禽呼肠孤病毒σA基因酵母双杂交诱饵载体的构建与鉴定

Construction and Identification of Bait Vector of Avian Reovirus σA Gene in Yeast Two-hybrid System

为了筛选与禽呼孤病毒σA基因相互作用的宿主蛋白,本试验应用酵母双杂交技术构建禽呼孤病毒σA基因的诱饵载体pGBKT7-σA。从禽呼肠孤病毒S1133标准毒株抽提RNA,采用RT-PCR方法扩增得到σA基因片段,将其连接到诱饵载体pGBKT7上,把通过测序的重组诱饵载体命名为pGBKT7-σA。将重组诱饵载体转化酿酒酵母Y2HGold后,把不同浓度的诱饵载体转化液涂布在营养缺陷型培养基上,观察该重组诱饵载体在酵母细胞中有无毒性作用和自激活现象。结果显示,酵母双杂交诱饵载体pGBKT7-σA构建成功,且对酿酒酵母无毒性和自激活现象。本研究为进一步利用酵母双杂交技术筛选与σA蛋白互作的宿主蛋白奠定了基础。

In order to screen the host protein that interacted withσA gene of avian reovirus,the bait vector pGBKT7-σA of avian reovirusσA gene in yeast two-hybrid system was constructed.Total RNA of avian reovirus S1133 standard strain was purified by using Trizol reagents.Avian reovirusσAgene was amplified by RT-PCR and was cloned into yeast two-hybrid bait vector pGBKT7.The recombinant bait vector was named as pGBKT7-σA.The recombinant bait vector was transformed into Saccharomyces cerevisiae Y2 HGold.And then the recombinant bait vector of different concentrations was applied to the nutrient deficient medium.The results showed that the yeast two-hybrid bait vector had been constructed successfully,and the recombinant bait vector pGBKT7-σA on yeast cells had no toxicity and self-activation.This study laid a foundation for further screening host protein interacted withσA protein by yeast two-hybrid technique.