鹅源新城疫病毒M蛋白酵母双杂交诱饵载体的构建及鉴定

Construction and identification of M bait vector of goose-origin Newcastle disease virus in yeast two-hybrid system

为了筛选鹅源新城疫病毒(JS/5/05/Go)M蛋白与鸡胚成纤维细胞作用的靶蛋白,应用酵母双杂交技术构建M蛋白的诱饵载体pGBKT7-M。采用RT-PCR方法从鹅源新城疫病毒中扩增了M基因片段,将其克隆到pGEM-T载体中,经测序鉴定正确后,定向克隆到酵母双杂交诱饵载体pGBKT7中,将重组诱饵载体转化酿酒酵母Y187,并验证其在酵母细胞中有无自激活和毒性作用。结果显示,成功构建了诱饵载体pGBKT7-M,并证明其对报告基因无自激活作用,且对酵母细胞无毒性。表明诱饵载体pGBKT7-M可用于酵母双杂交系统寻找与M蛋白相互作用的蛋白质。

The recombinant bait vector pGBKT7-M was constructed for screening the proteins which interact with M protein of goose-origin Newcastle disease virus（NDV）（JS5/05/Go） in chicken embryo fibroblasts.The M gene was amplified by RT-PCR from the goose-origin NDV and cloned into pGEM-T vector and sequenced.After being verified by sequencing,it was subcloned into the bait vector pGBKT7 of yeast two-hybrid system,and the recombinant vector was transformed into yeast cells Y187.Then self-activation and toxic action of the bait vector pGBKT7-M were tested.Results indicated that pGBKT7-M was successfully constructed and proven to be no self-activation and toxic to the host yeast.The pGBKT7-M could be used as a bait to screen the target proteins interacting with M protein in the yeast two-hybrid system.