/NheⅠ双酶切载体GV266和hTSHR基因,T4连接酶连接构建GV266-hTSHR表达质粒。转染GV266-hTSHR到293T细胞后,Western blot证明hTSHR表达。用Opti-MEM、Lipofectamine 2000、辅助质粒在293T细胞构建GV266包装质粒。qPCR测定包装病毒滴度。用GV266包装质粒、GV266-hTSHR表达质粒共转染293T细胞获得GV266-hTSHR-293T稳定表达株。绿色荧光蛋白(GFP)荧光鉴定稳定表达株。结果 GV266-hTSHR构建物阳性大肠杆菌克隆的DNA测序结果与hTSHR序列比对100%吻合。Western blot显示,过表达hTSHR的HEK293T细胞出现相对分子质量为62×103的目的条带。qPCR测定包装质粒滴度达到了2×108 TU/mL。GV266-hTSHR-293T细胞稳定表达株表达GFP荧光。结论构建了GV266-hTSHR表达质粒,获得了GV266-hTSHRHEK 293T稳定表达株,为研究抗甲状腺多肽提供了必要的实验材料。

Objective To establish a strain of eukaryotic cells with overexpressed human thyrotropin receptor （hTSHR） for the development of anti-thyroid drugs. Methods GV266 vector and hTSHR gene were digested by Age I /Nhe I . Plasmids expressing GV266-hTSHR were constructed using T4 ligase and then transfected into 293T cells. The expression of hTSHR was determined by Western blot. Packaging plasmids were built in the 293T cells with Opti-MEM, Lipofectamine 2000 and helper plasmid. The titer of the packaging plasmids was determined with qPCR. The packaging plasmids and the plasmids expressing GV266-hTSHR were co-transfected into 293T cells to obtain a strain of cells （GV266-/iTSHK-293T） with stable expression of hTSHR. The GV266-hTSHR- 293T stain was detected by green fluorescent protein （GFP） fluorescence. Results The DNA sequence of GV266- hTSHR matched that of hTSHR. The Western blot showed a 62 X 10^3 target band. The titer of packaging plasmids reached 2 X 10^8 TU/mL. The GV266-ATSHR-293T cells were visible under GFP fluorescence. Conclusion HEK 293T cells with stable expression of hTSHR was established.