奇异变形菌ppk1基因缺失株的构建与鉴定

发布时间：2018-03-16 12:45

本文选题：奇异变形菌　切入点：自杀质粒　出处：《中华医院感染学杂志》2015年05期 　论文类型：期刊论文

【摘要】：目的构建奇异变形菌(PMI)聚磷酸盐激酶1(polyphosphate kinase 1,PPK1)基因缺失株,为研究ppk1基因在PMI致尿路感染中的作用及其机制奠定基础。方法通过融合PCR将目的基因上游及下游同源臂连接成一个片段,并克隆入自杀质粒pCVD442,将重组质粒转化入大肠埃希菌SM10λpir中,再与PMI受体菌进行接合试验,通过氨苄西林、蔗糖筛选得到ppk1基因缺失株(△pk1),进行PCR和DNA测序鉴定;体外比较野生株和缺失株在低营养条件下的生存能力。结果利用融合PCR将目的基因上、下游片段连接成一个重组片段,并连接至pCVD442自杀质粒;重组自杀质粒进入PMI后出现了同源重组事件,同源片段发生替换,ppk1基因被敲除,通过PCR及测序分析证实基因组目的基因已缺失;经体外测定MOPS培养基中的生长曲线,发现敲除株与野生株相比,在低营养环境中生存能力明显降低。结论自杀质粒同源重组方法可用于PMI基因敲除株的构建,是研究PMI基因功能的重要手段,ppk1基因与PMI在低营养环境中的生存能力有关。
[Abstract]:Objective to construct a polyphosphate kinase 1 polyphosphate kinase 1 (PPK1) gene deletion strain of Proteus mirabilis. To study the role and mechanism of ppk1 gene in urinary tract infection induced by PMI. Methods the upstream and downstream homologous arms of the target gene were linked into a fragment by fusion of PCR. The recombinant plasmid pCVD442 was cloned into Escherichia coli SM10 位 pir and then conjugated with PMI receptor bacteria. The ppk1 gene deletion strain (pk1G) was screened by ampicillin and sucrose, and identified by PCR and DNA sequencing. Results the fusion PCR was used to link the target gene and downstream fragment into a recombinant fragment and ligated to the suicide plasmid of pCVD442. After the recombinant suicide plasmid entered into PMI, the homologous recombination event occurred, the homologous fragment was knockout, the genomic target gene was found missing by PCR and sequencing, and the growth curve of MOPS medium was determined in vitro. It was found that the survival ability of the knockout strain was significantly lower than that of the wild strain. Conclusion the suicide plasmid homologous recombination method can be used to construct the PMI gene knockout plant. It is an important tool to study the function of PMI gene. It is related to the survivability of PMI in low nutrition environment.
【作者单位】：广州医科大学附属第二医院检验科;
【基金】：国家自然科学基金资助项目(81200505)
【分类号】：R378

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