CAJ | 학술논문

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。
목적：이용Red중조계통구건장출혈형대장애희균（EHEC）O157：H7 ppk기인결실주,병연구기생물특성。방법설계병합성일대동원비인물，매조인물5'단여ppk기인동원，3'단여잡나매소항성기인동원，확증잡나매소항성기인편단；제비함유pKD46질립적EHEC O157：H7 EDL933w감수태세균，연후장해항성기인편단전격전화입제비적감수태균주중；이용pKD46개도적Red중조계통，통과동원중조체환EHEC O157：H7 EDL933w ppk기인，PCR화측서험증；통과혁란염색、측D600치、길모살염색비교EHEC O157：H7 EDL933w야생주화돌변주적형태결구、생장능력화점부성。결과구건료ppk기인결실병함유잡나매소항성적EHEC O157：H7 EDL933w돌변균주（EHEC O157：H7 EDL933wΔppk），초보탐색료EHEC O157：H7 EDL933w야생주화돌변주적생물학특성。결론본연구운용Red중조계통구건료장출혈형대장애희균O157：H7 ppk기인결실주，위진일보연구장출혈형대장애희균중ppk기인적조공궤제전정료기출。
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, OD60 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.