多药耐药阴沟肠杆菌临床株ampC基因的克隆和表达

Cloning and expression of ampC gene from a multidrug resistant isolate of Enterobacter cloacae

目的克隆一株临床分离的多药耐药阴沟肠杆菌的ampC基因,并研究其编码的AmpCβ-内酰胺酶的特性。方法聚合酶链反应(PCR)扩增阴沟肠杆菌ECLC074的ampC基因,将扩增的目标片段连接入pMD18-T进行双链测序后,进一步克隆入pACYC184质粒,用获得的pACYC184/ampC重组质粒转化大肠杆菌MC4100。采用琼脂稀释法测定阴沟肠杆菌ECLC074,大肠杆菌MC4100及大肠杆菌MC4100重组菌的抗生素敏感性,采用三维试验及等电聚焦电泳研究β-内酰胺酶的特性。结果DNA测序及限制性酶切分析结果表明,阴沟肠杆菌ECLC074的ampC基因已被克隆入pACYC184质粒,大肠杆菌MC4100重组菌和阴沟肠杆菌ECLC074均产生一种等电点为7.8的β-内酰胺酶,该酶具有AmpCβ-内酰胺酶的耐药表型特征。结论阴沟肠杆菌的ampC基因可以被克隆入pACYC184质粒,并能在大肠杆菌MC4100中表达,这为深入研究染色体AmpCβ-内酰胺酶创造了条件。

Objectives To clone the ampC gene from a multidrug resistant isolate of Enterobacter cloacae and identify the properties of the ampC β-lactamase encoded by the cloned gene. Methods The entire ampC gene of Enterobacter cloacae ECLC074 was amplified by polymerase chain reaction. The target amplification fragment was ligated with pMD18-T to be sequenced on both strands by the dideoxy chain termination method. The ampC gene was then cloned into plasmid pACYC184. The recombinant plasmid pACYC184/ampC was transformed into E. coli MC4100. Agar dilution susceptibility test was used to determine the antibiotics susceptibilitiy of Enterobacter cloacae ECLC074, E. coli MC4100 and the recombinant E. coli MC4100 strain. Three-dimensional extract test, and analytical isoelectric focusing overlay technique were used to identify the properties of β-lactamases, Results Sequencing and restriction analysis revealed that ampC gene of Enterobacter cloacae ECLC074 was cloned into pACYC184 plasmid. Both Enterobacter cloacae ECLC074 and the recombinant E. coli MC4100 strain produced a β-lactamase with an isoelectric point of 7.8 and a drug-resistant phenotype characteristic of an ampC β-lactamase. Conclusion ampC gene of Enterobacter cloacae can be cloned into pACYC184 plasmid and expressed in E. coli MC4100, this forms the basis for further study of chromosomal ampC β-lactamases.