Construction and identification of hp0532 gene mutant in Helicobacter pylori Cag-PAI

摘要

To construct a hp0532 gene-deleted mutant of Helicobacter pylori and study its impact on translocation of CagA protein. A recombinant fragment containing a kanamycin resistance gene that was flanked by the up- and down-stream portions of the gene hp0532 was amplified and engineered into pBluescript SK II-plasmid. To construct suicide plasmidpBlueKM40-/lhp0532 based on allelic exchange, Electrotrans formation of H pylori cells with pBshp0532-mutant resulted in isolation of kanamycinresistant H pylori transformants, which was identified by polymerase chain reaction and sequencing analysis. To investigate the function of hp0532 gene in Helicobacter pylori Cag-PAI we performed the coculture of H. pylori and gastric cell BGC-823; We managed to construct suicide plasmidpBlueKM40-/lhp0532. After being analyzed by restriction endonuclease, the mutant vector produced the expected band and we also obtained a coculture of H. pylori which can affect CagA protein translocation. 4 hours after the wild coculture was infected, CagA entered the cell. The hp0532 is an important virulence factors in H. pylori Cag-PAI, involved in CagA protein transportation.