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RNAi-DNA稳定表达载体的构建与应用

The construction and application of siRNA-DNA expression vector

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【作者】 周唏张鹏辉

【Author】 ZHOU Xi1,ZHANG Peng-hui2(1.Department of Ophthalmology,The First Affiliated Hospital, Chongqing University of Medical Sciences,Chongqing 400016, China;2.Department of Clinical Labortories,Chongqing University of Medical Sciences,Chongqing 400016,China)

【机构】 重庆医科大学附属第一医院眼科重庆医科大学医学检验系 重庆400016重庆400016

【摘要】 目的:构建siRNA的DNA稳定表达载体,为研究RNAi在哺乳动物内持续、稳定抑制靶基因表达奠定基础。方法:合成含靶向hTERT基因的siRNA转录模板的发夹结构,将载体质粒pGenesil-1用BamH1+HindIII进行双酶切后,T4DNA连接酶连接成重组质粒,转染到肝癌SMMC-7721细胞中进行稳定筛选、表达,检测稳定筛选前后hTERT基因表达变化。结果:重组质粒在大肠杆菌菌株JM109内扩增。提纯、纯化后用HindIII、EcoRI酶切鉴定及测序鉴定证明hTERT-siRNA转录模板完整、正确的插入到pGenesil-1质粒中,建立了稳定抑制hTERT基因的SMMC-7721细胞株,并在mRNA水平抑制了肝癌SMMC-7721细胞hTERT基因表达。结论:成功构建了siRNA-DNA稳定表达载体,能在哺乳动物细胞中表达,并初步应用于靶基因的抑制。

【Abstract】 Objective: To construct DNA stable expressed vector of siRNA for establishing material foundation of gene expression oflong inhibition effectively by RNAi in mammalian cells. Methods: The hairpin structure of siRNA transcript template targeting hTERTgene was synthesized. Then, both pGenesil- 1 empty vector and siRNA transcript template were digested by BamH1/ plus HindIII. Two di-gested fragments were ligated with T4 DNA ligase to be recombinant vector. The recombinant vector was then transfected into hapartocar-cinoma SMMC- 7721 cells to selected stably and detected the expression alteration of hTERT. Results: The recombination plasmid wasamplified in the E. coli. JM109. After the identification of redigesting by HindIII and EcoRI and sequencing, the reconstructive plasmidwas confirmed that contained the correct and full nucleotide sequence of hTERT- siRNA transcript template in pGenesil- 1 vector. ThemRNA and protein of hTERT gene were inhibited by RNAi in hapartocarcinoma SMMC- 7721 cells. Conclusion: The siRNA- DNA ex-pression vector was constructed successfully, could express in mammalian cells, and initiate employment of suppressing target gene.

【关键词】 siRNADNA稳定表达载体构建应用
【Key words】 siRNADNA stable expressed VectorConstructionApplication
  • 【文献出处】 现代医药卫生 ,Modern Medicine Health , 编辑部邮箱 ,2005年24期
  • 【分类号】Q78;
  • 【被引频次】6
  • 【下载频次】178
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