Construction and Function Analysis of the ipaH4.5 Gene Deletion Mutant of Shigella flexneri 2a Strain 301

Abstract:

A γ-Red recombination system was used to construct an ipaH4.5 deletion mutant of S. flexneri 2a 301. A recovery mutant was obtained by introducing a low-copy plasmid strain. PCR analysis confirmed that ipaH4.5 was successfully deleted in the mutant and restored in the complementary strain. The growth curves of wild-type, deletion mutant and recover mutant were measured. Some biochemical tests were investigated. Invasion tests were performed to evaluate the virulence of these three strains. Quantities of cytokines in the culture supernatants of murine macrophages cell line J774 after being infected was measured by enzyme-linked immunosorbent assays (ELISA). No significant difference of growth rates and basic biochemical events were observed among three strains, and no difference in invasion ability was observed either. However, More IL-1β and TNF-а were observed in murine macrophages infected with deletion mutant than wild type and recover mutant. So we conclude that ipaH4.5 can inhibit inflammation response of the host after Shigella flexneri 2a 301 enter into the cell.

Key words:shigella flexneri 2a, IpaH4.5 gene, gene deletion, Biological function, invasion assay