TEM-1型β内酰胺酶的原核表达纯化及活性验证  

Prokaryotic Expression, Purification and Biological Activity of β-Lactamase TEM-1

作　　者：李静 徐建余 宁年智 刘传杰[2] 黄洁[2] 刘雪超[2] 李涛[2] 王慧[2] 

机构地区：[1]华北理工大学,河北唐山063009 [2]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《生物技术通讯》2017年第3期319-323,共5页Letters in Biotechnology

基　　金：国家重点基础研究发展计划(2015CB554202)

摘　　要：目的:产β-内酰胺酶是大肠杆菌、肺炎克雷伯菌等对β内酰胺类抗生素耐药的主要机制之一,该酶能水解青霉素、头孢菌素、碳青酶烯类等多种抗生素,其中TEM型是发现最早且种类最多的β内酰胺酶。探讨TEM-1型β内酰胺酶原核表达载体的构建、酶的纯化及活性验证。方法:利用PCR技术,以大肠杆菌全基因组为模板钓取TEM-1基因,构建p ET22b(+)-TEM-1表达质粒,经测序鉴定后,转入大肠杆菌Transetta(DE3),IPTG诱导表达、亲和纯化,并通过抗生素水解实验验证其酶活性。结果:构建了可溶性表达TEM-1的工程菌,通过亲和纯化最终获得纯度达90%以上的TEM-1型β内酰胺酶,该酶可水解氨苄西林。结论:获得了对氨苄西林具有水解活性的TEM-1型β内酰胺酶,并对其进行了酶动力学参数检测,验证其抗生素水解特性,有利于对临床抗生素的合理应用做出指导。Objective： The production of hydrolytic enzyme β-lactamase is one of the most important mechanisms of Escherichia coli and Klebsiella pneumoniae to resist β-lactam antibiotics. TEM is the first reported β-lactamase and has most varieties of subtypes. Our study aim at the construction of TEM-1 prokaryotic expression vector, the purification and the biological activity measurement of this enzyme.Methods： By using microbiological technology, we amplified TEM-1 gene from a clinical E.coli strain and constructed the expression plasmid pET22b（＋）-TEM-1. The expression vector was confirmed by sequencing and was then transformed into E.coli Transetta（DE3） strain. The recombinant TEM-1 expressed by induction of IPTG, and then the product was purified. We investigated the activity of this enzyme by performing antibiotic hydrolyzing experiment.Results： TEM-1 was expressed in prokaryotic expression system. After purification, the purity of the target protein is up to 90% and the protein could hydrolyze ampicillin efficiently.Conclusion： We obtained the TEM-1 protein withe the verified ability of hydrolyzing ampicillin, and calculated its hydrolysis dynamical parameter. Studies about the characteristics of TEM-1 enzyme will contribute to the reasonable use of clinical antibiotics.

关 键 词：TEM-1 Β内酰胺酶 表达 纯化 酶动力学 

分 类 号：Q78[生物学—分子生物学]