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卡氏肺孢子胸腺嘧啶核苷酸合酶基因siRNA表达载体的构建和鉴定

Construction and identification of plasmid vector of short interfering RNA on pneumocystis carinii thymidylate synthase gene

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【作者】 唐滢浍姚云清谭燕王莉妮李文桂刘成伟陈雅棠

【Author】 TANG Ying-hui,et al (Department of Infection Diseases,the First Affiliated Hospital,Chongqing Medical University)

【机构】 重庆医科大学附属第一医院感染科

【摘要】 目的:构建和鉴定大鼠卡氏肺孢子(Pneumocystis carinii,PC)胸腺嘧啶核苷酸合酶(Thymidylate synthase,TS)基因的siRNA(Short interfering RNA)表达载体。方法:人工合成针对PCTS基因的1对shRNA(Short hairpin RNA)序列并定向克隆到空载体pTZU6+1上,构建siRNA表达重组质粒pPC-TS,并采用双酶切产物凝胶电泳和基因测序法鉴定。结果:酶切电泳和基因测序鉴定得到的产物与预期的目的基因一致。结论:成功构建卡氏肺孢子胸腺嘧啶核苷酸合酶基因siRNA表达载体。

【Abstract】 Objective:To construct and identify a plasmid vector of short interfering RNA(siRNA) on pneumocystis carinii (PC) thymidylate synthase. Methods: Short hairpin RNA oligonucleotides of thymidylate synthase were chemically synthesized and inserted into plasmid vector pTZU6+1 after annealing. The recombinant plasmid,pPC-TS,transformed into E. coil. TOP10 and amplified,was digested by restriction endonucleases Hind Ⅲ and EcoR Ⅰ and identified by gel electrophoresis and DNA sequencing. Results: Gel electrophoresis and DNA sequencing showed that the recombinant plasmid containing the correct and full shRNA oligonucleotide. Conclusion:The siRNA plasmid,pPC-TS,was constructed successfully.

【基金】 国家自然科学基金资助项目(30471512);重庆市医学科技计划项目(04-2-100)
  • 【文献出处】 重庆医科大学学报 ,Journal of Chongqing Medical University , 编辑部邮箱 ,2009年07期
  • 【分类号】R531.5;R346
  • 【被引频次】1
  • 【下载频次】44
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