酵母双杂交诱饵蛋白LASS1融合表达质粒的构建及鉴定

Construction and verification of bait protein LASS1-fusion expression plasmid in yeast two-hybrid system

目的构建酵母双杂交系统诱饵蛋白大鼠LASS1融合表达质粒,为进一步筛选大鼠脑神经元内与LASS1p相互作用的蛋白奠定基础。方法应用PCR方法获得大鼠LASS1基因2个片段,分别克隆入酵母双杂交系统诱饵蛋白质粒载体pGBKT7,转染酵母菌AH109并检测重组质粒的自激活现象及毒性。利用Western印迹检测重组质粒在AH109的表达情况。结果LASS1基因的PCR产物片段大小分别为Flag(111bp)、Slag(109bp);Western印迹结果表明2个重组质粒表达的蛋白均可以与抗c-Myc抗体在22kU处特异性反应;重组质粒转化酵母后无自激活作用。结论重组质粒pGBKT7-Flag、pGBKT7-Slag均能够在AH109内正确表达,可作为酵母双杂交系统诱饵蛋白使用。

Objective To construct bait protein LASSl-fusion expression plasmid in yeast two-hybrid system for further screening the protein interacting with LASS1p in rat cerebral nerves. Methods Two fragments of rat LASSI gene were amplified by PCR, and PCR products were cloned into the bait protein vector pGBKT7 of yeast two-hybrid system. Following the competent AH109 yeast transformed with the two recombinant plasmids, the toxicity and autonomous activation of the recombinant plasmid in AH109 were tested. Western blot analysis were performed to detect the recombinant protein expressed in AH109. Results The size of the two PCR products were Flag （111 bp） and Slag （ 109 bp） ; Western blot showed that the expression protein of the two recombinant plasmids reacted with e-Myc antibody and detected a 22 kU in the extracts of AH109 yeast. And no toxicity and autonomous activation was oberserved in AH109. Conclusions The two re-combinant plasmids pGBKTT-Flag and pGBKT7-Slag both express successfully in AH109, and the fusion protein can be used as the bait protein in yeast two-hybrid system.