CTX-M型阴沟肠杆菌的基因克隆及原核表达产物特性研究  

Cloning and Prokaryotic Expression of CTX-M β-lactamase of Enterobacter cloacae

作　　者：江洁华[1] 徐韫健[1] 廖伟娇[1] 李乃健[2] 

机构地区：[1]广州医学院第一附属医院检验科,广州510120 [2]广州医学院,06级检验本科广州510180

出　　处：《热带医学杂志》2008年第10期1004-1008,1027,共6页Journal of Tropical Medicine

基　　金：广东省自然科学基金(No.5002318);广东省科技厅项目(No.63077);广州市科技局(No.2007J1-C0141);广州市医药卫生科技(No.2007-YB-156)

摘　　要：目的对阴沟肠杆菌所产CTX-M型β-内酰胺酶进行基因克隆、原核表达重组质粒的构建及其特性研究。方法以产CTX-M型β-内酰胺酶的阴沟肠杆菌45号基因组DNA为模板,设计两对引物PCR扩增CTX-M,将其克隆入pGEM-T载体后测定该核苷酸序列,再将CTX-M基因克隆到pGEX-6P-3表达质粒并转入感受态BL21,选择阳性菌落进行药物敏感试验和酶动力学检测。结果PCR扩增出大小为876bp和1067bp的基因片段,与GenBank上CTX-M-9和CTX-M-3酶的基因序列同源性为100%。CTX-M-3型重组菌株药敏试验结果与原菌株相比,对青霉素类和头孢噻肟具有明显的水解作用,第三、四代头孢菌素对之较为稳定,动力学结果与酶稳定性结果基本一致。结论重组质粒pGEX-6P-3/CTX-M构建成功,CTX-M-3型重组菌株与原菌株比较,对β-内酰胺类抗生素的敏感性和稳定性增加,亲和力下降。Objective To clone, express and characterize the CTX-M β-lactamase of Enterobacter cloacae. Method The blaCTX-M was amplified by PCR and sequenced after being subcloned in pGEM-T vector. The blaCTX-M was then cloned into pGEX-6P-3 vector and transformed into E.coli BL21. Drug sensitivity, the stability and kinetic parameters of β-lactamase hydrolysis of the recombinant strain were examined. Result Sequences of blaCTX-M shared 100% amino acid identity with blaCTX-M-3 and blcCTX-M-9 published in GenBank. The recombinant strain was resistant to penicillin and cefetaxime, susceptible to the third and forth generation of cephalosporin. The results of kinetic parameters were the same as stability test. Conclusion Prokaryotic recombinant plasmid pGEX-6P-3/CTX-M had been constructed successfully. Compared with strain 45, the recombinant strain＇s susceptibility and stability to β-lactam antibiotics increased, but appetency decreased.

关 键 词：CTX-M型 克隆 重组质粒 酶动力学 

分 类 号：R378.2[医药卫生—病原生物学]