对虾白斑病毒VP15基因的RNA干扰体外研究  

siRNA Interference on VP15 Gene of Shrimp WSSV

作　　者：黎铭[1] 陈晓汉[1] 马春霞[2] 蒋伟明[1] 马宁[1] 熊建华[1] 彭金霞[1] 陈秀荔[1] 彭敏[1] 曾地刚[1] 李咏梅[1] 

机构地区：[1]广西壮族自治区水产研究所遗传育种与健康养殖重点实验室,广西南宁530021 [2]广西壮族自治区兽医研究所,广西南宁530001

出　　处：《水生态学杂志》2010年第5期56-61,共6页Journal of Hydroecology

基　　金：广西科学研究与技术开发项目(桂科基0731035);国家科技支撑计划项目(2008BADB9B03;2007BAD29B01-6);广西科学基金项目(桂科青0832069)

摘　　要：VP15是南美白对虾WSSV病毒的一个核衣壳蛋白基因。采用体外干扰实验筛选对VP15具有针对性的siRNA。实验构建了真核表达载体pEGFP-VP15,并将设计的siRNA以及pEGFP-VP15共转染BHK细胞。采用Western blot方法检测GFP-VP15融合蛋白的表达,半定量RT-PCR方法检验siRNA抑制VP15基因转录的效果。实验结果表明,pEGFP-VP15在PK细胞正常表达,设计的3对siRNA对GFP-VP15的mRNA转录均有不同程度的干扰效果;其中,siRNA1的干扰效果最为显著。实验结果为下一步对虾体内干扰WSSV病毒复制研究以及筛选更多的特异siRNA建立基础。VP15, a structure gene for virion nucleocapsid. In order to screen out specific siRNA for VP15, an vitro vector named pEGFP- VP15 was constructed, and then siRNA targeting VP15 and pEGFP- VP15 were cotransfected into PK cell. The expression of pEGFP- VP15 was verified by Western blot, and the interference effect to VP15 in transcription was detected by semi -quantitative reverse transcription and polymerase Chain reaction. Our study here demonstrates that pEGFP- VP15 can be expressed in PK cell correctelly, the mRNA transcription for GFP -VP15 was suppressed by three pairs siRNA specifically, and the siRNAI made the most remarkable effect. Our study results would provide a system for choosing specifieal siRNA for VP15 and also provide a foundation for further research such as suppressing the wssv reproduce in shrimp.

关 键 词：WSSV VP15 RNA 干扰 

分 类 号：Q503[生物学—生物化学]