人DOC-2氨基端PID结构域酵母双杂交诱饵载体的构建及自激活鉴定  

Cloning of phosphotyrosine interacting domain of human DOC-2 gene and construction of the bait vector in yeast two-hybrid system

作　　者：刘淑娟[1] 杨力军[2] 王涛[3] 吴元明[4] 

机构地区：[1]第四军医大学西京医院妇产科,陕西西安710032 [2]第四军医大学西京医院泌尿外科,陕西西安710032 [3]第四军医大学生物化学与分子生物学教研室,陕西西安710032 [4]第四军医大学病理生理教研室,陕西西安710032

出　　处：《西北国防医学杂志》2007年第2期101-103,共3页Medical Journal of National Defending Forces in Northwest China

基　　金：国家自然科学基金资助项目(30500538)

摘　　要：目的:获得人DOC-2氨基端磷酸酪氨酸作用结构域(PID)cDNA基因,构建酵母双杂交诱饵载体,并检测对报告基因的激活作用。方法:PCR扩增含人DOC-2编码区的全长cDNA片段,克隆入诱饵载体pGBKT7,再亚克隆得到含PID结构域的酵母双杂交诱饵载体pGBKT7-nDOC2。将重组质粒导入酵母菌AH109,检测其表达产物在酵母细胞中对报告基因有无激活作用。结果:成功构建人DOC-2氨基端磷酸酪氨酸作用结构域(PID)酵母双杂交诱饵载体,该段基因表达的蛋白对酵母菌AH109既无毒性,对报告基因也没有激活作用。结论:我们可利用构建的酵母双杂交诱饵载体来钓取人DOC-2氨基端PID结构域的相互作用蛋白,以利于对DOC-2基因功能进行研究。Objective：To construct the bait vector with the phosphotyrosine interacting domain （PID） of human DOC - 2 in yeast two - hybrid system and investigate the effect of its expression on the growth of yeast cell, and detect the activation of reporter genes. Methods： The coding cDNA sequence of human DOC - 2 gene was amplified and cloned into the pGBKT7 plasmid. After verified by endonucleases digestion analysis, the pGBKT7 - DOC2 was digested by a series of endonucleases, and the recombinant bait vector pGBKT7 - nDOC2 of yeast two - hybrid system was obtained. Then, the recombinant plasmids were transferred into yeast cell AH109, and their expression products were assayed to observe if they could activate the reporter genes. Results： The bait vector with the PID domain of human DOC -2 in yeast two - hybrid system was constructed successfully. The PiD domain of human DOC -2 was not toxic to AH109 and could not activate the reporter genes. Conclusion： The yeast two - hybrid GAL4 system could be used to fish the PID domain of human DOC - 2 interacting proteins arid to further study the function of human DOC -2 gene.

关 键 词：人DOC-2基因 磷酸酪氨酸作用结构域 酵母双杂交 诱饵载体 

分 类 号：Q78[生物学—分子生物学]