质粒介导AmpC酶的结构及传播机制研究  

Study on the genetic structure and transmission mechanism of a plasmid-mediated AmpC β-lactamase

作　　者：陆玮新[1] 魏泽庆[1] 俞云松[1] 陈亚岗[1] 李兰娟[1] 

机构地区：[1]浙江大学医学院附属第一医院传染科卫生部传染病重点实验室,杭州310003

出　　处：《中华医学杂志》2005年第43期3043-3047,共5页National Medical Journal of China

基　　金：国家自然科学基金资助项目(30270074);浙江省自然科学基金资助项目(M303812)

摘　　要：目的明确多重耐药肺炎克雷伯菌质粒介导的AmpC酶基因及周围序列的结构特征,阐明质粒介导AmpC酶传播机制。方法抽提接合菌的质粒,用限制性内切酶HindIII消化,酶切DNA片段的黏末端用Taq酶补平并在末端加单个的脱氧腺苷(A),与pGEM-T Easy载体进行T-A克隆。在含氨苄西林和头孢西丁的平板上筛选重组菌,抽提重组质粒,酶切分析,克隆片段用步移法测序。对重组菌进行药敏试验和等电点分析。结果克隆筛选到含5.2kb插入片段的重组质粒pT948,插入片段测序发现其含有一个ampC(blaDHA-1)基因和一个ampR调节基因。在blaDHA-1结构基因的侧翼发现一个插入序列IS26及Ⅰ类整合子的qacE△1和sulI基因。重组菌表达等电点为7.7,药敏试验显示对头孢西丁耐药,对头孢他啶的耐药性能被头孢西丁诱导。结论克隆到质粒介导的AmpC酶为DHA-1型,其相邻的插入序列IS26参与DHA-1的转移。Objective To clone the gene of plasmid-mediated AmpC β-1actamase from the plasmid of multiple-drug resistance Klebsiella pneumoniae producing plasmid-mediated AmpC β-1actamase and demonstrate its mechanism of transmission. Methods Plasmids of the transconjugant were extracted and digested with restriction endonuclease HindlII. Taq DNA polymerase was applied to fill the recessed 3＇ termini, and a single deoxyadenosine was added to the 3＇termimi of fragments. Then these fragments were ligated with pGEM-T Easy vector. E. coli DH5α containing recombinant plasmid was selected on MacConkey agar plates containing ampicillin and cefoxitin. Insert fragments were sequenced by primer walking. MIC determinations and isoelectric focusing electrophoresis （IFE） were utilized to analyze recombinant. Results The recombinant plasmid pT948 containing a 5.2-kb insert was obtained. The inserted fragment contained a blaDnA_t and a regulatory gene ampR. The insertion sequence （IS26） , qacE△l and sull genes of the I type integron were obtained near the blaDHA-1 gene. Recombinant expressed a [3-1actamase with pI of 7.7. MIC determinations showed that recombinant was resistant to cefoxitin and the resistance to ceftazidime could be induced by the cefoxitin. Conclusion The plasmid-mediated ampC gene cloned was identified as blaDHA-1. IS26 observed on the flanks of the blaDHA-1 maybe relate to the translocation of blaDHA-1 gene region from the chromosome to plasmid.

关 键 词：克隆 分子 质粒 DNA可移植因子 AMPC酶 

分 类 号：R346[医药卫生—基础医学]