石斑鱼神经坏死病毒RNA干扰的转染条件优化与效果分析

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

克隆了石斑鱼神经坏死病毒（Nervous necrosis virus,NNV）的衣壳蛋白（Major capsid protein,MCP）基因序列并构建了绿色荧光蛋白（EGFP）基因与MCP基因的融合真核表达载体pEGFP-MCP,设计了3对针对MCP基因序列的小片段干扰性RNA（Small interfering RNA,siRNA）序列（NNV-001、NNV-002、NNV-003）,开展了转染方法、转染剂量与转染效果的研究,用脂质体转染法将pEGFP-MCP和不同剂量的siRNA共转染导入黑头呆鱼肌肉（FHM）细胞.结果表明,用无血清培养基转染、4-6 h后换液的转染方法,比不换液、只将转染混合物代替同体积培养基的转染方法效率高;当质粒的转染量为50、70、100和150 ng时,100 ng的转染量为最合适;在pEGFP-MCP与siRNA共转染组,3对siRNA序列都有干扰效果,均能干扰绿色荧光蛋白的表达,其中NNV-002效果最好.当siRNA终浓度为200 nmol/L时,显示出了较好的沉默效果.

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 capsid 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 grouper nervous necrosis virus MCP gene 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.