斜纹夜蛾GST基因的原核表达载体构建、融合蛋白表达及Northern印迹分析

Construction and inducing expression of prokaryotic expression vector of glutathione S-transferase gene from Spodoptera litura and expression of GST mRNA with Northern Blot

谷胱甘肽-S-转移酶(GST)是生物体内重要的解毒酶系之一。根据斜纹夜蛾(Spodoptera litura)GST基因设计特异引物,从cDNA文库中扩增GST基因并克隆至pGEM-T载体,经鉴定后经SpeⅠ和EcoRⅠ双酶切后,与表达载体pPROEX HTb连接,转化感受态细胞E.coliDH5α,经PCR鉴定和双酶切鉴定得到阳性重组质粒pPROEX HTb-GST,在IPTG诱导下,获得融合蛋白的表达。经SDS-PAGE蛋白电泳鉴定,表达产物为25KD的GST融合蛋白。Northern杂交结果表明,2龄期斜纹夜蛾GST基因在mRNA水平上的表达量最大,3龄期次之,5龄期时的表达量最小。

Glutathione S-transferases（GSTs） were demonstrated to be involved in detoxification and xenobiotics in insects.GST gene was amplified from Spodoptera litura cDNA library using two specific primers.The PCR product was then cloned to pGEM-T vector and both the GST gene and pPROEX HTb backbone were digested by SpeⅠ＋EcoRⅠ.The GST fragment was cloned into pPROEX HTb by T4 ligase and transformed into E.coli DH5α.The positive recombinant plasmid named pPROEX HTb-GST was obtained.The GST protein was expressed distinctly after inducing by IPTG.Afterwards they were identified by SDS-PAGE electrophoresis.The prokaryotic recombinant expression plasmid pPROEX HTb-GST was correctly constructed and 25KD fusion protein was obtained in E.coli.Northern blotting result indicated that GST gene was expressed at the level of RNA.These provide experimental basis for further research on its biological function.