生物学杂志

生物学杂志

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携带NMU2R shRNA重组腺相关病毒载体的构建及其评价

  

  1. 1．重庆工商大学 环境与资源学院， 重庆 400067；2. 天然药物研究重庆高校市级重点实验室， 重庆 400067
  • 出版日期:2019-06-18 发布日期:2019-06-18
  • 通讯作者: 郑旭煦，博士，教授，研究方向为天然产物研究与开发，E-mail：xuxuzheng@ctbu.edu.cn
  • 作者简介:郭莉霞，博士，副教授，研究方向为天然产物活性物质药理药效研究，E-mail：80074241@qq.com
  • 基金资助:
    国家自然科学基金 (81603173)； 重庆市教委科学技术研究项目(KJ1600607)；重庆高校市级重点实验室开放课题(1456032)

Construction and appraisal of recombinant adeno-associated virus vector for expression shRNA targeting NMU2R mRNA

  1. 1. Environmental and Resources Institute， Chongqing Technology and Business University， Chongqing 400067;2. Key Laboratory of Natural Medicine Research of Chongqing Education Commission， Chongqing 400067， China
  • Online:2019-06-18 Published:2019-06-18

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摘要： 构建稳定表达NMU2R shRNA的重组腺相关病毒载体，制备并纯化靶向性沉默NMU2R表达的高滴度重组腺相关病毒。首先设计合成NMU2R shRNA，退火形成双链与pAAV-ZsGreen-shRNA质粒BamH I和Hind III双酶切产物相连接构建形成质粒pAAV-ZsGreen-rNMU2R shRNA，经双酶切和测序鉴定证实，重组质粒克隆正确，将重组质粒转染到AAV-293包装细胞中包装成腺相关病毒载体，成功制备重组腺相关病毒rAAV5-ZsGreen-rNMU2R shRNA，经测定纯化所得rAAV5病毒滴度为1×1012 vg/mL。最后将rAAV5-ZsGreen-rNMU2R shRNA感染大鼠嗜铬细胞瘤细胞PC12，检测NMU2R shRNA基因沉默效果，利用空病毒载体pAAV-ZsGreen-shRNA作对照，Real-time PCR及Western Blot方法测得NMU2R mRNA及蛋白表达水平与对照组相比显著下降。综上，成功构建了高滴度携带NMU2R shRNA重组腺相关病毒载体rAAV5-ZsGreen-rNMU2R shRNA。

关键词: neuromedin U 2受体, RNA干扰, 重组腺相关病毒

Abstract: To construct the recombinant adeno-associated virus vector carrying neuromedin U 2 receptor (NMU2R) short hairpin RNA (shRNA) for preparation of high-titer viruses, hairpin RNA was designed to target NMU2R rat mRNA, and was synthesized and cloned into pAAV-ZsGreen-shRNA plasmid which was double digested by BamH I and Hind III. The presence of the target sequence in the plasmid pAAV-ZsGreen-rNMU2R shRNA was confirmed by enzyme digestion, PCR and DNA sequencing analysis. And then the plasmid was transfected into AAV 293 cells. AAV 293 cells expression NMU2R shRNA were obtained and subsequently infected AAV packaging system to package the rAAV5-ZsGreen-rNMU2R shRNA. After purification, the functional and infectious virus was obtained and the titer of virus was 1×1012 vg/mL. Real-time PCR and western blot methods were used to detect the expression of NMU2R after infection with PC12 cells, and the empty viral vector pAAV-ZsGreen-shRNA was used as control. The results indicated that the expression of NMU2R mRNA and protein was decreased significantly after infection for 24 h and 48 h as compared with that of the control. In summary, a high-titer recombinant adeno-associated virus-5 vector carrying NMU2R shRNA has been constructed successfully.

Key words: neuromedin U 2 receptor, RNA interference, recombinant adeno-associated virus

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