自杀质粒同源重组方法构建弗氏志贺菌htrA可控表达菌株  

Arabinose-controlled expression of htrA in Shigella flexneri through suicide plasmidmediated homologous recombination

作　　者：杨晶[1] 陆丽兰 徐萍[1] 刘红胜[1] 冯尔玲[1] 王恒樑[1] 朱力[1] 

机构地区：[1]军事医学科学院生物工程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》2015年第5期354-356,402,共4页Military Medical Sciences

基　　金：国家自然科学基金资助项目(81171531;81125012;81373316);国家973计划资助项目(2011CB504901;2013CB910804)

摘　　要：目的构建弗氏志贺菌Htr A蛋白的可控表达菌株。方法构建自杀质粒同源重组载体,利用重组方法在基因组htr A前插入阿拉伯糖启动子,同时构建并导入外源表达Ara C蛋白的表达载体,实现对Htr A蛋白的可控表达;在此基础上,通过蛋白印迹实验,检测诱导前后全菌以及周质中Htr A蛋白的表达情况。结果测序结果表明,自杀质粒同源重组载体以及外源表达载体构建成功;蛋白印迹实验结果显示,对比阿拉伯糖诱导菌株,未诱导菌株中基本未检测到Htr A蛋白的表达。结论自杀质粒同源重组方式可在无阿拉伯糖时有效阻遏Htr A蛋白的表达;同时在诱导后,还可恢复Htr A蛋白的正常表达,有利于后续功能研究。Objective To achieve arabinose-controlled expression of HtrA strain and detect the expression of HtrA protein.Methods Arabinose promoter with htrA100 was amplified from pACD-htrA vector by PCR and cloned into pGP704 vector.Then, Shigella flexneri 2a strain 301 was transferred with the recombinant plasmid pGD-htrA and an AraC-expression vector .The expressions of HtrA in whole-cell and periplasmic space were detected by Western blotting .Results The suicide plasmid-mediated homologous recombinant vector and the inducible HtrA expression strain were successfully constructed.Without arabinose,HtrA protein was hardly detected ,but in the presense of arabinose , HtrA protein could be detected in whole-cell lysate and in periplasmic space lysate by Western blot .Conclusion Homologous recombination using suicide plasmid can significantly knock down the expression of HtrA protein .After being induced with arabinose , HtrA protein can be expressed normally .

关 键 词：志贺菌 弗氏 htrA基因 自杀质粒同源重组 阿拉伯糖操纵子 免疫印迹法 

分 类 号：R378.25[医药卫生—病原生物学]