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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

DOI: 10.13560/j.cnki.biotech.bull.1985.2015.08.016, PP. 108-113

Keywords: Wap65-2,酵母双杂交,表达,自激活,毒性

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Abstract:

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(Larimichthyscrocea)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Westernblot分析重组蛋白的表达。成功扩增LcWap65-2m,并克隆至pGBKT7载体中;Westernblot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。

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