副溶血弧菌生物膜相关基因突变株的构建  

Construction of biofilm formation related mutants in Vibrio parahaemolyticus

作　　者：李迎丽[1] 张义全[2] 闫小娟 刘梦颖[1] 杨瑞馥[2] 邱景富[1] 周冬生[2] 

机构地区：[1]重庆医科大学公共卫生与管理学院,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

出　　处：《中华预防医学杂志》2013年第5期439-443,共5页Chinese Journal of Preventive Medicine

基　　金：国家自然科学基金（31170127,30871370）;国家重点基础研究发展计划（2009CB522604）

摘　　要：目的构建副溶血弧菌生物膜相关基因突变株并进行验证。方法采用PCR方法扩增得到靶基因的上下游同源臂片段，然后以上下游同源臂片段为模板，PCR扩增得到同源臂融合片段。将同源臂融合片段经酶切处理后克隆到自杀质粒pDSl32中。通过结合转移的方式将携带有同源臂融合片段的重组质粒转入副溶血弧菌RIMD2210633中，利用同源重组得到突变株。用PCR方法筛选和鉴定突变株，同时对突变株进行表型分析，从而在分子水平和表型试验上验证突变株是否构建成功。结果构建得到了分别携带副溶血弧菌生物膜相关基因vbfa、crp、hns、swrZ、swrT、cpsR融合同源臂片段的重组质粒，通过PCR扩增，分别得到了大小为1190、1128、1136、953、1242、1112bp的片段；用重组质粒构建了相应的突变株（AvbfR、Acrp、Ahns、AswrZ、AswrT和AcpsR），进行PCR鉴定时，突变株得到了大小分别为1190、1128、1136、953、1242、1112bp的扩增产物，比阳性对照分别小610、739、421、542、427、1367bp；用靶基因内部的引物进行扩增时，无扩增产物；选其中一株突变株Ahns进行生物膜形成能力表型分析，结果表明突变株Ahns生物膜形成量与野生株相比明显增加。结论构建得到了6株副溶血弧菌生物膜相关基因突变株，并从分子和表型试验上验证了突变株构建正确。Objective To construct the mutants of biofilm related genes in Vibrio parahaemolyticus and confrm the mutants. Methods The homologous upstream and downstream flanking fragments of target gene were amplified by using PCR, and the fusion homologous fragment was amplified by using the two flanking fragments as template. Then the fusion homologous fragment was digested by restriction enzyme and cloned into suicide plasmid pDS132. The recombinant plasmid was transferred into Vibrio parahaemolyticus RIMD 2210633 through conjugation. The mutants were screened and identified by PCR and the phenotype of one mutant was analyzed in order to verify＂ that the mutants were constructed successfully. Results Six recombinant plasmids carrying the fusion homologous fragments of genes vbfR, crp, hns, swrZ, swrT and cpsR respectively were constructed and identified by PCR. The amplification products of 1190,1128,1136,953, 1242 and 1112 bp were obtained respectively. The six mutants （AvbfR, Acrp, Ahns, AswrZ, AswrT and AcpsR） were constructed using recombinant plasmids. Verified by PCR, the size of amplification products of mutants （1190,1128,1136,953,1242 and 1112 bp respectively） was less （610,739,421,542,427 and 1367 bp respectively） than the corresponding positive control. Meanwhile, none of the products was amplified using the primers locating on the target gene. One mutant Ahns was selected to test the ability of biofilm formation. The result showed that the ability of biofilm formation of mutant Ahns was increased compared with the wild type. Conclusion Six mutants of biofilm related genes in Vibrio parahaemolyticus were constructed and tested by molecular and phenotype experiment to confirm that the mutants were constructed successfully.

关 键 词：弧菌属 质粒 生物膜 基因敲除技术 突变 

分 类 号：R[医药卫生]