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为掌握鸡细胞中应激颗粒(stress granules,SGs)形成机制,以及鸡GTP酶激活蛋白SH3功能区结合蛋白1(G3BP1)在新城疫病毒(NDV)感染诱导SG形成过程中的作用,用NDV感染HeLa细胞,免疫荧光试验检测内源性G3BP1的定位,外源转染鸡G3BP1和各分段结构域,以不同应激处理之后观察与内源性SG标志物共定位现象,最后转染G3BP1之后感染NDV,以Western blot和TCID50检测病毒复制情况。结果显示:NDV感染能够诱导内源性G3BP1聚集形成SG,将鸡G3BP1分别转染至HeLa、DF-1和CEF后,以不同应激处理之后均能在细胞中观察到SG。将鸡G3BP1不同结构域分别转染HeLa细胞,以氧化应激和NDV分别处理,结果显示G3BP1的RNA结合结构域对聚集至关重要,而NTF2样结构域不能被招募到SGs中,并且抑制原有SGs的形成。最后证实G3BP1过表达能够增强病毒复制,说明SG在NDV复制过程中发挥促进作用。综合以上结果表明:鸡G3BP1在鸡SG形成过程中至关重要,鸡SG能够促进NDV复制,这为进一步研究NDV和禽源细胞互作奠定了基础。
To investigate the mechanism of stress granules (SGs) in chicken cells and the role of G3BP1, a chicken GTPase activator protein, in the induction of SG formation by Newcastle disease virus (NDV) infection, NDV HeLa cells were infected with HeLa cells. The localization of endogenous G3BP1 was detected by immunofluorescence assay. The G3BP1 and the segmented domains were transfected with different stress. The colocalization with endogenous SG markers was observed and the G3BP1 After infection NDV, Western blot and TCID50 detection of viral replication. The results showed that NDV infection could induce the aggregation of endogenous G3BP1 to form SG. After G3BP1 was transfected into HeLa, DF-1 and CEF respectively, SG could be observed in cells after different stress treatments. The different domains of chicken G3BP1 were transfected into HeLa cells separately and treated with oxidative stress and NDV respectively. The results showed that the RNA binding domain of G3BP1 was crucial for the aggregation, whereas the NTF2-like domain could not be recruited to SGs, There SGs formation. Finally, it was confirmed that G3BP1 overexpression enhanced the replication of virus, indicating that SG plays a promoting role in NDV replication. Taken together, the above results indicate that chicken G3BP1 is crucial in the formation of chicken SG, and chicken SG can promote the replication of NDV, which lays the foundation for further study on the interaction between NDV and avian cells.