PIAS-NY酵母双杂交诱饵载体的构建及其自激活活性分析  

Construction and identification of yeast two-hybrid bait vector pGBKT7-PIAS-NY

作　　者：郑英[1] 张露萍[1] 王海燕[1] 孙红亚[1] 梁虹[1] 王建军[1] 

机构地区：[1]扬州大学医学院组织学与胚胎学教研室,江苏扬州225001

出　　处：《中国现代医学杂志》2011年第32期3971-3974,共4页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:31071020);江苏省自然科学基金(No:BK2009192)

摘　　要：目的构建PIAS-NY(protein inhibitor of activated STAT-NY,PIAS-NY)的诱饵载体,为应用酵母双杂交系统筛选与PIAS-NY相互作用的蛋白建立实验基础。方法应用PCR法扩增PIAS-NY编码序列的片段,先将其克隆入pGEMT-easy内,经序列测定确认无误后,再将片段亚克隆入pGBKT7载体内,将构建好的诱饵载体pGBKT7-PIAS-NY转化到酵母细胞AH109及Y187中,进行诱饵载体的毒性及自激活活性分析。结果构建了PIAS-NY的诱饵载体,经过自激活活性的分析发现,该诱饵载体没有自激活活性,同时对两种酵母细胞的生长无毒性作用。结论成功构建了PIAS-NY的酵母双杂交诱饵表达载体,为进一步筛选与之相互作用的蛋白提供了实验基础。[Objective] To eonstruct the bait vector pGBKT7-PIAS-NY for screening its interacting proteins by yeast two-hybrid system. [Methods] The open reading frame of PIAS-NY was amplified and cloned into pGEMT-easy vector. After being verified by sequencing, the fragment was cloned into the bait expression vector pGBKT7. The bait vector pGBKTT-PIAS-NY was transformed into AH109 and Y187 yeast cells. Then toxicity and self-activation of the bait protein was tested. [Results] PIAS-NY was amplified and cloned into pGBKT7 successfully. The bait vector was transformed into AH109 and Y187. And the DNA-BD fusion has no toxicity and self-activation in these-yeast cells. [ Conclusions ] The bait vector of PIAS-NY was constructed successfully. It can be used for the following screening work by yeast two-hybrid system.

关 键 词：酵母双杂交 PIAS—NY 载体构建 

分 类 号：R339.2[医药卫生—人体生理学]