RNA干扰技术抑制新加坡石斑鱼虹彩病毒感染细胞多肽ICP46与绿色荧光蛋白融合基因的表达

The inhibition of expression of SGIV ICP46-GFP in FHM cell by RNA interferencing

新加坡石斑鱼虹彩病毒(singapore grouper iridovirus,SGIV)是一种严重的能引起全身性疾病的病原体,对石斑鱼养殖造成了重大的经济损失。将含有SGIV感染细胞多肽ICP46(infected cell polypeptides 46)基因的真核表达载体pEGFP-ICP46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ICP46-GFP融合蛋白主要分布于FHM细胞的细胞质中。根据SGIVICP46的序列,设计并体外化学合成了特异性干扰SGIVICP46的siRNA(siRNA-ICP46),与pEGFP-ICP46共转染到FHM细胞中,通过荧光显微镜观察不同时间点荧光强度的变化。转染后24～48h,实验细胞(共转染siRNA-ICP46和pEGFP-ICP46)和阳性对照细胞(共转染siRNA-GFP和pEGFP-ICP46)中的荧光微弱,发荧光的细胞数量较阴性对照(共转染siRNA-Negative和pEGFP-ICP46)少70%左右,但其后实验细胞和阳性对照细胞的荧光强度开始增强,在转染后72h其与阴性对照组已差别不大。说明体外化学合成的siRNA-ICP46转染后24～48h可有效抑制FHM细胞中外源导入SGIV ICP46基因的表达。

Singapore grouper iridovirus（SGIV）,as a causative agent of serious systemic disease,resulted in significant economic losses in grouper aquaculture.In this study,recombinant eukaryotic vector pEGFP-ICP46 which was inserted with SGIV ICP46（infected cell polypeptides 46） gene was transfected into fathead minnow（FHM） cells,and ICP46-GFP fusion protein was successfully expressed in the cytoplasm of FHM cells.Candidate siRNA targeting SGIV ICP46 gene（siRNA-ICP46） was designed and chemically synthesized.To investigate the inhibition effect of siRNA-ICP46,pEGFP-ICP46 and siRNA was co-transfected into FHM cells,and the green fluorescence was observed by fluorescence microscope at the indicated time points（24,36,48,60 and 72 h post transfection）.During 24-48 h after transfection,the green fluorescence in FHM cells co-transfected with siRNA-ICP46 /pEGFP-ICP46 were similar to that of positive control（co-transfected with siRNA-GFP/pEGFP-ICP46）,and both of them are about 70% weaker than that of negative control（co-transfected with siRNA-Negative/pEGFP-ICP46）,which show the siRNA-ICP46 can effectively inhibit the extrinsic SGIV ICP46 gene in FHM cells during 24-48 h after transfection.