两步PCR介导的Red重组技术快速敲除鼠疫耶尔森菌sRNA及染色体大片段

王仁霞1 刘荣娇1 李子微1 王瑞欢1 杨瑞馥2 韩延平2 邓仲良1

(1.湖南省南华大学公共卫生学院, 湖南衡阳 421001)
(2.军事医学科学院微生物与流行病研究所, 北京 100071)
【知识点链接】重组酶; 荚膜; 感受态; 转座子; 减毒

【摘要】【目的】利用Red同源重组系统, 通过二步PCR法建立一种适合鼠疫耶尔森菌s RNA和大片段染色体基因敲除的方法。【方法】第一步PCR先扩增出目的基因的上、下游同源臂 (600–1000 bp) 及卡那抗性盒, 再以上、下游同源臂及卡那抗性盒等摩尔混合物为模板, 通过融合PCR获得含上下游同源臂及卡那抗性盒的线性突变盒, 再将此突变盒的PCR产物电转到含有p KD46质粒的鼠疫201菌株, 在阿拉伯糖的诱导下, p KD46质粒表达Red重组酶, 促使卡那抗性盒替换目的基因, 最后对获得的重组克隆进行PCR鉴定。【结果】本研究通过两步PCR法构建600–1000 bp的同源臂, 提高了同源重组效率, 并将鼠疫菌s RNA Ryh B1 (108 bp) 和Ryh B2 (106 bp) 和染色体大片段47-2 (10.4 kb) 、47-3 (21.6 kb) 、47-3a (9.2 kb) 及47-3b (6.1 kb) 成功敲除。【结论】基于Red重组系统构建的二步法突变技术, 是一种简单、高效的精确修饰鼠疫菌s RNA及大片段染色体的方法, 适合于鼠疫菌全基因组的基因敲除, 为鼠疫菌基因表达与调控、致病和毒力等研究提供有力的工具。

【关键词】 鼠疫耶尔森菌; Red重组系统; sRNA; 大片段染色体; 基因敲除;

【基金资助】 湖南省教育厅重点项目 (15A165) Supported by the Hunan Provincial Department of Education Key Projects (15A165)

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This Article

ISSN:0001-6209

CN: 11-1995/Q

Vol 57, No. 07, Pages 1126-1137

July 2017

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摘要

  • 1 材料和方法
  • 2 结果和分析
  • 3 讨论
  • 参考文献