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.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
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)
