| CMV启动子调控的VEGF-shRNA表达载体的构建及有效干扰序列的筛选 |
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| 引用本文： | 刘晓艳,方红,陈鸿超,蒋筱凌. CMV启动子调控的VEGF-shRNA表达载体的构建及有效干扰序列的筛选[J]. 浙江大学学报(医学版), 2010, 39(2). DOI: 10.3785/j.issn.1008-9292.2010.02.012 |
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| 作者姓名： | 刘晓艳 方红 陈鸿超 蒋筱凌 |
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| 作者单位： | 浙江大学医学院,附属第一医院皮肤科,浙江,杭州,310003 |
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| 基金项目： | 国家自然科学基金资助项目，浙江省卫生厅课题 |
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| 摘 要： | 目的:构建针对人VEGF基因的小干扰RNA(siRNA)的表达载体,实现CMV启动子调控,转染肿瘤细胞后观察其对VEGF基因的干扰作用,并筛选出有效的VEGF-shRNA.方法:设计三对VEGF靶向的发夹状shRNA,依据设计合成两条互补的寡核苷酸链,退火后连接入质粒pDC311-SV40-RC中,转化扩增后进行序列测定.用脂质体包裹转染人骨肉瘤细胞U-2 OS,分别培养3d和7d后收集细胞培养上清液,ELISA检测上清中VEGF蛋白的表达.结果:将针对VEGF基因的siRNA的双链寡核苷酸片段成功克隆到pDC311-SV40-RC载体,经过阳性菌落PCR鉴定与测序,结果正确;转染U-2 OS细胞后,ELISA方法检测蛋白表达水平证实干扰序列3能有效降低VEGF的表达.结论:成功构建了CMV启动子调控的针对VEGF基因的siRNA载体,转染肿瘤细胞后可抑制VEGF的表达,并筛选出有效的干扰序列.
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| 关 键 词： | RNA,小分子干扰 血管内皮生长因子A 转染 启动区(遗传学) 寡核苷酸类 序列分析 |
Construction of VEGF gene-targeted small interfering RNA mediated by CMV promoter and its silencing effect |
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| LIU Xiao-yan,FANG Hong,CHEN Hong-chao,JIANG Xiao-ling. Construction of VEGF gene-targeted small interfering RNA mediated by CMV promoter and its silencing effect[J]. Journal of Zhejiang University. Medical sciences, 2010, 39(2). DOI: 10.3785/j.issn.1008-9292.2010.02.012 |
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| Authors: | LIU Xiao-yan FANG Hong CHEN Hong-chao JIANG Xiao-ling |
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| Affiliation: | LIU Xiao-yan,FANG Hong,CHEN Hong-chao,JIANG Xiao-ling(The First Affiliated Hospital,College of Medicine,Zhejiang University,Hangzhou 310003,China) |
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| Abstract: | Objective: To construct VEGF gene-targeted small interfering RNA(siRNA) and its expression vector driven by CMV promoter and to investigate its interference effect.Methods: The VEGF gene-targeted hairpin siRNA was designed,two complementary oligonucleotide strands were synthesized.After annealing,two-strand oligonucleotide was inserted into pDC311-SV40-RC vector,which was then identified by PCR and sequenced.Then human U-2 OS cell line was transfected with the vector using lipofectamine method.Finally,ELISA... |
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| Keywords: | RNA,small interfering Vascular endothelial growth factor A Transfection Promoter regions (Genetics) Oligonucleotides Sequence analysis |
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