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

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。

Screening proteins interacting with Gankyrin by using yeast double hybridization system

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.