| Nogo-66受体RNA干扰质粒的构建及其干扰效率鉴定 |
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| 引用本文： | 范有明,蔡明春. Nogo-66受体RNA干扰质粒的构建及其干扰效率鉴定[J]. 第三军医大学学报, 2007, 29(13): 1285-1288 |
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| 作者姓名： | 范有明 蔡明春 |
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| 作者单位： | 汕头大学香港中文大学联合汕头国际眼科中心,汕头,515041;第三军医大学高原军事医学系病理生理学及高原生理学教研室,全军高原医学重点实验室,重庆,400038 |
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| 基金项目： | 中国博士后科学基金，国家自然科学基金 |
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| 摘 要： | 目的 构建及筛选高效率针对Nogo-66受体(Nogo-66 receptor, NgR) 进行RNA干扰(RNAi)的质粒.方法 RT-PCR克隆NgR,插入表达质粒中,构建NgR-GFP融合蛋白表达质粒.根据NgR序列,设计4对针对NgR mRNA进行RNAi的寡核苷酸.退火后,插入短发夹RNA(short hairpin RNA, shRNA)表达质粒中,构建shRNA表达质粒.将NgR-GFP融合蛋白表达质粒与shRNA表达质粒共转染AAV-293细胞.通过对GFP表达量的观察及Western blotting定量检测NgR-GFP融合蛋白表达量,鉴定shRNA表达质粒对NgR的干扰效率.结果 针对NgR进行RNAi的4个序列中,有1个序列的干扰效率大于90%以上.结论 成功构建了1个针对NgR的高效RNAi表达质粒.
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| 关 键 词： | Nogo-66受体 RNA干扰 质粒 |
| 文章编号： | 1000-5404（2007）13-1285-04 |
| 修稿时间： | 2007-01-222007-04-03 |
Construction and identification of plasmid mediated RNA interference targeting Nogo-66 receptor |
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| FAN You-ming,CAI Ming-chun. Construction and identification of plasmid mediated RNA interference targeting Nogo-66 receptor[J]. Acta Academiae Medicinae Militaris Tertiae, 2007, 29(13): 1285-1288 |
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| Authors: | FAN You-ming CAI Ming-chun |
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| Affiliation: | 1.Joint Shantou International Eye Center of Shantou University and Chinese University of Hong Kong, Shantou 515041; 2 Department of Pathophysiology, College of High Altitude Military Medicine, Third Military Medical University, Chongqing 400038, China |
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| Abstract: | Objective To construct and identify of a plasmid mediated high efficiency of RNA interference(RNAi) against Nogo-66 receptor(NgR).Methods After cloning of NgR by RT-PCR,the fragments were inserted into pcDNA3.1/CT-GFP-TOPO to produce plasmid expressing NgR-GFP fusion protein.Four pairs of oligonucleotide were designed according to NgR sequence and annealed.The resulting fragments were ligated into short hairpin RNA(shRNA) expressing plasmid.The plasmids expressing NgR-GFP fusion protein and shRNA were co-transfected into AAV-293 cells.RNAi efficiency against NgR was observed under fluorescent microscope and calculated by Western blotting.Results The interference efficiency of one sequence was above 90%.Conclusion A plasmid mediated high RNAi efficiency against NgR is constructed successfully. |
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| Keywords: | Nogo-66 receptor RNA interference plasmid |
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