大黄鱼Wap65-2基因成熟肽区酵母双杂交诱饵载体的构建及鉴定

摘要

The mature peptide ofWap65-2 gene(LcWap65-2m)in large yellow croaker(Larimichthys crocea)was used to construct the yeast two-hybrid bait vector, and its self-activation and toxic effect were measured.LcWap65-2m from the liver cDNA of large yellow croaker was amplified, and cloned into the plasmid pGBKT7. After verification by PCR amplification, enzyme digestion and sequencing, the bait vector pGBKT7-LcWap65-2m was transformed into the yeast strain Y187. The self-activation and toxic effect of the recombinant protein were detected in selective nutrition medium, and the expression of the bait protein LcWap65-2m was analyzed by Western blot.LcWap65-2m was successfully amplified and inserted into the plasmid vector pGBKT7. Western blot analysis showed that the bait protein LcWap65-2m was expressed in yeast cells;phenotypic screening tests revealed that the bait protein LcWap65-2m had no self-activation activity and toxic effect. In conclusion, the yeast two-hybrid bait vector pGBKT7-LcWap65-2m was successfully constructed, which laid the foundation for screening the protein interacted with the bait protein Wap65-2 using the yeast two-hybrid system.%用大黄鱼Wap65-2基因成熟肽区构建酵母双杂交诱饵质粒，并对诱饵质粒进行自激活活性及毒性检测。从大黄鱼（Larimichthys crocea）肝组织cDNA中扩增Wap65-2基因成熟肽片段（LcWap65-2m），将其克隆至pGBKT7载体中，获得诱饵载体pGBKT7-LcWap65-2m，经PCR、酶切和测序验证无误后，转化至酵母菌株Y187中，在营养缺陷培养基中观察重组蛋白的毒性作用和自激活活性，同时利用Western blot分析重组蛋白的表达。成功扩增LcWap65-2m，并克隆至pGBKT7载体中；Western blot检测结果表明，pGBKT7-LcWap65-2m在酵母细胞中表达诱饵蛋白LcWap65-2m；表型筛选结果显示，LcWap65-2m无毒性作用和自激活活性。成功构建了LcWap65-2m酵母双杂交诱饵载体，为进一步利用酵母双杂交技术筛选与Wap65-2相互作用的蛋白奠定基础。