采用Red重组系统敲除铜绿假单胞菌弹性蛋白酶基因

发布时间：2018-06-29 01:41

本文选题：铜绿假单胞菌 + 弹性蛋白酶　；参考：《中国人兽共患病学报》2013年02期

【摘要】：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。
[Abstract]:Objective to knockout the elastase gene of Pseudomonas aeruginosa and obtain a strain of Pseudomonas aeruginosa with no elastase activity. Methods the Red recombinant enzyme gene of 位 phage was amplified by PCR from pKD46 plasmid and cloned into E. coli and Pseudomonas aeruginosa shuttle plasmid pUCP polyclonal site. The PAO1 / pUCP-Red gene knockout system was constructed. Conventional gene manipulation was performed to construct a linear target fragment of gentamicin resistance gene, which was homologous to elastase gene upstream and downstream, and was transformed into pUCP-Red- / PAO1 receptive state by electroporation. Gentamicin and Carbenicillin resistant plates were used to screen the positive recombinant bacteria, and the knockout of elastase gene was identified by PCR and elastase assay. Results A strain of Pseudomonas aeruginosa with no elastase activity was obtained by constructing Red recombination system. Conclusion this study successfully knockout the elastase gene of Pseudomonas aeruginosa. The obtained strains with no elastase activity will provide materials and basis for further study on the detailed mechanism of elastase in the pathogenicity of Pseudomonas aeruginosa.
【作者单位】：第三军医大学附属新桥医院医学实验技术中心;
【基金】：国家自然科学基金(No.30970115)资助~~
【分类号】：R378

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