靶向传染性法氏囊病病毒VP1基因siRNA对病毒复制的抑制  

Inhibition on replication of infectious bursal disease virus by siRNA targeting VP1 gene

作　　者：欧阳伟[1] 王永山[1] 刘小娟[1,2] 张海彬[2] 

机构地区：[1]江苏省农业科学院兽医研究所农业部动物疫病诊断与免疫重点开放实验室国家兽用生物制品工程技术研究中心,江苏南京210014 [2]南京农业大学动物医学院,江苏南京210095

出　　处：《中国兽医学报》2011年第10期1404-1409,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30571371);江苏省自然科学基金资助项目(BK2008352;BK2009041;BK2010471);江苏省农业科技创新基金资助项目(CX(08)106)

摘　　要：根据传染性法氏囊病病毒(IBDV)VP1基因保守区序列,选择设计了3个靶向VP1的小干扰RNA序列(siR-NA):VP1-siRNA668、VP1-siRNA1034和VP1-siRNA2250,化学合成DNA序列,体外转录合成siRNA,转染鸡胚成纤维细胞(CEF)后接种IBDV,分析siRNA对病毒复制的影响。结果显示,病毒接种后72h,3个VP1-siRNA转染组未出现明显的细胞病变(CPE),病毒滴度分别为102.75,102.00,101.75 TCID50/0.1mL,而siRNACON转染和单纯IBDV接种对照组均出现明显CPE,病毒滴度均为106 TCID50/0.1mL;用荧光定量RT-PCR分析VP1基因水平,3个VP1-siRNA转染组比对照组分别降低了73.10%,81.79%,90.28%。结果表明,本试验选择设计的3个siRNA具有明显抑制IBDV复制功能,其中2 250位点的siRNA抑制效果最明显,推测该区域是VP1基因的重要功能区,是新型抗IBDV药物与疫苗设计的重要靶标。Three short interfering RNA（siRNA） sequences,named VP1-siRNA668,VP1-siRNA1034 and VP1-siRNA2250 based on conserved regions in the VP1 gene of the infectious bursal disease virus（IBDV） were selected and synthesized via in vitro transcription.The chicken embryo fibroblast（CEF） was transfected with the 3 siRNA and then infected with IBDV,respectively.The viral titers of CEF cultures were 102.75,102.00 and 101.75 TCID50/0.1 mL in CEF cultures transfected with VP1-siRNA668,VP1-siRNA1034 and VP1-siRNA2250,respectively,which were significantly lower than 106TCID50/0.1 mL in siRNACON and IBDV control groups.As compared to the control,the inhibitory rates were 73.10%,81.79% and 90.28% in the real-time fluorescence quantitative RT-PCR assay for detecting VP1 gene,respectively.The results indicated that the 3 siRNA could effectively inhibit IBDV replication in vitro,in which 2 250 the highest site of the siRNA inhibitory effect,suggesting that is a major functional region of VP1 gene and an important target for designing novel drug and vaccine against IBDV.

关 键 词：RNA干扰(RNAI) 小干扰RNA(siRNA) 传染性法氏囊病病毒(IBDV) VP1基因 

分 类 号：S852.65[农业科学—基础兽医学]