大肠杆菌头孢哌酮抗性基因亚克隆及序列初步分析

Sub cloning and Preliminary Sequence Analysis of the Gene Encoding of a Cefoperazone Hydrolyzing β lactamase Isolated from Escherichia Coli

为了确定耐药质粒ｐ Ｆ Ｃ所编码β内酰胺酶衍化类型，用从临床分离出的大肠杆菌耐药质粒 ｐ Ｆ Ｃ头孢哌酮抗性基因，经分段亚克隆，构建重组质粒ｐ Ｆ Ｂ１、ｐ Ｆ Ｂ２、ｐ Ｆ Ｂ３ ，对上述 ３ 个重组质粒分别测序获得的抗性基因序列经 Ｉｎｔｅｒｎｅｔ互联网同源性检索，表明该抗性基因部分序列，与肺炎克雷伯氏菌编码超广谱β内酰胺酶 Ｔ Ｅ Ｍ５２ 的抗性基因序列存在高度同源性（９７％ ），提示ｐ Ｆ Ｃ头孢哌酮抗性基因所编码的 β内酰胺酶可能衍化自 Ｔ Ｅ Ｍ型。

Escherichia coli HX88108,which is resistant to cefoperazone(CPZ),was isolated from a severely infected patient.We studied genetical basis of β lactamase produced in E.coli HX88108 by pFL25,one of the recombinant plasmid of pFC.Largescale pFL25 plasmid was extracted,purified,and cleaved with restriction endonuclease EcoRⅠ,SalⅠ,PvuⅠ,then subcloned into vector pUC19 as 1.9 kb, 0.9 kb, 0.65 kb fragments respectively.Recominant plasmids were selected by α complementation and determined by restriction endonuclease analysis.DNA sequencing was performed by the dideoxy polymerase chain termination method.Partial nucleotide sequence(1 78 nucleotide position)of the gene was found to be highly homologous (97%)with the gene coding for TEM 52 extended spectrum β lactamase of K.pneumonise,suggesting that the β lactamase coded by the cefoperazone resistant gene might be derived from TEM type β lactamase.