慢病毒介导的靶向NDV P基因shRNA对NDV复制的抑制作用  

Lentivirus Delivery of the Short Hairpin RNA Targeting NDV P Gene Inhibits Production of the Newcastle Disease Virus in Chicken Embryo Fibroblasts and Chicken Embryos

作　　者：杨少华[1] 许传田[1] 张琳[1] 黄艳艳[1] 黄庆华[1] 胡北侠[1] 张秀美[1] 

机构地区：[1]山东省农业科学院畜牧兽医研究所山东省畜禽疫病防治与繁育重点实验室,山东济南250100

出　　处：《病毒学报》2016年第1期39-45,共7页Chinese Journal of Virology

基　　金：公益性行业(农业)科研专项(201303033;201003012);现代农业产业技术体系建设专项资金(CARS-42-Z12)

摘　　要：小干扰RNA(siRNA)诱导的RNA降解可以特异性地抑制病毒感染,它作为一种有效的抗病毒治疗方法正被广泛研究。为了探讨慢病毒介导的shRNA对NDV复制的抑制效果,从而为新城疫病毒的抗病毒研究奠定基础,本研究以新城疫病毒(Newcastle disease virus,NDV)P基因为靶基因,构建了靶向NDV P基因的shRNA重组慢病毒表达载体RNAi-341和RNAi-671。将其与辅助细胞共转染293T细胞,获得包装好的重组慢病毒;在鸡胚成纤维细胞(Chicken embryo fibroblast,CEF)和SPF鸡胚上进行了干扰实验,并通过荧光定量PCR和病毒滴度测定检测shRNA对NDV的抑制效果。结果发现,RNAi-341和RNAi-671均能抑制FLAG-P蛋白在293T细胞中的瞬时表达。在CEF细胞感染后16h后,NDV的病毒滴度分别降低了66.6倍和30.6倍;在鸡胚感染48h后,RNAi-341和RNAi-671组NDV病毒的增殖量分别减少99%和98%。RNAi-341与RNAi-671不仅能抑制P基因的转录,还能显著降低NP、M、F、HN和L基因的转录水平。与RNAi-671相比,RNAi-341的抑制效果更好。研究结果表明,慢病毒介导的靶向P基因的shRNA具有抗病毒作用,能够抑制NDV在CEF和鸡胚中的复制,从而为临床防治NDV提供一个新方法。Small interfering ribonucleic acid（siRNA）-induced RNA degradation can inhibit viral infection,and has been investigated extensively for its efficacy as antiviral therapy.The potential therapeutic role of lentiviral-mediated short hairpin ribonucleic acid（shRNA）to Newcastle disease virus（NDV）replication in vivo has been explored less often.We constructed two recombinant lentiviral vectors containing shRNA against the phosphoprotein（P）of the NDV,RNAi-341 and RNAi-671.Recombinant shRNA lentivirus vectors were co-transfected into 293 Tcells,along with helper plasmids,to package the recombinant shRNA lentivirus.Lentivirus-based shRNAs were titrated and transduced into NDV-susceptible chicken embryo fibroblasts（CEFs）and chick embryos.Antiviral activity against the NDV strain was evaluated by virus titration and real-time reverse transcription-polymerase chain reaction.RNAi-341 and RNAi-671 strongly suppressed transient expression of a FLAG-tagged P fusion protein in 293 Tcells.RNAi-341 and RNAi-671 NDV reduced virus titers by 66.6-fold and 30.6-fold,respectively,in CEFs 16 hafter infection.RNAi-341 and RNAi-671 reduced virus titers in specific pathogen-free chick embryos by 99%and 98%,respectively,48 hafter infection.Both shRNAs inhibited accumulation of not only P-gene mRNA,but also nucleocapsid,M-,F-,HN-,and L-gene mRNA.RNAi-341 silenced P-gene mRNA more potently than RNAi-671.These results suggest that shRNAs silencing the P gene had substantial antiviral properties and inhibited NDV replication in CEFs and chick embryos.

关 键 词：新城疫病毒 SHRNA P基因 慢病毒载体 

分 类 号：S852.65[农业科学—基础兽医学] R373.9[农业科学—兽医学]