摘要
目的利用酵母双杂交技术研究甲型H3N2流感病毒截短型PB1-F2蛋白与人类宿主蛋白的相互作用,为该病毒蛋白的功能研究和致病机制提供理论依据。方法以本实验室分离和鉴定的甲型H3N2流感病毒A/Guangdong/7028/2010为模版,构建pGBKT7-PB1-F2重组载体,利用Y2HGold酵母双杂交系统,从人类通用cDNA文库中筛选与其相互作用的蛋白。结果成功构建含诱饵蛋白基因的pGBKT7-PB1-F2重组载体,转化酵母自激活和毒性实验显示为阴性;酵母双杂交实验显示Y2HGold和Y187酵母的结合率为5.22%,符合实验要求;经筛选和验证后,得到3个与截短型PB1-F2蛋白有相互作用的阳性克隆,分别为钾/钠ATP酶β1亚基、热休克蛋白40和白介素-2受体γ亚基。结论初步推断截短型H3N2流感病毒PB1-F2蛋白可能影响流感病毒在宿主细胞中的复制功能和凋亡调控。
Objective To study the interaction between truncated PB1-F2 of influenza A virus subtype H3N2 and host proteins by yeast two-hybrid system, and understand the function and mechanism of PBI-F2 protein. Methods We focused on the truncated PB1-F2 of influenza A virus subtype H3N2 and use A/Guangdong/7028/2010 as template which isolated and identified by our laboratory. PB1-F2 gene was cloned into bait plasmid pGBKT-7 and confirmed by DNA sequencing. Yeast two-hybrid library screening （Y2HGold） was used to study the interaction between PB1-F2 and host proteins through Univeral Human （Normalized） cDNA library. Results pGBKT7-PB1-F2 was successfully constructed and confirmed by DNA sequencing, there were no auto-activation and toxicity for the pGBKTT-PB1-F2 as bait vector. The mating efficiency of Y2HGold and Y187 yeast was 5.22%. Three positive clones interacting with PB1-F2 were obtained and identified by yeast two-hybrid screening. They were NA/K-ATPase β1 subunit,HSP40 and interleukin 2 receptor γ subunit, respectively. Conclusion Host proteins NA/K-ATPase β1 subunit, HSP40 and interleukin 2 receptor γ subunit may affect the virus replication and cell life cycle through interaction with the viral protein PB1-F2.
出处
《热带医学杂志》
CAS
2012年第6期657-660,F0004,共5页
Journal of Tropical Medicine
基金
国家重大传染病防治科技重大专项(2009ZX10004-213)
广东省重大传染病专项
