GPR40 shRNA表达载体构建及其稳定转染βTC6细胞系的建立

Construction of GPR40 shRNA Expression Vectors and Establishment of its Stably Transfected βTC6 Cell Lines

目的构建G蛋白偶联受体40(Gprotein coupled receptor 40,GPR40)shRNA表达载体质粒,获得干扰质粒稳定转染的小鼠βTC6细胞系。方法将合成的寡核苷酸链退火形成双链,连接入pSilencer4.1-CMVneo表达载体,并测序鉴定。脂质体法把重组质粒转染至βTC6细胞,通过G418筛选建立稳定转染的βTC6细胞系,采用蛋白印迹(Western blotting)技术检测GPR40蛋白表达。结果构建了GPR40 shRNA重组真核表达载体,并成功地下调了βTC6细胞的GPR40表达。结论成功构建了GPR40 shRNA真核表达载体,获得稳定表达GPR40 shRNA的βTC6细胞系。

Objective To construct the G protein coupled receptor 40（GPR40）shRNA expression vectors and obtain the stably transfected βTC6 cell lines.Methods Two target gene segments were synthesized and cloned respectively into pSilencer4.1-CMV neo expression vector to construct two recombinant eukaryotic vectors which were identified by DNA sequencing.The recombinant plasmids were transfected into βTC6 cells by lipofectanite.The resistant βTC6 cells were selected by G418.The expression of GPR40 was detected by western blot.Results The GPR40 shRNA recombinant eukaryotic plasmid vector was constructed.Furthermore,the recombinant plasmid knocked down GPR40 protein in βTC6 cells.Conclusion A recombinant expression plasmid of GPR40 shRNA is successfully constructed and a βTC6 cells line stably expressing GPR40 shRNA is estabished.