对虾白斑综合症病毒vp15基因的RNA干扰研究

RNA interference on vp15 gene of shrimp white spot syndrome virus(WSSV)

VP15是对虾白斑综合症病毒(WSSV)的一种核衣壳蛋白,以往的研究表明VP15属DNA结合蛋白,具有潜在的浓缩包装病毒基因组的功能.利用RNAi技术将vp15基因沉默,来研究vp15基因沉默后对WSSV增殖及结构的影响.体外合成了vp15基因的特异双链RNA(vp15-dsRNA)和非特异双链RNA(gfp-dsRNA),分别与WSSV混合共注射淡水原克氏螯虾.在感染后24、36、48 h,每个实验组分别取病虾步足肌肉,样品用Trizol试剂盒提取RNA用于RT-PCR检测、用病毒检测试剂盒提取DNA模板用于荧光定量PCR分析.另外每组随机选取1只虾,取中肠,用于树脂包埋组织超薄切片分析.逆转录PCR分析结果显示vp15-dsRNA能特异性敲除vp15基因转录表达,实时荧光定量PCR分析结果显示vp15-dsRNA干扰vp15基因的表达能有效抑制WSSV病毒粒子的增值.此外,利用树脂包埋组织超薄切片电子显微镜技术观察发现vp15基因被干扰后病毒粒子数量上明显降低并且引起病毒粒子包装异常.通过vp15基因特异dsRNA干扰,有效抑制了WSSV病毒增殖,此外进一步阐明VP15蛋白的功能及其在病毒组装方面的重要作用,对进一步了解病毒组装以及病毒防治具有重要的意义.

VP15 is a nucleocapsid protein of white spot syndrome virus （WSSV）. Previous studies showed that VP15 is a DNA binding protein and has potential function of viral genome condensation. In this research, we use RNAi technology to silence vpl5 gene and study the effect of vp15 gene silencing on the WSSV proliferation and structure. We in vitro synthesized vp15 genespecific doublestranded RNA （vpl5dsRNA） and nonspecific doublestranded RNA（gfpdsRNA） ,which were respectively mixed with WSSV and injected with the Crayfish. At 24,36,48 h postinfection, shrimp muscle of each experimental group were collected for subsequent experiments. Total RNA was extracted with Trizol kit for subsequent RTPCR experiment, and DNA templates were extracted with virus detection kit for quantitative PCR analysis. Another one shrimp in each group were randomly selected, taken the midgut tissue for ultrathin sections electron microscopy analysis. RTPCR analysis revealed that vpl5-dsRNA could specifically knockout vpl5 gene transcription, and realtime fluorescence quantitative PCR analysis showed that vp15-dsRNA can interfere with the vp15 gene expression and effectively inhibited WSSV proliferation. In addition ,Ultra thin section electron microscopy showed that vp15 dsRNA interference can significantly inhibit the viral multiplication and cause abnormal packaging. In this study, we used vpl5 gene specific dsRNA interference, effectively inhibited WSSV virus multiplication,which further elucidates the function of VP15 protein and the important role in virus assembly. It is of great significance for further understanding of virus assembly and virus prevention.