Objective   To construct an enterohaemorrhagic Escherichia coli (EHEC) strain with tccP gene knockout based on O157∶H7 strain

which gene mediates attaching and effacing (A/E) lesions.    Methods   According to EHEC O157∶H7 gene sequence in the GenBank

PCR primers were designed and then the DNA fragments outside tccP were amplified. The PCR products was linked to chloramphenicol-resistant gene Cam and then subcloned into plasmid pBluescriptSKⅡ(-). After digested by double enzymes

the homologous recombination DNA fragments was used to replace tccP wild-type gene. PCR

RT-PCR

as well as Western blotting were used to identify tccP gene knockout from the DNA

RNA and protein levels respectively.    Results   DNA

RNA and protein levels confirmed that the Cam fragment had been successfully replaced tccP gene and integrated into the O157∶H7 genome.    Conclusion   EHEC O157∶H7 tccP knockout strain is successfully constructed

which is helpful in the research on the pathogenic mechanism of the bacteria.