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目的探讨日本脑炎病毒抑制IFN-α介导的JAK-STAT信号转导通路过程中发挥关键作用的病毒特异蛋白,为阐明日本脑炎病毒抑制Ⅰ型IFN介导的JAK-STAT信号转导通路的作用机制奠定基础。方法分别构建日本脑炎病毒编码的7种非结构蛋白基因(NS1,NS2A,NS2B,NS3,NS4A,NS4B和NS5)的重组真核表达载体。采用间接免疫荧光法和Western blotting观察它们在细胞内的表达及定位情况。利用含萤火虫荧光素酶(Luc)报告基因的重组载体pISRE-Luc,观察表达病毒不同非结构蛋白的细胞内IFN-α介导的JAK-STAT信号转导通路的活化程度。构建融合表达红色荧光蛋白的STAT1重组真核表达载体pRed-STAT1,利用该重组载体在表达病毒非结构蛋白的细胞内观察融合蛋白Red-STAT1在IFN-α作用下的细胞内定位情况。同时,对表达病毒非结构蛋白的细胞内IFN-α介导的STAT1分子的磷酸化水平进行检测。结果日本脑炎病毒的7种非结构蛋白均可在哺乳动物细胞内正确表达,而且表达蛋白均位于细胞质中。在这7种非结构蛋白中,NS5可阻断STAT1分子的核转运及抑制STAT1分子的磷酸化水平,从而抑制IFN-α介导的细胞内JAK-STAT信号转导通路的活化。结论日本脑炎病毒的非结构蛋白NS5对Ⅰ型IFN系统的信号转导通路具有显著的抑制作用,可能是一种Ⅰ型IFN系统的拮抗蛋白。
To investigate the role of Japanese encephalitis virus in inhibiting the IFN-α mediated JAK-STAT signal transduction pathway play a key role in the virus-specific protein, in order to elucidate Japanese encephalitis virus type Ⅰ IFN-mediated JAK-STAT signal transduction pathway The mechanism of action to lay the foundation. Methods Recombinant eukaryotic expression vectors of seven nonstructural protein genes (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) encoded by Japanese encephalitis virus were constructed. Indirect immunofluorescence and Western blotting were used to observe their expression and localization in the cells. The level of activation of IFN-α-mediated JAK-STAT signal transduction pathway, expressing different nonstructural proteins of the virus, was observed using the recombinant vector pISRE-Luc containing the firefly luciferase (Luc) reporter gene. The STAT1 recombinant eukaryotic expression vector pRed-STAT1 fused with the red fluorescent protein was constructed, and the intracellular localization of the fusion protein Red-STAT1 under the action of IFN-α was observed in the cells expressing the virus non-structural protein. At the same time, intracellular IFN-α-mediated phosphorylation of STAT1 molecules expressing viral nonstructural proteins was examined. Results The seven non-structural proteins of Japanese encephalitis virus could be correctly expressed in mammalian cells, and the expressed proteins were located in the cytoplasm. Among the seven non-structural proteins, NS5 blocked the nuclear translocation of STAT1 and the phosphorylation of STAT1, thereby inhibiting IFN-α-mediated activation of the JAK-STAT signal transduction pathway. Conclusion NS5, a non-structural protein of Japanese encephalitis virus, has a significant inhibitory effect on the signal transduction pathway of type Ⅰ IFN system and may be an antagonist of type Ⅰ IFN system.