Construction of hp0523gene mutant in Helicobacter pylori Cag-PAI and the influence on the ability of CagA protein translocation?

Abstract:

Abstract:[Objective] To construct the hp0523 gene mutant of Helicobacter pylori and investigate the function of hp0523 gene. [Methods] We designed and amplified the upstream homologous fragment and downstream homologous fragment of hp0523 gene via PCR method. We constructed the suicide plasmid pBlueKM40-?hp0523 based on allelic exchange. We introduced the suicide plasmid pBlueKM40-?hp0523 into Helicobacter pylori 11637 by electroporation and screened the mutant based on antibiotic selection. We checked the mutant using the PCR and gene sequenced. We performed the coculture of Helicobacter pylori and gastric cell BGC-823 and detected the ability of CagA’s translocation and expression via Western blot. [Results] We constructed the suicide plasmid pBlueKM40-?hp0523 successfully and got the hp0523 deletion mutant. PCR and gene sequenced results showed the gene hp0523 was deleted. The results of CagA translocation assay showed that hp0523 interrupted the translocation of CagA. The comparison between wild-type and mutant showed that hp0523 affected the expression of CagA. [Conclusions] We constructed the hp0523 deletion mutant of Helicobacter pylori NCTC11637. This study suggests that hp0523 gene is an important virulence factor, which may be a component of the apparatus for CagA’s translocation.