柯萨奇病毒A16型2 B基因的酵母双杂交诱饵载体的构建及其特性鉴定

Establishment of Yeast Two-Hybrid Bait Vector of CV A16/2 B and I-dentification of Its Characteristics

目的：构建柯萨奇病毒A16的2B基因的酵母诱饵表达载体，用于筛选与2B相互作用的蛋白。方法 PCR扩增2B全序列，克隆入pGBKT7？BD，将构建好的 pGBKT7？2B转化到酵母细胞 Y2HGold；用Western印迹分析诱饵蛋白的表达，检测诱饵蛋白有无毒性和自激活效应。结果成功克隆了pGBKT7？2B并转化至Y2 HGold中，转化细胞Y2 HGold [pGBKT7？2B]可以表达诱饵蛋白2B，2B蛋白对转化细胞无细胞毒性和自激活效应。结论成功构建了酵母诱饵表达载体pGBKT7？2B，可用于酵母双杂交筛选与2B蛋白相互作用的宿主靶蛋白。

Objective To construct the yeast bait expression vector pGBKT7？2 B of coxsack？ievirus （ CV ） A162 B gene for screening the target proteins that interact with the bait protein 2B. Methods The fragments of 2B were amplified by PCR, and then cloned into the bait expres？sion vector pGBKT7. The bait vector pGBKT7？2B, verified by sequencing, was transformed into Y2 HGold yeast cells. The expression of bait protein 2 B was analyzed by Western blotting. The cytotoxic？ity and transcriptional activation of the bait protein were detected. Results pGBKT7 was successful？ly cloned and transformed into Y2 HGold. The transfected cells could express bait protein 2B, which exhibited neither cytotoxicity nor transcriptional activation. Conclusion The bait expression vector pGBKT7？2B was constructed successfully, which can be utilized to screen the target proteins that interact with CV A162 B.