肠出血性大肠杆菌O157∶H7前噬菌体CP-933Y缺失突变株的构建

发布时间：2018-03-23 00:26

本文选题：肠出血性大肠杆菌　切入点：前噬菌体　出处：《生物技术通讯》2016年06期 　论文类型：期刊论文

【摘要】：目的:利用Red重组系统敲除肠出血性大肠杆菌O157∶H7前噬菌体片段CP-933Y,进而构建CP-933Y缺失突变株。方法:以肠出血性大肠杆菌O157∶H7菌株为模板,加入酶切位点PCR扩增前噬菌体CP-933Y上、下游各600 bp的同源臂序列;酶切后分别连接到p UC19-kan质粒的卡那霉素(包含FRT位点)抗性基因两侧,构建中间是卡那霉素抗性基因标记含有目的基因上、下游同源序列的线性片段;导入含有p KD46质粒的O157∶H7菌株中,利用Red编码的同源重组酶使该片段与目的基因上、下游发生同源重组,卡那霉素抗性基因置换菌株中CP-933Y前噬菌体片段,最后导入p CP20质粒去除卡那霉素抗性标记基因。结果:经PCR及测序验证,O157∶H7菌株中前噬菌体片段CP-933Y被敲除,敲除株与野生株具有相似的生长曲线。结论:构建了大肠杆菌O157∶H7前噬菌体CP-933Y缺失株,为进一步研究前噬菌体CP-933Y的功能奠定了基础。
[Abstract]:Objective: to construct a CP-933Y deletion mutant using Red recombinant system to knockout the O157:H7 prophage fragment CP-933Y. methods: the Escherichia coli enterohemorrhagic O157:H7 strain was used as a template, and the phage CP-933Y was added to the pre-amplification phage CP-933Y with the restriction site of PCR. The downstream homologous arm sequence of 600bp was ligated to the side of kanamycin (including FRT site) resistance gene of p UC19-kan plasmid, and the gene marker of kanamycin resistance gene contained the target gene. The linear fragment of downstream homologous sequence was introduced into the O157:H7 strain containing p KD46 plasmid, and the homologous recombination occurred between the fragment and the target gene by using the homologous recombination enzyme encoded by Red. The CP-933Y prophage fragment of kanamycin resistance gene replacement strain was introduced into p CP20 plasmid to remove kanamycin resistance marker gene. Results: the CP-933Y fragment of prephage fragment was knocked out by PCR and sequencing. Conclusion: the deletion strain of Escherichia coli O157:H7 prophage CP-933Y was constructed, which laid a foundation for further study on the function of prophage CP-933Y.
【作者单位】：广西医科大学;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;
【基金】：国家重点基础研究发展计划(2015CB554202) 国家自然科学基金(81401643)
【分类号】：Q78;R378.21

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