Preliminary study of functions of ompW gene in Salmonella paratyphi A / 中华微生物学和免疫学杂志

ABSTRACT

ObjectiveTo construct ompW- and ompW+ mutants of Salmonella paratyphi A with λRed system,and then study the function of the gene preliminarily.Methods Homologous regions were amplified from the genome Salmonella paratyphi A 50973,and then connect with kana fragment from plasmid pET22b-kan to construct a recombinant vector.The resultant fragments were amplified and transferred into 50973 with the help of λRed system after its concentration.Then the ompW- mutant was obtained PCR identification.Connect the recombinase expression plasmid pACU184 with full fragment of ompW regulatory region and coding region,then transfer the connection product into the mutant,the ompW+ mutant was obtained after double digest identification.Full cells of the wild,ompW- and ompW+ mutants were samples for SDS-PAGE and Western blot to detect the expression of protein OmpW.Biochemical identification of wild strain and mutant strains was conducted,so did the growth curves of the wild and the ompW- mutant.Choose BALB/c mice as a model to determine median lethal dose LD50 of the wild and mutant strains in order to observe the correlation between ompW gene and bacterial virulence.ResultsompW gene was knocked out in Salmonella paratyphi A 50973,also the ompW+ mutant was constructed; The wild and ompW+ mutant express the protein OmpW,while the ompW- mutant lost the protein.Each of the wild and mutant strains was Salmonella paratyphi A,and no obvious difference could be observed for their growth curves.LD50 for each strain was also similar.Conclusion The ompW gene has no correlation with the virulence in S.paratyphi A 50973,but the contribution of the mutants made an important foundation for the further study of functions of the gene ompW in Salmonella paratyphi.