| 靶向EphB4基因的shRNA真核表达载体的构建与鉴定 |
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| 引用本文： | 张艳荣,董万利,胡锦,惠国桢.靶向EphB4基因的shRNA真核表达载体的构建与鉴定[J].苏州大学学报(自然科学版),2008,28(3). |
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| 作者姓名： | 张艳荣 董万利 胡锦 惠国桢 |
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| 作者单位： | [1]苏州大学附属第一医院神经内科,江苏苏州215006 [2]上海交通大学附属第六人民医院神经外科,上海200233 [3]苏州大学附属第一医院神经外科,江苏苏州215006 |
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| 基金项目： | 国家自然科学基金
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上海市科技发展基金 |
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| 摘 要： | 目的 构建和鉴定靶向EphB4基因的短发夹RNA(shRNA)真核表达载体.方法 体外合成一对互补并编码靶向EphB4基因的特异性shRNA序列的寡核苷酸,克隆到经Bbs Ⅰ酶切线性化的psiRNA人H1启动子质粒载体中,经酶切和测序鉴定所构建的重组载体是否正确;采用脂质体转染的方法将构建的重组载体导入人恶性胶质瘤细胞系U251中,采用逆转录-聚合酶链反应(RT-PCR)检测转染细胞EphB4mRNA的表达变化.结果 经酶切和测序证明,pH1-EphB4-shRNA序列正确;转染pH1-EphB4-shRNA载体后,U251细胞EphB4基因的电泳条带明显减弱,在磷酸甘油醛脱氢酶(GAPDH)参比下较空载体组下降60%.结论 靶向EphB4基因的shRNA真核表达载体构建成功,转染U251细胞之后获得稳定表达,并可特异性沉默EphB4基因的表达,为进一步研究EphB4基因在恶性胶质瘤的发生发展中的作用提供了实验基础.
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| 关 键 词： | EphB4 短发夹RNA 恶性胶质瘤 载体构建 靶向 基因 shRNA 真核表达载体构建 构建与鉴定 Expression Vector Identification 实验基础 作用 发展 发生 恶性胶质瘤 研究 稳定表达 GAPDH 甘油醛脱氢酶 磷酸 电泳 转染细胞 测序 |
Construction and Identification of pH1-EphB4-shRNA Expression Vector |
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| ZHANG Yan-rong,DONG Wan-li,HU Jin,HUI Guo-zhen.Construction and Identification of pH1-EphB4-shRNA Expression Vector[J].Suzhou University Journal of Medical Science,2008,28(3). |
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| Authors: | ZHANG Yan-rong DONG Wan-li HU Jin HUI Guo-zhen |
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| Institution: | ZHANG Yan-rong1,DONG Wan-li1,HU Jin2,HUI Guo-zhen3(1.Dept of Neurology,3.Dept of Neurosurgery,the First Hospital Affiliated to Suzhou University,Jiangsu Suzhou 215006,China,2.Dept of Neurosurgery,the 6th People Hospital Affiliated to Shanghai Jiaotong University,Shanghai 200233,China) |
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| Abstract: | Objective To construct short hairpin RNA(shRNA) eukaryotic expression vector,targeting gene EphB4 which may play an important role in glioma pathogenesis.Methods A specific DNA oligonucleotide targeting gene EphB4 on appropriate site was synthesized and inserted into Bbs I linearized psiRNA-H1 vector.The sequence of the plasmid was identified by DNA sequencer and restriction endonuclease digestion.The recombinant plasmid was transfected into the glioma cell lines U251 by lipofection.After transfected 48 hou... |
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| Keywords: | EphB4 shRNA malignant glioma vector construction |
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