猪传染性胃肠炎病毒M基因酵母双杂交诱饵载体的构建及鉴定

Construction and Identification of a Bait Vector with TGEV Protein in the Yeast Two-Hybrid System

筛选与猪传染性胃肠炎病毒(TGEV)M蛋白相互作用的宿主蛋白,构建M蛋白的诱饵载体pGBKT7-M.首先利用PCR技术从重组表达载体pFastBacTM-M中扩增得到M基因,定向克隆至载体pMD19-T simple,验证正确后克隆至酵母双杂交系统诱饵表达载体pGBKT7,经双酶切、PCR反应及测序验证其正确插入后,利用PEG/LiAc法将构建好的重组诱饵载体pGBKT7-M及空载体pGBKT7分别转化到酵母Y2HGold感受态细胞中,检测其对酵母菌株是否有毒性作用和自激活活性及诱饵蛋白在酵母细胞内的表达情况.结果表明:扩增得到M基因738bp,成功构建了诱饵表达载体pGBKT7-M,此诱饵载体对酵母细胞无细胞毒性和自激活活性.Western blot检测到5×104左右的蛋白条带,显示诱饵蛋白在酵母细胞内稳定表达.

A bait vector for M protein（pGBKT7-M）was constructed for screening the host proteins of the interactions between TGEV and M protein.First,the gene from the structural proteins M was amplified from pFastBacTM-M by PCR and cloned into the vector pMD19-T simple.After being verified,it was directly cloned into the bait vector pGBKT7 of the yeast two-hybrid system.Then the recombinant plasmid was identified by PCR,restriction enzyme digestion and sequence analysis and transformed into the yeast cells Y2 HGold by the PEG/LiAc method.Toxicity and self-activation of the bait protein were detected using different droupout minimal bases,and the bait vector pGBKT7-M was shown to have no toxicity to the yeast cells and no self-activation phenomenon to the report genes.The above results indicated that the constructed bait vector pGBKT7-M can be used for screening the host proteins interacting with TGEV M protein using the yeast two-hybrid system.