酵母双杂交筛选与人巨细胞病毒US28相互作用的蛋白  

Screening of the host proteins that interact with human cytomegalovirus US28 protein by yeast two-hybrid system

作　　者：于洪敏[1] 金新[2] 张欢欢[1] 张茂林[1] 段铭[1] 关振宏[1] 

机构地区：[1]吉林大学人兽共患病教育部重点实验室,人兽共患病研究所,吉林长春130062 [2]吉林农业大学动物科学学院,吉林长春130118

出　　处：《中国热带医学》2016年第1期6-9,33,共5页China Tropical Medicine

基　　金：国家重点基础研究发展计划(973计划)(No.2012CB518901)

摘　　要：目的利用酵母双杂交技术筛选与人巨细胞病毒US28蛋白相互作用的宿主蛋白,可为研究US28蛋白的作用机制提供依据。方法构建p GBKT7-US28诱饵重组载体,检测其在酵母细胞中的表达和自激活作用,然后利用酵母双杂交系统筛选人脑文库中与US28相互作用的蛋白质。对获得的阳性克隆进行PCR鉴定、测序及序列比对分析,并通过酵母共转化实验再次确认蛋白的相互作用。结果 p GBKT7-US28在酵母中成功表达US28融合蛋白;酵母双杂交筛选并验证,获得了5个与US28蛋白相互作用的宿主蛋白。结论初步鉴定得到5个与US28相互作用的细胞蛋白,为探索US28蛋白的新功能以及揭示巨细胞病毒的致病机理和疾病治疗奠定基础。Objective To screen the cellular proteins which interact with US28 protein of human cytomegalovirus （HCMV） by using yeast two-hybrid system, and to provide a theoretical basis for the explorement of US28 function. Methods The bait plasmid pGBKT7-US28 was constructed and transformed into yeast cells. The expression and self-activation capacities of bait protein were tested, and then a screening was performed using a pre-transformed human brain eDNA library to screen the interaction partners of US28. To confirm the putative positive clones by PCR, DNA sequencing and bioinformatic analysis were performed. Subsequently, the retransformation experiments were also performed to further verify the interactions in yeast cells. Results The US28 protein of bait plasmid pGBKT7-US28 was constructed and expressed in yeast successfully. Finally, five positive clones interacting with US28 were obtained via yeast two-hybrid screening and verification. Conclusion The results showed that five positive clones interacting with US28 were obtained, which would contribute to probe novel functions of US28 at molecular level, and provide useful evidence to explain the pathogenic mechanism and treatment of HCMV infection.

关 键 词：酵母双杂交 US28蛋白 蛋白相互作用 人巨细胞病毒 炎症 

分 类 号：R-33[医药卫生]