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

目的敲除铜绿假单胞菌弹性蛋白酶基因,获得无弹性蛋白酶活性的铜绿假单胞菌菌株。方法采用PCR从pKD46质粒上扩增λ噬菌体的Red重组酶基因,并将其克隆到大肠杆菌和铜绿假单胞菌穿梭质粒pUCP多克隆位点上,电击转化铜绿假单胞菌PAO1感受态细胞,构建PAO1/pUCP-Red基因敲除体系。常规基因操作构建两端与弹性蛋白酶基因上、下游同源,中间为庆大霉素抗性基因的线性打靶片段;并将其电击转化pUCP-Red/PAO1感受态;采用庆大霉素和羧苄青霉素抗性平板初步筛选阳性重组菌;通过PCR、RT-PCR及弹性蛋白酶活性检测方法,鉴定菌株弹性蛋白酶基因的敲除情况。结果本研究通过构建Red重组系统,获得了无弹性蛋白酶活性的铜绿假单胞菌菌株。结论本研究成功敲除了铜绿假单胞菌弹性蛋白酶基因,所获无弹性蛋白酶活性的菌株将为系统深入研究弹性蛋白酶在铜绿假单胞菌致病性中的详细作用机理提供材料和基础。

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 se- quences of elastase gene upstream and downstream locuses respectively was obtained by conventional cloning methods. And the fragment was electroporated into PAO1/pUCP-Red competent cells and screened on LB plate containing gentamycin and earben- 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.