鹅细小病毒VP2和VP3蛋白酵母双杂交诱饵载体的构建及验证

Construction and Identification of Bait Vectors with VP2 and VP3 Gene of Goose Parvovirus in Yeast Two-Hybrid System

为筛选与鹅细小病毒VP2和VP3蛋白相互作用的鹅胚成纤维细胞蛋白,构建VP2和VP3蛋白的诱饵载体pGBKT7-VP2和pGBKT7-VP3。从pGEX-4T-VP1质粒中PCR扩增VP2和VP3基因,克隆至pMD-18T载体中,经测序验证鉴定后定向克隆到酵母双杂交载体pGBKT7中。将2个重组诱饵载体经PCR、酶切和测序验证后分别转化酵母菌H2YGold中,检测其在酵母细胞中有无自激活和毒性作用。结果表明:成功构建了pGBKT7-VP2和pGBKT7-VP3诱饵载体,且其对报告基因无自激活作用,对酵母细胞无毒性。由此说明,诱饵载体pGBKT7-VP2和pGBKT7-VP3可用于酵母双杂交系统筛选与VP2和VP3蛋白相互作用的细胞结合蛋白。

Two bait vectors, pGBKT7-VP2 and pGBKTT-VP3,were constructed for screening cellu- lar proteins interacting with VP2 and VP3 proteins of goose parvovirus from yeast two-hybrid cDNA library of goose embryo fibroblast cells in this study. VP2 and VP3 genes were amplified by PCR from plasmid pGEX-4T-VP1 and cloned into pMD-18T vector. After being verified by sequencing, they were subcloned into vector pGBKT7 of yeast two-hybrid system.Then the recombinant plasmids identified by PCR, enzyme digestion and sequencing were transformed into yeast cells H2YGold.The two bait vectors＇ self-activation to reporter genes and cytotoxicity to the yeast cells were tested. The results showed that the two bait plasmids, pGBKT7-VP2 and pGBKT7-VP3, were successfully con- structed and proved to be no self-activation to reporter genes and no cytotoxicity to the yeast cells.They could be used to screen cellular proteins interacting with VP2 and VP3 proteins in the yeast two-hybrid system.