副溶血性弧菌基因敲除方法的建立及应用  

Establishment of a suicide vector-based gene knockout method in studies of Vibrio parahaemolyticus

作　　者：刘霞[1,2] 高鹤[2,3] 杨琳[1,2] 张义全[2] 谭亚芳[2] 郭兆彪[2] 黄新祥[1] 杨瑞馥[2] 周冬生[2] 

机构地区：[1]江苏大学医学技术学院,江苏镇江212013 [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 [3]中国疾病预防控制中心传染病预防控制所,传染病预防控制国家重点实验室,北京102206

出　　处：《中国实验动物学报》2011年第3期188-192,276,共6页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金(30930001、30900823、30771179);973项目(2009CB522600)

摘　　要：目的摸索出一套副溶血性弧菌基因敲除的可靠方案,副溶血性弧菌致病相关基因的敲除对深入研究其致病机制有重要意义。方法通过融合PCR技术将目的基因上下游同源臂融合并克隆到自杀载体pDS132上,将重组质粒转化大肠杆菌S17λpir中,再接合转移到副溶血性弧菌菌株内,经pDS132质粒上sacB基因的反向筛选得到突变株。结果成功构建了副溶血性弧菌RIMD2210633菌株ΔopaR,ΔtoxR和ΔaphA三个基因突变株。结论通过自杀载体同源重组成功获得精确敲除的无痕突变株更有利于基因功能的研究,使后续副溶血性弧菌突变株与野生株的对比研究成为可能。Objective To establish a suicide vector-based gene knockout method in studies of Vibrio parahaemolyticus.Methods The upstream and downstream flanking DNA fragments of target gene were fused by PCR,and then cloned into the suicide vector pDS132.The recombinant plasmid was introduced into E.coli strain SM17λpir and transferred into V.parahaemolyticus by conjugation.The sacB gene in pDS132 was sucrose-sensitive and used to screen the deletion mutants.Results The opaR,toxR and aphA mull mutants of V.parahaemolyticus were successfully constructed.Each target gene was exactly deleted,and the mutants constructed were unmarked.Conclusions The gene knockout method is stable with a good efficiency,and can be used for gene function studies of V.parahaemolyticus.

关 键 词：自杀载体 同源重组 无痕突变 

分 类 号：Q95-33[生物学—动物学]