摘要
为研究A型流感病毒的重要毒力因子PB1-F2对蛋白激酶R(PKR)的调控机制,通过纯化GST-PB1-F2融合蛋白,利用GST pull-down试验证实了PB1-F2与PKR发生相互作用;采用免疫荧光试验进一步验证了二者的相互关系;实时荧光定量PCR表明PB1-F2能负调控PKR及IL-6的mRNA水平;采用siRNA干扰PKR基因的表达后,PB1-F2抑制IL-6的mRNA转录作用更显著。进一步运用蛋白免疫印迹方法检测了PB1-F2对PKR及其底物eIF-2α蛋白磷酸化水平的影响,显示过表达PB1-F2后,PKR及eIF-2α的磷酸化表达水平明显下调。本研究表明PB1-F2与PKR相互作用而抑制PKR磷酸化活化,并通过PKR调控IL-6的表达,为探索PB1-F2的未知生物学功能提供了重要信息。
To investigate the regulation mechanism of the crucial virulence factor PB1-F2 of influenza A virus （IAV） on protein kinase R （PKR）,GST-PB1-F2 fusion protein was purified and the interaction between PB1-F2 and PKR was verified by GST pull-down assay in vitro.Then the interactional relationship between them was further conformed by immunofluorescence experiment.The results of real-time quantitative PCR showed that PB1-F2 decreased the mRNA transcription of PKR and IL-6.The mRNA level of IL- 6 was also reduced when PKR gene was knocked down with PKR siRNA,which illustrated PB1-F2 might in part at least repress IL-6 mRNA expression through inhibition of PKR. The Western blot experiment was utilized to explore the effect of PB1-F2 on the phosphorylation of PKR and eIF-2a protein.The results demonstrated that the phosphorylation of PKR and eIF-2a was significantly down-regulated in response to PB1-F2 overexpression.This study provided the critical information for exploring the biological function of PB1-F2 protein.
出处
《动物医学进展》
北大核心
2017年第5期1-5,共5页
Progress In Veterinary Medicine
基金
国家重点基础研究发展计划(973计划)(2012CB518901)
