酵母双杂交筛选与蛋白激酶CK2α′相互作用蛋白--泛素-52氨基酸融合蛋白的鉴定  

Identification of Ubiquitin-52 Amino Acid Fusion Protein as Interacting Partner of Protein Kinase CK2 α′ Subunit by Two-hybrid Screening

作　　者：陈碧[1] 骆艳婷[1] 刘新光[1] 梁念慈[1] 

机构地区：[1]广东医学院生物化学与分子生物学研究所,湛江524023

出　　处：《中国生物化学与分子生物学报》2008年第9期826-832,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家重点基础研究发展规划(973计划)项目子课题项目(No.2007CB507403);国家自然科学基金项目(No.30672205);广东省自然科学基金项目(No.05011579);广东省高校自然科学研究重点项目(No.06Z015);湛江市2006年科技招标项目~~

摘　　要：蛋白激酶CK2是一种真核细胞中普遍存在的信使非依赖性丝/苏氨酸蛋白激酶.为研究CK2α′亚基在精子发生中的作用机制,将构建于pACT2质粒的人睾丸cDNA文库和人蛋白激酶CK2α′为诱饵蛋白进行酵母双杂交实验.以初步筛选与人蛋白激酶CK2α′相互作用蛋白的阳性候选克隆,筛选获得8个阳性克隆,其中1个与人泛素-52氨基酸融合蛋白基因(UBA52)的cDNA序列有高度同源性(100%).GST pull-down实验在细胞外进一步证实了CK2α′与UBA52之间存在相互作用.本实验证明,人泛素-52氨基酸(UBA52)融合蛋白是人CK2α′亚基的相互作用蛋白,它们之间的相互作用对精子发生机制的影响尚不清楚,进一步分子机制研究正在进行中.Protein kinase CK2 is a ubiquitous eukaryotic messenger-independent protein serine/threonine kinase.To investigate the role of CK2 in spermatogenesis,a bait plasmid has been constructed by subcloning the human CK2α′ cDNA fragment into the yeast two-hybrid vector pGBKT7.Both of the bait pGBKT7-HCKα′ and a human testis cDNA library vectors were then cotransformed into AH109 competent cells to screen the interacting protein of human CK2α′ subunit by yeast two-hybrid system.The result showed that eight positive clones were selected for interaction with human CK2α′subunit.One of the potential binding partners showed high homology to human ubiquitin-52 amino acid fusion protein gene（100%）.GST pull-down assay confirmed that CK2α′ interacted with human ubiquitin-52 amino acid fusion protein in vitro.The interaction between CK2α′ and UBA52 was demonstrated both in a yeast two-hybrid system and in an in vitro assay.The importance of such interaction in spermatogenesis is unclear.Further investigation is required to address this issue.

关 键 词：人蛋白激酶CK2α′ 酵母双杂交系统 睾丸 人泛素-52氨基酸融合蛋白(UBA52) GST pull-down 

分 类 号：Q555.7[生物学—生物化学] R339.21[医药卫生—人体生理学]