| 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 |
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| 引用本文： | 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(3):42-48. |
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| 作者姓名： | 谢吉国 闫秀英 简纪常 吴灶和 |
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| 作者单位： | 1. 广东海洋大学水产学院,广东省水产经济动物病原生物学及流行病学重点实验室暨广东省高等学校水产经济动物病害控制重点实验室,广东湛江524025
2. 仲恺农业工程学院,广东广州510000 |
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| 基金项目： | 国家973计划(2009CB118704) |
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| 摘 要： | 根据草鱼呼肠孤病毒(Grass Carp Reovirus,GCRV)的vp5基因设计特异性引物,并扩增其开放阅读框序列(open reading frame,ORF),然后连接至pGBKT7载体上构建酵母双杂交诱饵载体pGBKT7-vp5,转化至酵母菌Y2HGold,并进行PCR鉴定以及自激活性和毒性检测。PCR、酶切以及测序表明,pGBKT7-vp5重组诱饵载体构建成功。自激活性和毒性检测显示,阳性重组菌株pGBKT7-vp5/Y2HGold和阴性对照菌株pGBKT7/Y2HGold在SD/-Trp平板上出现大小相一致的乳白色菌落,且在SD/-Trp/X-α-Gel、SD/-Trp/AbA+/X-α-Gel平板上生长出蓝色菌落,而在SD/-Trp/-Ade/-His无菌落出现。表明重组菌株pGBKT7-vp5/Y2HGold表达的融合蛋白激活了报告基因AUR1-C和MEL1,没有激活报告基因ADE2和HIS3并且对宿主菌无毒性。该重组诱饵载体可用于酵母双杂交系统进一步筛选cDNA文库中与其相互作用的蛋白。
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| 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 |
Construction, Transformation, and Self-Activity Research of Bait Recombined Vector with vp5 Gene |
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| XIE Ji-guo,YAN Xiu-ying,JIAN Ji-chang,WU Zao-he.Construction, Transformation, and Self-Activity Research of Bait Recombined Vector with vp5 Gene[J].Journal of Zhanjiang Ocean University,2012(3):42-48. |
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| Authors: | XIE Ji-guo YAN Xiu-ying JIAN Ji-chang WU Zao-he |
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| Institution: | 1,2(1.Fisheries College of Guangdong Ocean University,Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals&Key Laboratory of Diseases Controlling for Aquatic Economic Animals of Guangdong Higher Education Institutions,Zhanjiang 524025,China;2.Zhongkai University of Agriculture and Engineering,GuangZhou,510225,China) |
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| 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-vp5,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. |
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| Keywords: | Grass Carp Reovirus(GCRV) yeast two-hybrid system bait vector self-activation |
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