禽呼肠孤病毒σA基因酵母双杂交诱饵载体的构建与鉴定

发布时间：2018-02-05 16:41

本文关键词： 禽呼肠孤病毒 σA基因酵母双杂交诱饵载体　出处：《中国畜牧兽医》2017年03期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:In order to screen host proteins interacting with 蟽 A gene of avian reovirus. The bait vector pGBKT7- 蟽 A of avian reovirus 蟽 A gene was constructed by yeast two-hybrid technique. RNA was extracted from the standard strain of avian reovirus S1133. The 蟽 A gene fragment was amplified by RT-PCR and ligated to the decoy vector pGBKT7. The recombinant bait vector was named pGBKT7- 蟽 A. the recombinant bait vector was transformed into Saccharomyces cerevisiae Y2HGold. The transformation solution of different concentration of bait carrier was coated on nutrition-deficient medium to observe the non-toxicity and self-activation of the recombinant bait vector in yeast cells. The results showed that the recombinant bait vector had no toxicity and self-activation. Yeast two-hybrid bait vector pGBKT7- 蟽 A was successfully constructed. This study laid a foundation for the further screening of host proteins interacting with 蟽 A protein by yeast two-hybrid technique in the absence of toxicity and self-activation of Saccharomyces cerevisiae (Saccharomyces cerevisiae).
【作者单位】：广西大学动物科学技术学院;广西壮族自治区兽医研究所广西兽医生物技术重点实验室;
【基金】：国家自然科学基金项目(31660715、31160512) 国家"万人计划"领军人材专项(2016-37) 广西特聘专家专项(2011B020)
【分类号】：S852.65

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