Deletion of Helicobacter pylori vacuolating cytotoxin gene by introduction of directed mutagenesis

Abstract

Aim: To construct a vacA-knockout Helicobacter pylori mutant strain, whose only difference from the wild strain is its 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 DMA 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 gastric cells co-cultured with cell-free supernatants from H pylori wild strain or the mutant. Conclusion: We constructed a vacA-knockout strain of H pylori through direct mutagenesis, which creates an important precondition for the future research on virulence comparison with gene expression analysis.