酵母双杂交筛选PA28γ相互作用蛋白  

Identification of human liver proteins interaction with PA28γ by yeast two-hybrid screening

作　　者：覃世逆[1] 王穗海[1] 徐伟文[1] 

机构地区：[1]南方医科大学生物技术学院,广东广州510515

出　　处：《热带医学杂志》2014年第6期714-716,共3页Journal of Tropical Medicine

基　　金：国家863课题资助项目(2012AA020205)

摘　　要：目的通过酵母双杂交筛选与蛋白酶体激活因子3(PA28γ)相互作用的肝细胞蛋白,为研究其在肝癌的发生发展中的作用机制提供新依据。方法通过酵母双杂交技术,构建PGBKT7-PA28γ蛋白酶体激活因子酵母双杂交诱饵载体,以PA28γ为诱饵,在人类肝细胞cDNA文库中筛选与之相互作用的蛋白,并将部分阳性基因构建表达载体后与PA28γ共转染于AGS细胞进行初步验证。结果筛选出32个阳性克隆,其中有5个与肝癌细胞周期及凋亡密切相关,如ZPR9。将PWPI-PA28γ和Pcmv-myc-ZPR9共转染AGS细胞,检测发现PA28γ含量与ZPR9负相关。结论成功筛选到一些与肝癌细胞周期及凋亡相关的蛋白,为研究其机制提供了良好的线索和基础。Objective Identify PA28γ which is a proteasome activator interaction proteins by yeast two-hybrid screening for studying its effect on cell cycle and apoptosis in liver cancer cell. Methods We screened the human liver cDNA library by yeast two-hybrid and got some positive clones.First,we cloned PA28γ to pGBKT7 as bait for autoactivation assay in yeast Y2HGold,then the yeast was mated with Y187 which contains the human liver cDNA.We also constructed some expression vectors for preliminary validation. Results After PCR screening,we got 32 positive yeast clones,including 5 genes which are closely related to cell cycle and apoptosis,e.g.ZPR9.We also constructed pCMV-myc-ZPR9vector. PWPI-PA28γ and pCMV-myc-ZPR9 were then cotransfected to AGS cells,and detected the expression by Westernblot.A negative correlation was found between PA28γ and ZPR9 expression. Conclusion We identified PA28γ interaction proteins through yeast-two hybrid screening, it will be useful for further study on it＇s role in liver cancer development.

关 键 词：PA28γ 蛋白酶体 酵母双杂交 

分 类 号：R392.11[医药卫生—免疫学]