酵母双杂交系统筛选和验证与RARα-V相互作用的蛋白

目的 利用酵母双杂交技术筛选与RARα-V相互作用的蛋白,研究RARα-V的作用靶点及其生物学功能.方法 构建诱饵质粒pGBKT7-RARα-V,利用酵母双杂交技术从K562细胞cDNA文库中筛选与RARα-V相互作用蛋白的基因序列,并通过酵母回转试验与GST pull-down技术进行验证.结果 成功构建诱饵质粒,且没有毒性、渗漏和自激活现象;利用酵母双杂交技术筛选到16个能与RARα-V相互作用的蛋白质;经酵母回转试验得到8个阳性克隆;并经GST pull-down技术在体外验证了RARα-V与JTV-1蛋白的相互作用.结论 在细胞内RARα-V与多种蛋白有相互作用,白血病的发病机制可能与这些蛋白相互作用所致的生物学功能改变有关.

Screening and identification of proteins interacting with RARα-V via yeast two-hybrid system

Objective To screen the protein interacting with retinoic acid receptor variant protein (RAReα-V) via the yeast two-hybrid technique(YTHT) and to find out the targets protein and studv its bio-logical function. Methods The bait vector of pGBKT7-RARα-V was constructed for screening proteins inter-acting with RARα-V in K562 cell eDNA expression library via Yia YTHT.The protein-protein interaction was confirmed with re-transformation in yeast and GST pull-down in vitro.Results The bait vector was success-fully constructed without toxicity,leakage and self-activation.Sixteen proteins were screened bv YTHT and eight positive clones were identified by retransformation in yeast.The interaction between RARα-V and JTV-l was confirmed by GST pull-down in vitro. Conclusions There are some kinds of proteins interacting with RARα-V in cell.The biological dysfunction caused by certain protein-protein interaction may be involed in the pathogenesis of leukemia.