Construction and characterization of enterohemorrhagic Escherichia coli O157:H7 ppk- deleted strain / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To construct enterohemorrhagic Escherichia coli (EHEC) O157 H7 ppk gene deletion strains and study its biological characteristics.</p><p><b>METHODS</b>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.</p><p><b>RESULTS AND CONCLUSION</b>We established a ppk-deleted EHEC O157H7 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.</p>