南方医科大学学报

南方医科大学学报 ›› 2014, Vol. 34 ›› Issue (06): 904-.

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肠出血型大肠埃希菌O157:H7 ppk基因缺失株的构建及其生物学特性分析

韩朋，孙琦，赵素慧，张其威，万成松   

  • 出版日期:2014-06-20 发布日期:2014-06-20

Construction and characterization of enterohemorrhagic Escherichia coli O157:H7 ppkdeleted
strain

  • Online:2014-06-20 Published:2014-06-20

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摘要： 目的利用Red重组系统构建肠出血型大肠埃希菌（EHEC）O157:H7 ppk基因缺失株,并研究其生物特性。方法设计并合
成一对同源臂引物，每条引物5’端与ppk基因同源，3’端与卡那霉素抗性基因同源，扩增卡那霉素抗性基因片段；制备含有pKD46
质粒的EHEC O157:H7 EDL933w感受态细菌，然后将该抗性基因片段电击转化入制备的感受态菌株中；利用pKD46 介导的
Red重组系统，通过同源重组替换EHEC O157:H7 EDL933w ppk基因，PCR和测序验证；通过革兰染色、测D600值、吉姆萨染色比
较EHEC O157:H7 EDL933w野生株和突变株的形态结构、生长能力和黏附性。结果构建了ppk基因缺失并含有卡那霉素抗性
的EHEC O157:H7 EDL933w突变菌株（EHEC O157:H7 EDL933wΔppk），初步探索了EHEC O157:H7 EDL933w野生株和突变
株的生物学特性。结论本研究运用Red重组系统构建了肠出血型大肠埃希菌O157:H7 ppk基因缺失株，为进一步研究肠出血
型大肠埃希菌中ppk基因的调控机制奠定了基础。

Abstract: Objective To construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its
biological characteristics. Methods The gene fragment of kanamycin resistance was amplified using a pair of homologous arm
primers whose 5’ and 3’ ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157: H7
EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene
was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was
confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD600
value and Giemsa staining. Results and Conclusion We established a ppk-deleted EHEC O157:H7 EDL933w strain with
kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate
further study of the regulatory mechanism of ppk gene.