靶向鸭肠炎病毒NP基因RNAi对DEV增殖的影响

摘要

The aim of the present study was to explore the influence of nucleocapsid protein (NP)to the proliferation of duck enteritis virus (DEV).We designed and constructed pSilencer-DEV-NP according to the DEV NP gene sequence in GenBank.After these pSilencers were transfected into duck embryo fibroblast (DEF) cells in three different ways,the expression of pSilencer DEV-NP to DEV proliferation in DEF cells was analyzed by fluorescence microscope and FQ-PCR.The results showed that the green fluorescence was observed by microscope in DEF cells transfected with pSilencer-DEV-NP-1,pSilencer-DEV-NP-2,pSilencer-DEV-NP-3 and pSilencerDEV-NP-4,respectively.The silencing efficiency of pSilencer-DEV-NP-1 to 4 on DEV proliferation were 81.10％,72.69％,77.35％ and 67.69％,respectively.FQ-PCR was used to amplify and calculate.The results showed that pSilencer-DEV-NP-1 could silence the DEV proliferation in DEF cells by three transfection ways,but the silencing efficiency was different.The silencing effect of pSilencer-DEV-NP-1 was the best at 60 h after transfection and its efficiency was 69.20 ％.The second highest efficiency occurred in the way of simultaneously DEV infection and pSilencer transfection,the best situation at 48 h was 63.79％.The worst efficiency was 52.58％ at 24 h after the operation with the method of pSilencer-DEV NP-1 transfection after DEV infection.These results suggest that the nucleocapsid protein has a certain effect on DEV proliferation,which lays a theoretical foundation for clarification of the mechanism of DEV replication and proliferation.%为探究核衣壳蛋白(NP)对鸭肠炎病毒(DEV)增殖的影响,笔者根据GenBank上DEV-NP基因序列,设计并构建pSilencer-DEV-NP,采用三种不同方式处理转染鸭胚成纤维(DEF)细胞后,应用荧光显微镜法和FQ-PCR法分析pSilencer-DEV-NP对DEV增殖的影响,结果显示:经荧光显微镜法观察,pSilencer-DEV-NP 1～4在DEF细胞中均呈现绿色荧光;FQ-PCR法扩增并计算,pSilencer-DEV-NP-1～4对DEV增殖的沉默效率分别为81.10％、72.69％、77.35％和67.69％;采用三种方法处理,pSilencer-DEV-NP-1对DEF细胞中DEV的增殖均有沉默作用,但沉默效率不尽一致,其中先后转染后感染时pSilencer-DEV-NP-1的沉默效果最好,在处理60 h时沉默效率高达69.20％;同时转染和感染时pSilencer-DEV-NP-1的沉默效果次之,在处理48 h时沉默效率最好仅达63.79％;先感染后转染时pSilencer-DEV-NP-1的沉默效果较差,在处理60 h时沉默效率最高仅为52.58％.上述研究结果提示,核衣壳蛋白对鸭肠炎病毒增殖具有一定的影响,为阐明核衣壳蛋白在鸭肠炎病毒复制与增殖的作用机制奠定了理论基础.