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NS1蛋白是流感病毒编码的一种小分子多功能蛋白,可在病毒的复制过程中抑制宿主细胞的抗病毒免疫应答。为研究不同亚型流感病毒的NS1蛋白在细胞内的定位差异,分别用H1N1亚型WSN、PR8和CA04毒株,H9N2亚型SD毒株及H7N9亚型AH01毒株感染A549、MDCK细胞系以及构建的可表达不同亚型流感病毒NS1蛋白的p CMV-Myc-NS1质粒转染293T细胞,用激光共聚焦显微镜观察发现不同亚型流感病毒在不同细胞系和时间点的定位差异,感染后24 h时WSN和PR8毒株的NS1主要定位于细胞质中,而CA04和SD毒株主要定位于细胞核内。另外,观察过表达的WSN、SD和AH01毒株NS1的细胞定位,转染后24 h时WSN毒株NS1定位于细胞质中,而SD和AH01毒株主要定位于细胞核中。经氨基酸序列比对,对WSN毒株NS1蛋白进行关键氨基酸点突变,结果显示单一位点的改变未导致NS1蛋白细胞定位的改变,其细胞定位的差异不是由单一位点决定的。综上所述,分析不同亚型中的NS1的定位差异,这对进一步了解NS1蛋白同宿主细胞不同区域的蛋白的相互作用、流感病毒的调节机制以及病毒感染细胞中天然免疫反应具有一定的指导意义。
The NS1 protein is a small molecule multifunctional protein encoded by the influenza virus that inhibits the host cell’s anti-viral immune response during viral replication. To investigate the intracellular localization of NS1 proteins of different subtypes of influenza virus, A549, MDCK cell lines were infected with H1N1 subtype WSN, PR8 and CA04 strains, H9N2 subtype SD strain and H7N9 subtype AH01 strain respectively The constructed pCMV-Myc-NS1 plasmid expressing different subtypes of influenza virus NS1 protein was transfected into 293T cells. Confocal laser scanning microscopy was used to detect the differences in localization of different subtypes of influenza viruses at different cell lines and time points. After infection, NS1 of WSN and PR8 strains mainly located in the cytoplasm, while CA04 and SD strains mainly located in the nucleus. In addition, the cell localization of NS1, overexpressed WSN, SD and AH01 strain NS1 was observed. At 24 h after transfection, WSN strain NS1 localized in the cytoplasm whereas SD and AH01 strains predominantly localized in the nucleus. Amino acid sequence alignment of the key amino acid point mutations in the NS1 protein of the WSN strain showed that a single site change did not result in a change in the cell localization of the NS1 protein and the difference in cell localization was not determined by a single locus. In summary, the differences in localization of NS1 in different subtypes are analyzed, which is helpful to further understand the interaction between NS1 protein and different regions of host cells, the regulatory mechanism of influenza virus and the innate immune response in infected cells significance.