广东海洋大学学报2012，Vol.32Issue(3)：42-48,7.
草鱼呼肠孤病毒vp5基因诱饵重组载体的构建、转化与自激活作用检测
Construction, Transformation, and Self-Activity Research of Bait Recombined Vector with vp5 Gene
摘要
Abstract
The specific primers were designed according to vp5 gene of the Grass Carp Reovirus (GCRV), and the open reading frame of GCRV vp5 gene was cloned and ligated to the pGBKT7 vector to construct the bait vector in yeast two-hybrid system, pGBKT7-ypJ, and then transformed into the competent yeast Y2HGold. Whether the recombinant bait vector is toxic to the yeast and its self-activation in yeast two-hybrid system was measured. PCR reaction, restriction enzyme digestion and sequencing analyses showed that the recombinant bait vector pGBKT7-vp5 was successfully constructed. Self-activitation and toxicity analyses revealed that the positive recombinant pGBKT7-vp5/ Y2HGold had the same size white colony on SD/-Trp plate as the negative control pGBKT7/Y2HGold. It also has appeared blue colony on SD/-Trp/X-α-Gel and SD/-Trp/AbA+/X-α-Gel plate, but none colony appeared on SD/-Trp/-Ade/-His plate. All results indicated that the fusion protein expressed by recombinant pGBKT7-vp5/Y2HGold activated the report gene AUR1-C and MEL1, but inactivated the gene ADE2 and HIS3 either non-toxic to Y2HGold。 So the report gene Ade2 and His3 can be used in yeast two-hybrid system to further screen the cDNA library and to trap the gene interacting with it.关键词
草鱼呼肠孤病毒/酵母双杂交系统/诱饵载体/自激活性Key words
Grass Carp Reovirus (GCRV)/ yeast two-hybrid system/ bait vector/ self-activation分类
农业科技引用本文复制引用
谢吉国,闫秀英,简纪常,吴灶和..草鱼呼肠孤病毒vp5基因诱饵重组载体的构建、转化与自激活作用检测[J].广东海洋大学学报,2012,32(3):42-48,7.基金项目
国家973计划(2009CB118704) （2009CB118704）
