应用酵母双杂交技术筛选与癌性锚蛋白重复序列相互作用的蛋白  

Screening proteins interacting with Gankyrin by using yeast double hybridization system

作　　者：包玉洁[1] 戴强[1] 杨文燕[1] 程芃[1] 王碧君[1] 张燕捷[1] 

机构地区：[1]上海交通大学医学院附属第三人民医院消化内科,上海201900

出　　处：《内科理论与实践》2011年第4期295-297,共3页Journal of Internal Medicine Concepts & Practice

基　　金：国家自然科学基金(项目编号:30900672);上海市青年科技启明星(项目编号:10QA1404300);上海市教委优秀青年教师专项基金(项目编号:jdy09074)

摘　　要：目的:筛选与癌性锚蛋白重复序列(Gankyrin)相互作用的蛋白。方法:构建诱饵(Bait)质粒pGB-Psmd10-1,采用β-半乳糖苷酶滤膜分析检测其自身转录活性,以Bait质粒筛选人Hela细胞MATCHMAKER cDNA文库,共转染重复验证阳性克隆,并测序鉴定。结果:成功构建Bait质粒,经验证对报告基因LacZ无自激活作用。以Bait质粒筛选人Hela细胞cDNA文库,获得83个克隆,其中5个克隆经重复验证确定为阳性克隆,测序结果显示其cDNA为Psmc4的3段不同剪接体(NM_006503.2,NM_153001.1)。结论:在人Hela细胞中,Psmc4为与Gankyrin相互作用的蛋白。Objective To screen proteins interacting with Gann ankyrin repeat （Gankyrin）. Methods Full length Gankyrin gene was subcloned into the pGB vector to construct Bait plasmid pGB-Psmdl0-1, and Bait＇s self-activating effect was detected by β-galactosidase colony lift filter assay. Bait was employed to screen Hela MATCHMAKER cDNA library. The positive clones were confirmed by β-galactosidase colony-lift filter assay after co-transfection with Bait and furthermore the obtained plasmids cDNA sequences were compared with the isogenous sequences in GenBank by Blast software via Internet. Results Recombinant plasmid of pGB-Psmdl0-1 was constructed correctly and Bait fusion protein had no activation effect on autonomous reporter gene LacZ. Eighty-three clones were screened from the cDNA library and 6 clones were obtained by the print analysis of 13-galactosidase colony-lift filter assay. Among them, 5 library plasmids were confirmed to be positive clones by co-transfection with Bait plasmids. The five obtained plasmids cDNA sequences were compared with the isogenous sequences in GenBank, and finally 3 recorded cDNA sequences were obtained, encoding 3 different splicer of Psmc4. Conclusions Psmc4 was the protein in Hela cells interacting with Gankyrin and the results bring some new clues for studying the pathogenic effect of Gankyrin.

关 键 词：癌性锚蛋白重复序列 酵母双杂交 Psmc4 HELA细胞 

分 类 号：R730.4[医药卫生—肿瘤]