Wang Chunyan, Guo Liang, Yu Dayi, Hua Xiuguo, Yang Zhibiao, Yuan Congli, Cui Li
Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China.
Animal Disease Control Center of Min Hang District, Shanghai, China.
Hepat Mon. 2014 Jan 18;14(1):e13902. doi: 10.5812/hepatmon.13902. eCollection 2014 Jan.
Hepatitis E virus (HEV) is a major causative agent of acute clinical hepatitis in adults through much of Asia, the Middle East and Africa. Open reading frame 3 (ORF3) encodes around 120 amino acids of phosphorylation protein that associates with the cytoskeleton, while its precise biological function is still unknown.
In order to understand the function of ORF3 protein (pORF3) in depth, HEV ORF3 interacting proteins were screened in human hepatocytes cDNA library using two-hybrid system techniques and further verification of the interactions were carried out through co-immunoprecipitation (Co-IP).
The Cyto-Trap two-hybrid system technology, a classical method for analyzing protein interactions, was used to screen the pORF3 interacting proteins from human hepatocytes cDNA library.
Through the Cyto-Trap two-hybrid system, eight proteins interacting with pORF3 were winnowed. The Co-IP results confirmed that hepsin which is reported to function as the inhibitor of several tumors reacted with pORF3.
Out of eight screened proteins interacting with pORF3, hepsin was confirmed to have specific interactions with pORF3.
戊型肝炎病毒（HEV）是亚洲、中东和非洲大部分地区成人急性临床肝炎的主要病原体。开放阅读框3（ORF3）编码约120个氨基酸的磷酸化蛋白，该蛋白与细胞骨架相关，但其确切生物学功能尚不清楚。
为深入了解ORF3蛋白（pORF3）的功能，利用双杂交系统技术在人肝细胞cDNA文库中筛选HEV ORF3相互作用蛋白，并通过免疫共沉淀（Co-IP）进一步验证相互作用。
采用经典的分析蛋白质相互作用的方法——细胞捕获双杂交系统技术，从人肝细胞cDNA文库中筛选与pORF3相互作用的蛋白。
通过细胞捕获双杂交系统，筛选出8种与pORF3相互作用的蛋白。免疫共沉淀结果证实，据报道作为几种肿瘤抑制剂发挥作用的组织蛋白酶与pORF3发生反应。
在筛选出的8种与pORF3相互作用的蛋白中，证实组织蛋白酶与pORF3存在特异性相互作用。
