中山大学学报自然科学版 ›› 2011, Vol. 50 ›› Issue (1): 110-114.

• 研究论文 • 上一篇    下一篇

RNA干扰技术抑制SGIV ICP18绿色荧光融合蛋白表达的研究

夏立群1,2 , 张红莲1,2 , 梁海鹰1 , 秦启伟3   

  1. (1. 广东海洋大学水产学院,广东 湛江 524025;2.中山大学生命科学学院∥有害生物控制与资源利用国家重点实验室,广东 广州 510275;3.中国科学院南海海洋研究所∥海洋生物资源可持续利用重点实验室,广东 广州 510301)
  • 收稿日期:2009-11-01 修回日期:1900-01-01 出版日期:2011-01-25 发布日期:2011-01-25
  • 通讯作者: 秦启伟

The Inhibition of Expression of SGIV ICP18GFP in FHM Cell by RNA Interferencing

XIA Liqun 1,2, ZHANG Honglian 1,2, LIANG Haiying 1 , QIN Qiwei 3   

  1. (1. College of Fishery, Guangdong Ocean University, Zhanjiang 524025, China;2. State Key Laboratory of Biocontrol,Sun Yatsen University, Guangzhou 510275, China;3. South China Sea Institute of Oceanology,Guangzhou 510301,China)
  • Received:2009-11-01 Revised:1900-01-01 Online:2011-01-25 Published:2011-01-25

摘要: 将含有新加坡石斑鱼虹彩病毒(Singapore grouper iridovirus,SGIV)ICP18基因的真核表达载体pEGFP-ICP18转染到胖头鲤细胞(Fathead minnow cells,FHM)中进行融合表达,用荧光显微镜观察到ICP18GFP融合蛋白呈点状分布于FHM细胞的细胞质中。根据SGIV ICP18的序列,设计并体外化学合成了特异性干扰SGIV ICP18的siRNA(siRNAICP18),与pEGFPICP18共转染到FHM细胞中,通过荧光显微镜观察不同时间点的荧光强度变化。与序列非特异性siRNA(siRNAnegative)阴性对照相比,转染后24~48 h,共转染siRNAICP18和pEGFPICP18的实验细胞中发荧光的细胞数量较阴性对照少60%~80%左右,说明体外化学合成的siRNA-ICP18可有效抑制FHM细胞中外源导入SGIV ICP18基因的表达。

关键词: RNA干扰, 绿色荧光蛋白, 石斑鱼虹彩病毒SGIV, ICP18

Abstract: Singapore grouper iridovirus (SGIV) is a major pathogen resulting in heavy economic losses to grouper aquaculture. In this study, recombant eukaryotic vector pEGFPICP18 which inserted with SGIV ICP18 gene was transfected into Fathead minnow (FHM) cells, and ICP18GFP fusion protein was successfully expressed in FHM cells with a finely punctate cytoplasmic pattern. Candidate siRNA targeting SGIV ICP18 gene (siRNAICP18) was designed and chemically synthesized.To investigate the inhibition effect of siRNAICP18, pEGFPICP18 and siRNA were cotransfected into FHM cells, and the green fluorescence was observed by fluorescence microscope after transfection. The green fluorescence in FHM cells cotransfected with pEGFPICP18 and siRNAICP18 were 60%~80% fewer than that of negative control, which show the siRNAICP18 can effectively silence the extrinsic SGIV ICP18 gene in FHM cells during 24~48 h after transfection.

Key words: RNA interference, green fluorescent protein, singapore grouper iridovirus (SGIV), ICP18

中图分类号: