Gao Yulong, Liu Wei, Gao Honglei, Qi Xiaole, Lin Huan, Wang Xiaomei, Shen Rongxian
Division of Avian Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
Antiviral Res. 2008 Aug;79(2):87-94. doi: 10.1016/j.antiviral.2007.11.007. Epub 2007 Dec 26.
Infectious bursal disease (IBD) leads to considerable economic losses for the poultry industry by inducing severe immunosuppression and high mortality in chickens. The objective of this study was to determine if RNA interference (RNAi) could be utilized to inhibit IBDV replication in vitro. We selected 3 short interfering RNA (siRNA) sequences (siVP1(618), siVP1(1,115), and siVP1(2,571)) based on conserved regions in the vp1 gene of the infectious bursal disease virus (IBDV). When the Vero cells were transfected with siRNA, synthesized via in vitro transcription, and then infected with IBDV, siVP1(2,571) was discovered to be the most effective site for inhibiting IBDV replication. For long-term expression of siRNA and due to its suitability for large-scale preparation, the mouse U6 promoter was amplified using primers designed according to the siVP1(2,571) sequence. The resulting products were then subcloned into pEGFP-C1 to construct the shRNA expression vector pEC2571-shRNA. The shRNA-transfected Vero cells were then infected with IBDV. As compared to the control, the inhibitory rate in the pEC2,571-shRNA-transfected group was 87.4%. Indirect immunofluorescence and real-time polymerase chain reaction (PCR) confirmed that VP1 expression decreased at both the protein and RNA levels as compared to that in the controls. The results presented here indicate that DNA vector-based RNAi could effectively inhibit IBDV replication in vitro.
传染性法氏囊病（IBD）通过诱导鸡严重免疫抑制和高死亡率给家禽业造成相当大的经济损失。本研究的目的是确定RNA干扰（RNAi）是否可用于在体外抑制传染性法氏囊病病毒（IBDV）的复制。我们基于传染性法氏囊病病毒（IBDV）vp1基因的保守区域选择了3个小干扰RNA（siRNA）序列（siVP1(618)、siVP1(1115)和siVP1(2571)）。当用体外转录合成的siRNA转染Vero细胞，然后用IBDV感染时，发现siVP1(2571)是抑制IBDV复制的最有效位点。为了长期表达siRNA，并且由于其适合大规模制备，使用根据siVP1(2571)序列设计的引物扩增小鼠U6启动子。然后将所得产物亚克隆到pEGFP-C1中以构建shRNA表达载体pEC2571-shRNA。然后用IBDV感染转染了shRNA的Vero细胞。与对照组相比，pEC2571-shRNA转染组的抑制率为87.4%。间接免疫荧光和实时聚合酶链反应（PCR）证实，与对照组相比，VP1在蛋白质和RNA水平的表达均降低。此处给出的结果表明，基于DNA载体的RNAi可有效在体外抑制IBDV复制。
