摘要
寨卡病毒(Zika Virus)属于黄病毒科中的黄病毒属,虽然很早就已经被人类所发现,但是一直到2015年在南美巴西的大规模爆发,才引起了广泛的关注.寨卡病毒对人类的感染往往引起包括小头畸形和格林-巴利综合征在内的多种症状.寨卡病毒的基因组为单链正链RNA,其基因组可以编码翻译并剪切加工出3个结构蛋白,分别为膜蛋白,囊膜蛋白和核衣壳蛋白,以及7个非结构蛋白(NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5).相关研究已经证明,NS1蛋白与同属黄病毒属的登革病毒的发病有紧密的联系,而且根据其蛋白结构推测其可能与寨卡病毒穿越血脑屏障有关.因此鉴别NS1与细胞内的相互作用蛋白对于发现寨卡病毒在细胞内的转运,转录,以及装配都有重大的意义.在此,该课题构建并在HEK293细胞中表达包含Flag和Strep两种标签的NS1融合蛋白,通过免疫沉淀的方法将与NS1结合的蛋白利用标签蛋白进行分离,利用高分辨生物质谱技术,对蛋白进行分析鉴定.通过分别带有Flag与Strep标签的相互作用蛋白分析,发现了16个两种标签共同的结合蛋白,其进一步的通路分析证明这些蛋白于与病毒转录、病毒复制和免疫反应多个通路有关,相关的研究结果为今后进一步研究寨卡病毒的复制机制以及开发抗病毒药物提供了重要的参考价值.
Zika Virus belongs to the genus Flavivirus in the Flaviviridae family. Although it has been discovered by humans for a long time, it has caused widespread concern until the massive outbreak of Brazil in South America in 2015. The Zika virus genome contain a single-stranded positive-stranded RNA, and its genome can encode and cleave three structural proteins which includes membrane glycoprotein, envelope protein and capsid protein and 7 non-structural proteins(NS1, NS2 A, NS2 B, NS3, NS4 A, NS5). Here, we constructed and expressed NS1 fusion protein tagged with Flag and Strep in HEK293 cells, further we separated the protein bound to NS1 using immunoprecipitation assay. The binding proteins were analyzed and identified with high resolution mass spectrometry. Through the analysis of interacting proteins, 16 binding proteins shared by the two tags were found. Further pathway analysis confirmed that these proteins belong to multiple pathways related to viral transcription and viral replication. This research results provide important reference value for further research on Zika Virus replication mechanism and vaccine development.
引文
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