4株产TEM型超广谱β内酰胺酶肠杆菌科细菌的研究  

Study on four Enterobacteriaceae strains producing TEM-type extended-spectrum β-lactamases

作　　者：李家斌[1] 李惠[1] 李旭[1] 余鑫之[1] 马亦林[2] 俞云松[2] 

机构地区：[1]安徽医科大学第一附属医院传染病科,合肥230022 [2]浙江大学医学院附属第一医院传染病科

出　　处：《中国抗感染化疗杂志》2004年第6期324-327,共4页Chinese Journal of Infection and Chemotherapy

基　　金：安徽省教育厅自然科学基金 (编号 :2 0 0 3kj2 0 1)

摘　　要：目的 :鉴定临床分离的 3株大肠埃希菌和 1株肺炎克雷伯菌所产的TEM型 β内酰胺酶编码基因的序列 ,并进行原核表达 ,了解其相关特性。方法 :聚合酶链反应 (PCR)扩增TEM型β内酰胺酶编码基因片段 ,克隆入 pGEM Teasy载体 ,表达于感受态细胞大肠埃希菌DH5α中 ,双脱氧链终止法测定核苷酸序列 ,确定基因型 ;其基因与原核表达载体 (pET 2 8c)连接 ,并在感受态细胞大肠埃希菌DH5α中进行原核表达 ,提取质粒 ,用PCR进行表达鉴定 ,用超广谱 β内酰胺酶 (ESBLs)表型确认试验鉴定表达菌株的表型 ,另外对这些临床菌株进行接合试验。结果 :PCR扩增结果显示这 4株细菌产生的TEM型β内酰胺酶编码基因片段含 981个核苷酸 ,其表达酶的性质均为ESBLs,临床菌株的质粒接合试验均为阳性。这 4株临床菌株所产生的TEM型 β内酰胺酶的编码基因已被GenBank命名为TEM 10 (GenBank注册号 :U95 36 3)。 结论 :安徽地区这 4株细菌所产TEM型 β内酰胺酶均为TEM 10 ,在国内我们首次从临床分离株中发现TEM 10。Objective: To characterize the encoding g ene sequences of TEM-type extended spectrum β-lactamases produced by 3 Escheri chia coli strains and one Klebsiella pneumoniae strain, identify their gen otypes, express the genes in prokaryotic expression system, and study the releva nt properties of these TEM-type β-lactamases. Methods: Th e encoding genes of TEM-type β-lactamases produced by 4 isolates were amplifi ed by PCR. The purified PCR products were ligated with pGEM-T easy vectors, exp ressed in E. coli DH 5α, and sequenced by Sanger's dideoxy chain termi nation method. The newly obtained β-lactamases belonged to TEM-type as compar ed with amino acid sequences in the GenBank. Plasmids were extracted from the pr okaryotic expression system and PCR was performed to determine whether the expre ssion was successful. Phenotypes of the TEM enzymes were determined by ESBLs phe notype confirmation test. Conjugation test was performed to determine whether t heir genes are present in plasmid or chromosome. Results: Th e encoding genes of β-lactamases were belonged to TEM-type by PCR. The PCR pr oducts of the four strains had 981 nucleotides. These enzymes were characterized as ESBLs by ESBLs phenotype confirmation test. Transconjugants were successfull y selected from the four parental strains by conjugation tests. The TEM-type β -lactamase produced by the 4 strains was named as TEM-10 (U95363) by GenBank. Conclusions: The β-lactamase produced by 3 Escherichia c oli strains and one Klebsiella pneumoniae strain isolated from 4 patients in Anhui Pro vince was TEM-10. It was the first report of TEM-10 type β-l actamase in China. [

关 键 词：超广谱Β内酰胺酶 序列分析 接合试验 原核表达 

分 类 号：R378.2[医药卫生—病原生物学]