摘要
目的 研究产CTX M 14型超广谱 β 内酰胺酶 (ESBLs)功能性基因片段的结构特征。方法 质粒电转移及质粒谱分析以定位CTX M 14型基因 ;耐药质粒酶切基因文库 (鸟枪法 )技术及其计算机辅助在线分析以了解此酶功能性基因片段的分子结构特征 ;对Asn 10 4→Glu、Gly 2 32→Ala、Ser 2 37→Ala和Asp 2 4 0→Glu进行定点突变研究。结果 CTX M 14基因定位在 85kb可转移多重耐药的质粒上 ;采用鸟枪法克隆到 1.8kb、2 .4kb、3.1kb产CTX M 14的功能性基因片段 ,序列分析显示其定位在耐药质粒上转座子基因结构中 ;Asn 10 4→Glu突变可显著降低CTX M 14型ESBLs对头孢噻肟的水解能力。结论 CTX M 14基因定位在耐药质粒上转座子基因结构中 ;Asn 10 4可能是CTX M型ESBLs重要的活性位点。
Objective To investigate the structural characteristic of functional gene fragment coding CTX-M-14-type extended-spectrum beta-lactamases (ESBLs). Methods Plasmid electroporation and plasmid profile analysis were carried out for CTX-M-14 gene location. Shotgun cloning experiment with restriction enzyme-digested drug resistance plasmid and analysis aided by computer on the internet were implemented for cloning the functional gene fragment producing CTX-M-14 ESBLs and analysing its gene structural characteristic. Amino acid residues mutation of Asn-104→Glu, Gly-232→Ala, Ser-237→Ala and Asp-240→Glu was made by site-directed mutagenesis. Results CTX-M-14 ESBLs gene is located on a 85 kb self-conjugative multiple drug resistance plasmid. The functional gene fragments producing CTX-M-14, 1.8 kb, 2.4 kb and 3.1 kb in size, were cloned by shotgun cloning experiment. DNA sequencing and it was shown that CTX-M-14 gene was located on transferable gene elements transposon of the plasmid. The replacement of the catalytic residue Asn-104 by Glu resulted in a marked decrease in catalytic activity for cefotaxime. Conclusion CTX-M-14 gene is located on transferable elements transposon of the plasmid. Asn-104 is an critical residue for hydrolysis of oxyimino-beta-lactams in CTX-M-type ESBLs.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2003年第10期800-804,共5页
Chinese Journal of Microbiology and Immunology
