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AIM:To construct a vacA-knockout Helicobacter pylorimutant strain,whose only difference from the wild strain isits disrupted vacA gene.METHODS AND RESULTS:A clone containing kanamycinresistance gene used for homologous recombination wasconstructed in a directional cloning procedure into pBluescriptⅡ SK,and then transformed into vacA~+ H pylori by electroporation.Colonies growing on the selective media containingkanamycin were harvested for chromosomal DNA extraction,and the allelic exchange was determined by polymerase chainreactions and sequencing.Loss of vacuolating activity ofthe vacA-knockout strain was confirmed by examining thegastric cells co-cultured with cell-free supernatants from Hpylori wild strain or the mutant.CONCLUSION:We constructed a vacA-knockout strain ofH pylori through direct mutagenesis,which creates animportant precondition for the future research on virulencecomparison with gene expression analysis.
AIM: To construct a vacA-knockout Helicobacter pylorimutant strain, whose only difference from the wild strain is disrupted vacA gene. METHODS AND RESULTS: A clone containing kanamycin resistance gene used for homologous recombination was constructed in a directional cloning procedure into pBluescript II SK, and then transformed into vacA ~ + H pylori by electroporation. Colonies growing on the selective media containing kanamycin were harvested for chromosomal DNA extraction, and the allelic exchange was determined by polymerase chain reactions and sequencing. Loss of vacuolating activity of the vacA-knockout strain was confirmed by examining the gastrictric cells co-cultured with cell-free supernatants from Hpylori wild strain or the mutant. CONCLUSION: We constructed a vacA-knockout strain of Hpylori through direct mutagenesis, which creates animportant precondition for the future research on virulencecomparison with gene expression analysis.