人巨细胞病毒UL55编码蛋白结合蛋白的筛选与鉴定

Screening and Identification of Proteins Interacted with UL55 Protein

从人胎脑c DNA文库中筛选和鉴定出与人巨细胞病毒(Human cytomegalovirus,HCMV)UL55编码蛋白结合的蛋白。将UL55基因编码区克隆到诱饵载体p GBKT7中,在证实UL55蛋白不具有自激活作用的前提下,采用Match-maker GAL酵母双杂交系统筛选人胎脑c DNA文库中与UL55蛋白结合的宿主蛋白,用酵母双杂交回转实验验证UL55蛋白与获得的蛋白结合的可靠性。将酵母双杂交筛选出的文库蛋白烯醇化酶1(enolase1,ENO 1)构建到p GEX-4T-2载体上,利用GST pull-down技术体外验证ENO 1与HCMV UL55蛋白的结合。并依据所筛选出蛋白的生物学功能分析UL55蛋白可能的生物学功能。结果显示有10种蛋白与HCMV UL55编码蛋白结合。应用GST pull-down技术检测到ENO 1与HCMV UL55相互结合的蛋白条带。成功地筛选出10种与UL55蛋白相互结合的宿主蛋白,GST pull-down实验进一步表明ENO 1可以与HCMV UL55蛋白直接结合,为进一步研究UL55蛋白的功能提供了新的线索。

The objective of this study was to screen the binding proteins of human cytomegalovirus（ HCMV） UL55protein（ p UL55） from an human fetus brain c DNA library and identify the binding ability between protein ENO 1and HCMV p UL55 in vitro. Coding region of HCMV UL55 gene was cloned into the p GBKT7 vector. GAL4 yeast two-hybrid was used to screen the binding proteins of p UL55 from the human fetal brain c DNA library. After confirming the UL55 coding sequence have no the self-activaty,Yeast two hybrid rotary experiment was used to validate the reliability of interactions. Binding proteins were searched by the Blast network service at the National Center for Biotechnology Information using the sequences of the selected clones. The putative binding protein of p UL55,ENO 1,expressed by recombinant p GEX-4T-2 vector was further identified by GST pull-down technology.Ten proteins were identified to bind p UL55 from the human fetal brain c DNA library by yeast two-hybrid assay. The direct interaction between ENO 1 and p UL55 protein in vitro was confirmed by GST pull-down technology. At least10 host proteins showed binding abilities to p UL55. Among them,the direct interaction between ENO 1 and p UL55 was demonstrated by GST pull-down experiments. The results provide new clues for further study on biological functions of UL55 protein.