大鼠RGS4基因的克隆及在原核中的表达

Cloning of rat RGS4 gene and its expression in Escherichia coli

目的构建pGEX4T-1-RGS4载体,并检测融合蛋白GST-RSG4在原核中的表达水平。方法采用RT-PCR方法,将克隆SD大鼠G蛋白信号转导调节因子(regulator of G protein signaling4,RGS4)基因全长编码区的cDNA连接到原核表达载体pGEX4T-1中,IPTG诱导原核表达,通过Western blot检测融合蛋白GST-RSG4的表达水平。结果成功构建了pGEX4T-1-RGS4载体;经IPTG的诱导,RGS4可与GST以融合蛋白的形式高效表达。pGEX4T-1-RGS4在原核内表达产物相对分子质量约为50×103,与预期融合蛋白大小一致。结论构建的重组质粒能够在大肠杆菌中高效表达。

Objective To construct pGEX4T-1-RGS4 and detect the GST-RSG4 fusion protein expressed in Escherichia coli. Methods The cDNA of RGS4 was amplified from total RNA extracted from the liver issue of SD rat by RT-PCR and inserted into pGEXdT-1 vector. The GST-RSG4 fusion protein was induced by IPTG and its expression level was analyzed by Western blot. Results The pGEX4T-1-RGS4 vector was successfully constructed. Under the induction of IPTG, the RGS4 and GST expressed efficiently in the mode of fusion protein. The molecular weight of pGEX4T-1-RGS4 expressed in E. coli was 50 000, consistent with the expectation. Conclusion The recombinant plasmid expressed efficiently in E. coll. This is the first step for studying the relationship between RGS4 and the structure domains of GR though GST-pulldown method.