华南农业大学学报2011，Vol.32Issue(2)：93-98,6.
石斑鱼神经坏死病毒RNA干扰的
Preliminary Study on RNA Interference Against Red-Spotted Grouper Nervous Necrosis Virus-Optimization of Transfection Condition and Comparison of Interference Effect Based on FHM Cell
摘要
Abstract
Grouper is one of the most valuable commercial marine fish and viral nervous necrosis (VNN) is the most harmful disease of this fish. Capsid protein ( major caps id protein,MCP) of NNV is a major pathogenic factor and currently there is no effective drug available for its treatment. This paper is intended to provide a preliminary study of the RNAi control technology for NNV. The gronper nervous necrosis virus MCP sene was cloned and eukaryotic expression vector pEGFP-MCP was constructed with a green fluorescent protein (EGFP) fusion gene and the MCP gene. Three pairs of siRNA (NNV-001 ,NNV-002,NNV-003) against the gene sequence designed, were transfected into FHM cells( muscle cells of fathead minnow)with Lipofectamine2000 in order to detect EGFP expression and comparison of transfection method, dose and efficiency. The results showed that the transfection efficiency with serum-free medium after 4 - 6 h is higher than the rate of transfection with mixture medium. 100 ng was found to be the most appropriate amount when the plasmid was transfected with 50,70,100 and 150 ng respectively. In the cotransfection of siRNA and pEGFP,all of the three pairs of siRNA sequences had interference effects on EGFP expression and pair 002 was found to be the best. Better interference effects were obtained at the siRNA concentration 200 nmol/L.关键词
石斑鱼/神经坏死病毒/衣壳蛋白基因/RNA干扰Key words
grouper/ the fish viral nervous necrosis/ major capsid protein gene/ RNA interference分类
农业科技引用本文复制引用
黄桂菊,喻达辉,柳明,潘俐玲,王晓宁,曾令兵,龙华..石斑鱼神经坏死病毒RNA干扰的[J].华南农业大学学报,2011,32(2):93-98,6.基金项目
中央级公益性科研院所基本科研业务费专项资金(2007TS02) （2007TS02）
农业部淡水鱼类种质资源与生物技术重点开放实验室开放课题(LFB20070610) （LFB20070610）
中国水产科学研究院淡水生态与健康养殖重点开放实验室开放课题(2007FEA0214) （2007FEA0214）
