癌变·畸变·突变 ›› 2010, Vol. 22 ›› Issue (3): 175-178.doi: 10.3969/j.issn.1004-616x.2010.03.004

• 论著 • 上一篇    下一篇

负链RNA病毒反向遗传通用载体的构建与鉴定

王革非;张驰;曾祥兴;张衡;陈幼莹;李卫中;李康生   

  1. 汕头大学医学院微生物学与免疫学教研室,广东高校免疫病理重点实验室,广东 汕头 514041
  • 收稿日期:2009-07-09 修回日期:2009-12-28 出版日期:2010-05-30 发布日期:2010-05-30
  • 通讯作者: 李康生

Construction of universal reverse genetic vector for minus RNA viruses

WANG Ge-fei;ZHANG Chi;ZENG Xiang-xing;ZHANG Heng;CHEN You-ying;LI Wei-zhong;LI Kang-sheng   

  1. Department of Microbiology and Immunology, Key Immunopathology Lab of Guangdong Province,Shantou University Medical College,Shantou 515041, Guangdong, China
  • Received:2009-07-09 Revised:2009-12-28 Online:2010-05-30 Published:2010-05-30
  • Contact: LI Kang-sheng

摘要: 目的: 用于负链RNA病毒的重组技术称为反向遗传技术,其核心需要构建一个具有双向启动子的反向遗传通用载体,可以分别正向转录翻译病毒复制所需酶系和负向转录病毒负链RNA。为此,构建负链RNA病毒反向遗传通用的载体。 方法: 为提高转染和表达效率,首先改造pcDNA3载体,通过酶切删除pcDNA3载体中非必需序列,在保留Amp抗性的前提下,通过酶切去除pcDNA3载体中非必需的Kan抗性表达盒与CMV启动子前区。利用长引物合成含有反向pol I启动子与多克隆位点的基因片段。将合成后的片段酶切、补平粘端、连接,反向插入至经改造后的pcDNA3载体中,构建反向遗传通用表达载体。采用PCR检测、酶切鉴定以及DNA测序进行验证。 结果: PCR检测、酶切鉴定以及DNA测序结果表明,载体构建正确,合成及插入序列与预期设计完全一致。 结论: 成功构建具有CMV与pol I双向启动子的反向遗传通用载体,为建立负链RNA病毒反向遗传平台提供了研究基础。

关键词: 反向遗传, RNA病毒, 载体

Abstract: OBJECTIVE: To construct a system for reverse genetics of minus RNA viruses, the universal reverse genetic vector was constructed. METHODS: Using pcDNA3 as plasmid backbone, non-essential Kanamycin resistant expression cassettes and CMV promoter upstream region were removed. Then the fragment including reverse pol I promoter and multiple clone site were synthesized by PCR and inserted into the reconstructed pcDNA3. The modified pcDNA3 and universal reverse genetic vector were identified by restriction enzyme. RESULTS: Sequencing showed that the universal reverse genetic vector which had CMV and pol I bidirectional promoter was constructed successfully. CONCLUSION: This research established reverse genetics system of minus RNA viruses and provided research basis for further development of minus RNA viruses.

Key words: reverse genetics, RNA virus, vector

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