| 斜带石斑鱼神经坏死病毒CP基因 shRNA干扰载体的构建及效果评价 |
| |
| 引用本文： | 黄桂菊,陈健光,喻达辉,曾令兵,龙华.斜带石斑鱼神经坏死病毒CP基因 shRNA干扰载体的构建及效果评价[J].水产学报,2012,36(3):343-349. |
| |
| 作者姓名： | 黄桂菊 陈健光 喻达辉 曾令兵 龙华 |
| |
| 作者单位： | 中国水产科学研究院南海水产研究所,农业部海水养殖生态与质量控制重点开放实验室;中国水产科学研究院南海水产研究所,农业部海水养殖生态与质量控制重点开放实验室;中国水产科学研究院南海水产研究所,农业部海水养殖生态与质量控制重点开放实验室;中国水产科学研究院长江水产研究所,农业部淡水鱼类种质资源与生物技术重点开放实验室;中国水产科学研究院长江水产研究所,农业部淡水鱼类种质资源与生物技术重点开放实验室 |
| |
| 基金项目： | 广东省海洋渔业科技推广专项项目(A201001H06);中央级公益性科研院所基本科研业务费专项资金项目(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);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) |
| |
| 摘 要： | 利用Invitrogen公司的在线生物学软件分析斜带石斑鱼神经坏死病毒CP基因,设计针对CP基因不同位置的小发卡RNA(short hairpin RNA,shRNA)干扰序列其结构特征为正链(19 nt)-环(4 nt)-负链(19 nt)]。化学合成这些序列,并退火连接为双链干扰片段,将双链干扰片段定向克隆到干扰载体pENTRTM/U6中,构建shRNA干扰载体pshRNA-124、pshRNA-896和pshRNA-NNV。然后,用脂质体转染法分别将3种shRNA干扰载体和pEGFP-CP基因共转染导入黑头呆鱼(FHM)肌肉细胞,荧光显微镜观察细胞荧光强度,分析荧光抑制效率,Real-time RT-PCR检测CP基因mRNA的表达水平变化。结果表明,在pEGFP-CP与shRNA干扰载体共转染组,pshRNA-124、pshRNA-896、pshRNA-NNV的荧光抑制效率分别为47%、68%、51%。3种shRNA干扰载体都有干扰效果,均能干扰绿色荧光蛋白的表达,其中pshRNA-896干扰效率最好。Real-time RT-PCR检测表明,干扰质粒pshRNA-124、pshRNA-896、pshRNA-NNV对pEGFP-CP基因的沉默效率分别约为60%、96%和55%,与对照组相比差异显著(P<0.05)。研究表明,靶向斜带石斑鱼神经坏死病毒CP基因的shRNA干扰载体构建成功,为进一步运用RNA干扰技术进行CP基因的功能研究奠定了基础。
|
| 关 键 词： | 斜带石斑鱼 神经坏死病毒 CP基因 shRNA干扰载体 |
| 收稿时间： | 8/30/2011 5:41:33 PM |
| 修稿时间： | 2011/11/10 0:00:00 |
Construction and evaluation of short hairpin RNA interference vector targeting CP gene of Epinephelus coioides nervous necrosis virus |
| |
| HUANG Gui-ju,CHEN Jian-guang,YU Da-hui,ZENG Ling-bing and LONG Hua.Construction and evaluation of short hairpin RNA interference vector targeting CP gene of Epinephelus coioides nervous necrosis virus[J].Journal of Fisheries of China,2012,36(3):343-349. |
| |
| Authors: | HUANG Gui-ju CHEN Jian-guang YU Da-hui ZENG Ling-bing and LONG Hua |
| |
| Institution: | 2,3(1.The Key Laboratory of Mariculture,Ecology and Quality Control,Ministry of Agriculture,South China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510300,China; 2.The Key Laboratory of Freshwater Fish Germplasm and Biotechnology,Ministry of Agriculture,Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Jingzhou 434000,China; 3.The Key Laboratory of Freshwater Ecology and Healthy Aquaculture,Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Jingzhou 434000,China) |
| |
| Abstract: | The short hairpin RNA interference sequences targeting CP gene of red-spotted grouper nervous necrosis virus were designed by using online designing software of Invitrogen Co.,Ltd.The structure consisted of 19 nt sense strand,-4 nt loop and-19 nt antisense strand.These sequences are synthesized,annealed,and cloned into pENTRTM/U6 to construct the shRNA interference vectors named pshRNA-124,pshRNA-896 and pshRNA-NNV,respectively.Then the expression vector of pEGFP-CP and each of the three shRNA interference vectors are co-transfected into FHM cells by using Lipofectamine 2000.Then the expression and inhibition of EGFP are observed by using fluorescent microscope.The mRNA expression level of CP gene is anaylised by using real time RT-PCR.The result shows that pshRNA-124,pshRNA-896 and pshRNA-NNV all suppress the EGFP expression with inhibitory efficiency of 47%,68% and 51%,respectively.The pEGFP-CP gene silencing efficiencies of pshRNA-124,pshRNA-896 and pshRNA-NNV at mRNA level are 60%,96% and 55%,respectively,significantly higher than that of control(P<0.05).It demonstrates that all of the three shRNAi vectors have interference effects and can interfere with the expression of EGFP,of which pshRNA-896 is the best one.The result above suggests that shRNAi vectors targeting CP gene of red-spotted grouper nervous necrosis virus has been successfully constructed,which will be a basis for further study of the function of CP gene with RNAi technique. |
| |
| Keywords: | Epinephelus coioides nervous necrosis virus capsid protein gene short hairpin RNA interference vector |
| 本文献已被 CNKI 等数据库收录！ |
| 点击此处可从《水产学报》浏览原始摘要信息 |
| 点击此处可从《水产学报》下载免费的PDF全文 |
|
