肺炎克雷伯菌基因无痕敲除方法的建立  

A method for constructing unmarked deletion mutants of Klebsiella pneumoniae

作　　者：高钰双[1] 冯甜[1] 邱景富[1] 杨瑞馥[2] 周冬生[2] 李迎丽[1] 

机构地区：[1]重庆医科大学公共卫生与管理学院,重庆400016 [2]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《军事医学》2013年第6期431-434,共4页Military Medical Sciences

基　　金：国家自然科学基金资助项目(31071093,31170129);重庆市科委自然科学基金资助项目(CSTC 2010BB5103,CSTC 2010BB5354)

摘　　要：目的建立可行的肺炎克雷伯菌(Klebsiella pneumoniae)基因无痕敲除方法。方法 PCR扩增得到目的基因上下游同源臂融合片段,将其克隆到自杀载体pKO3-km,构建重组质粒。提取重组质粒,通过电转化转入肺炎克雷伯菌NTUH-K2044中,在43℃条件下培养筛选得到重组质粒整合到细菌基因组上的重组子,再利用质粒pKO3-km上的sacB基因在含蔗糖的平板上反向筛选得到突变株。结果与结论成功构建突变株,且突变株基因组上没有任何抗性筛选标记残留。该研究为肺炎克雷伯菌基因功能研究提供了基因敲除技术。Objective To establish a practical method for constructing an unmarked deletion mutant of the target gene.Methods The upstream and downstream flanking DNA fragments of the target gene were amplified by PCR.The two flanking fragments were fused by PCR and then cloned into the suicide vector pKO3-km.The recombinant plasmid was transformed into Klebsiella pneumoniae NTUH-K2044 by electroporation.Integration（at 43℃） and excision（at 30℃） of the plasmid resulted in the generation of mutant strains which were further confirmed by PCR.Results and Conclusion The unmarked deletion mutant was constructed using the method in this study,which could be used for further function study of the target gene.

关 键 词：克雷伯菌 肺炎 cAMP受体蛋白 基因敲除 自杀载体 同源重组 

分 类 号：R378.1[医药卫生—病原生物学]