Abstract: Objective To construct enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains with delection espF gene and its
nucleotide fragment and with espF gene complementation. Methods A pair of homologous arm primers was designed to
amplify the gene fragment of kanamycin resistance, which was transformed into EHEC O157:H7 EDL933w strain via the
PKD46 plasmid by electroporation. The replacement of the espF gene by kanamycin resistance gene through the
PKD46-mediated red recombination system was confirmed by PCR and sequencing. The entire coding region of espF along
with its nucleotide fragment was amplified by PCR and cloned into pBAD33 plasmid, which was transformed into a mutant
strain to construct the strain with espF complementation. RT-PCR was used to verify the transcription of espF and its nucleotide
fragment in the complemented mutant strain. Results and Conclusion We established EHEC O157:H7 EDL933w strains with
espF gene deletion and with espF gene complementation. Both espF and its nucleotide fragment were transcribed in the
complemented mutant strain. The two strains provide a basis for further study of the regulatory mechanism of espF.