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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 protein of human H3N2 influenza virus and human host protein by yeast two-hybrid technique and to provide a theoretical basis for the functional study and pathogenesis of the virus protein. Methods The pGBKT7-PB1-F2 recombinant vector was constructed with the template of A / Guangdong / 7028/2010 isolated and identified by our laboratory. The Y2HGold yeast two-hybrid system was used to screen and screen human universal cDNA library Of protein. Results The pGBKT7-PB1-F2 recombinant vector with bait protein gene was constructed successfully. The self-activation and virulence of transformed yeast showed negative results. Yeast two-hybrid assay showed that the binding rate of Y2HGold and Y187 was 5.22%, which accorded with the experimental requirements. After verification, three positive clones with interaction with truncated PB1-F2 protein were obtained, which were potassium / sodium ATPase β1 subunit, heat shock protein 40 and interleukin-2 receptor γ subunit. Conclusion It is preliminarily concluded that the PB1-F2 protein of truncated H3N2 influenza virus may affect the replication of influenza virus in host cells and the regulation of apoptosis.