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

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.