酵母双杂交筛选与单剪接型2．2kb乙型肝炎病毒剪接特异性新蛋白相互作用的肝细胞蛋白  

Yeast two-hybrid system screening liver cell proteins interacting with the novel protein encoded by the 2. 2 kb singly spliced variant of hepatitis B virus genome

作　　者：陈婉南[1] 陈金烟[1] 黄清玲[1] 林建银[1] 林旭[1] 

机构地区：[1]福建医科大学分子医学研究中心,福建省高校感染与肿瘤重点实验室,福州350004

出　　处：《中华微生物学和免疫学杂志》2010年第3期228-233,共6页Chinese Journal of Microbiology and Immunology

基　　金：基金项目：福建省科技厅青年人才创新项目（2008F3046）;福建省高等学校科技创新团队培育计划基金（FNU-RT001）;国家自然科学基金（30970163）

摘　　要：目的筛选与单剪接型2．2kb乙型肝炎病毒（HBV）剪接特异性新蛋白相互作用的肝细胞蛋白。方法PCR扩增单剪接型2．2kbHBV剪接特异性新基因TPss并克隆于诱饵载体pGBKT7，在证实TPss蛋白不具有自激活作用的前提下，以酵母双杂交系统筛查与TPss蛋白相互作用的肝细胞蛋白，进而通过哺乳动物细胞双杂交实验验证候选肝细胞蛋白与TPss蛋白在Huh7和HepG2肝细胞中的相互作用。结果构建酵母双杂交诱饵载体pGBKT7-TPss，Westernblot显示其在酵母中表达TPss蛋白。酵母双杂交筛选及哺乳动物细胞双杂交证实TPss蛋白可与4种肝细胞蛋白相互作用，即组织蛋白酶B、微粒体环氧化物水解酶、组织蛋白酶D与纤维蛋白原γ链。结论TPss可与多种肝细胞蛋白相互作用。Objective To screen the liver cell proteins interacting with the novel protein encoded by the 2.2 kb singly spliced variant of hepatitis B virus genome. Methods The splicing-specific gene TPss generated by the 2.2 kb singly spliced variant of HBV genome was amplified by PCR and cloned into the bait vector pGBKT7. After exclusion of self-activation capacities of TPss protein, a two-hybrid library screening was performed using a pre-transformed human liver cDNA library to screen the liver cell proteins interacting with TPss. Mammalian two-hybrid assay was also done to further confirm the interactions between the bait and prey proteins in Huh7 and HepG2 hepatocytes. Results TPss gene with the size of 336 bp was successfully amplified and cloned, and the TPss protein expressed well in AH109 yeast cells. Four liver cell pro- teins interacting with TPss, i. e. , cathepsin B, epoxide hydrolase 1, cathepsin D and fibrinogen gamma chain, were screened by yeast two-hybrid assay and further confirmed by mammalian two-hybrid assay. Conclusion TPss could interact with liver cell proteins.

关 键 词：乙型肝炎病毒 RNA剪接 酵母双杂交 

分 类 号：R3[医药卫生—基础医学]