中国人兽共患病学报2013，Vol.29Issue(2)：129-132,137,5.DOI:10.3969/cjz.j.issn.1002-2694.2013.02.005
采用Red重组系统敲除铜绿假单胞菌弹性蛋白酶基因
Generation of a Pseudomonas aeruginosa elastase gene targeted deletion mutant by Red recombination system
摘要
Abstract
The aim of this study is to obtain the elastase activity negative strain by knocking out the elastase gene in Pseudomonas aeruginosa PAO1. Three genes of Red recombination system from λ phage were amplified and cloned into Esche-richia-Pseudomonas shuttle vector pUCP, and the pUCP-Red vector was transformed into PAO1 competent cells by electropo-ration. Then the recombinant DNA fragment which contains gentamycin antibiotic cassette flanked by two 80-bp homology sequences of elastase gene upstream and downstream locuses respectively was obtained by conventional cloning methods. And the fragment was electroporated into PAOl/pUCP-Red competent cells and screened on LB plate containing gentamycin and carben-icillin. The elastase gene knocked-out strain was verified by the methods of PCR, RT-PCR and enzyme activity assays. The elastase activity negative strain was successfully obtained in this study by using the Red recombination system. The elastase gene knocked-out strain obtained in this study provides the basis and materials for systemic study of pathogenicity mechanism of elastase in Pseudomonas aeruginosa.关键词
铜绿假单胞菌/弹性蛋白酶/基因敲除/Red重组系统Key words
Pseudomonas aeruginosa / elastase/ gene knock-out/ Red recombination system分类
医药卫生引用本文复制引用
余华,熊浚智,何晓梅,盛哈蕾,蔡文强,谢玮,张克斌..采用Red重组系统敲除铜绿假单胞菌弹性蛋白酶基因[J].中国人兽共患病学报,2013,29(2):129-132,137,5.基金项目
国家自然科学基金(No.30970115)资助 （No.30970115）
