禽流感病毒NP基因的酵母双杂交饵载体表达及自激活检测

Expression of nucleoprotein of avian influenza virus bait vectors and detection of its self-activation in yeast two-hybrid system

目的构建H5N1亚型禽流感病毒NP基因的酵母双杂交诱饵载体,验证其在酵母中的表达并检测其自激活作用。方法以PCR法从pGEMT/H5NP扩增NP基因编码区序列,将其定向克隆到PGBKT7载体,经测序鉴定后,PEG/Li-Ac法转化酵母菌株AH109,用表型筛选法及颜色筛选法检测其自激活作用同时Westernblot验证诱饵蛋白的表达。结果获得NP编码区基因,并成功构建酵母双杂交系统中的诱饵载体pGBKT7-NP,对宿主细胞酵母菌株AH109无毒性和自激活作用,并能在酵母细胞中稳定表达。结论诱饵载体pGBKT7-NP可用于GAL4酵母双杂交系统钓取与禽流感病毒核蛋白相互作用的蛋白。

To express of nucleoprotein（NP） of avian influenza virus （H5N1） in yeast two-hybrid system and to detect its self-activation in this system, the complete encoding gene of NP was amplified from a plasmid pGEMT/H5NP by PCR and it was inserted into pGBKT7 vector according to the reading frame. The bait vectors were transformed into the yeast strain AH109 by PEG/LiAc method and its self-activation was tested by both the phenotype assay and the color assay,then expression of Ga14-NP fusion proteins identified by Western blot. In this way, the bait vectors of NP was constructed correctly and its expression in yeast were verified by Western blotling. The bait vector PGBKT7-NP was nontoxic to yeast and did not have selfactivating function. Therefore,yeast two-hybrid GAL4 system 3 can be utilized to fish NP-interacting protein.