大肠杆菌16SrRNA甲基化酶基因rmtB缺失株的构建及其耐药性分析  

Construction of rmtB in-frame deletion mutation of Escherichia coli and comparative analysis of its aminoglycosides resistance

作　　者：区炳明 陈琳[1,2,3] 宋玉洁[1,2] 王小舟[1,2] 张倩[1,2] 陈秀芳[4] 朱国强[1,2] 

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009 [3]江苏农牧科技职业学院,江苏泰州225300 [4]江苏省金坛市畜牧兽医站,江苏金坛213241

出　　处：《中国预防兽医学报》2015年第6期448-452,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(30571374;30771603;31072136;31270171);教育部创新团队和江苏高校优势学科建设工程资助;第54批中国博士后科学基金(2013M541740)

摘　　要：为探索大肠杆菌(E.coli)16S r RNA甲基化酶耐药基因rmt B在菌体形成氨基糖苷类抗生素抗性中的作用,本研究以rmt B阳性菌E.coli 3A11-16作为亲本株,经PCR扩增其上下游序列作同源臂序列,分别克隆于p BluescriptⅡKS+中,构建p Blue-Δrmt B,其rmt B基因中缺失的30 bp由质粒中多克隆位点的18 bp替换,以XbaⅠ/KpnⅠ酶切p Blue-Δrmt B,目的片段亚克隆于自杀性载体p DMS197中,构建p DMS-Δrmt B,电转化至野生菌E.coli3A11-16,筛选获得rmt B基因缺失株E.coli 3A11-16Δrmt B。同时将rmt B基因插入p BR322中转化于基因缺失株构建其回补株。微量稀释法分别测定rmt B亲本株、基因缺失株和回补株对氨基糖苷类抗生素的耐药性。PCR和测序结果显示rmt B基因缺失株构建正确,证明通过自杀性载体能够对质粒中的基因进行改造。药敏试验显示相对于亲本株,基因缺失株对4,6-二脱氧链霉胺类氨基糖苷抗生素耐药性降低至质控菌水平,表明16S r RNA甲基化酶rmt B基因是介导大肠杆菌对4,6-二脱氧链霉胺类氨基糖苷类抗生素高度耐药的重要基因。本实验通过构建rmt B基因缺失株,为进一步研究rmt B阳性菌生物学功能及其在E.coli中的耐药机制奠定了基础。To investigate the role of 16S rRNA methylase gene of rmtB for resistance to aminoglycosides, the up-and down stream homologous sequences of rmt/3 were amplified by PCR from E.coli 3A11-16, and cloned into the pBluescript lI KS＋ to construct the pBlue-ArmtB （with the 30 bp in-frame deletion and replaced by a 18 bp MCS sequence from the vector）. Then, the rmtB in-frame deletion fragment （ArmtB） was subcloned into pDMS197 within the Xba I and Kpn I sites to construct pDMS-ArmtB, which was introduced into the wild type strain E.coli 3A11-16 by electroporation, and the E.coli/ArmtB mutation strain was generated via allelic exchange between the target gene and pDMS-2xrmtB and counter-selection on LA containing 10% sucrose and NaCl-fi＇ee to excision pDMS197 plasmid. In addition, the complemented strain was established by transforming the recombinant plasmid pBR322-rmtB into the mutation strain. Their resistance characteristics against aminoglycosides were tested by microdilution susceptibility testing. The results suggested that the resistance against 4,6-Disubstituted 2-deoxystreptamines of the mutation strain dramatically decreased to the level of the control strain E.coli C600, compared to the wild-type and complement strains. We concluded that the rmtB gene encoded RmtB exclusively mediates E.coli to possess high level resistance against 4,6-Disubstituted 2-deoxystre-ptamines. The construction of rmtB deletion E.coli is benifical for analyzing the biological characteristics of the rmtB positive strain, which laid a foundation for further study of resistance mechanism of rmtB.

关 键 词：rmtB 自杀性载体 rmtB基因缺失株 接合性质粒 氨基糖苷类 

分 类 号：S852.61[农业科学—基础兽医学]