Constructing in-frame deletion irp6 gene mutant of enteroaggegative escherichia coli with yersinia HPI

  Objective   To construct the recombinant suicide vectors of irp6 gene in yersinia HPI and use it to construct in-frame deleted mutant of EAggEC.

  Methods   According to the sequence of irp6 gene of yersinia enterocolitica, the PCR primers were designed to amplify irp6 gene, then the PCR fragment was cloned into pMD18T-vector.The recombinant plasmid containing irp6 gene was mutated by precise deletion of middle fragment of irp6 gene in vitro. Then chloramphenicol gene was inserted into it.The fragment containing in-frame deleted irp6 gene which was inserted by chloramphencol gene was subcloned into suicide vector pKNG101, named pKH6.Moreover, EAggEC 17-2 was used to construct irp6 gene mutant by homologous recombination.

  Results   Irp6 gene of EAggEC 17-2 was exchanged by pKH6 by conjunction mobilization with the selection of sucrose.

  Conclusion   pKH6 which was constructed from the sequence of yersinia enterocolitica resulted in the construction of the in-frame deletion irp6 gene mutant EAG6 of EAggEC 17-2 successfully. The research on irp6 gene function would be carried out with EAG6.