Screening and identification of interacting intestinal proteins of Ctenopharyngodon idella with OmpU of Vibrio mimicus

Abstract:

[Objective] In order to determine interaction between outer membrane protein U (OmpU) of Vibrio mimicus and the proteins of intestinal tissue of grass carp. [Methods] By construction of recombinant expression plasmid pET-32a-OmpU, we obtained the soluble fusion His-OmpU protein through IPTG induction and affinity purification. Western blot analysis showed the expressed His-OmpU protein could be combined by polyclonal antibody of OmpU. The proteins of intestinal tissue of grass carp interacting with OmpU was screened by His-tag pull down assay and identified by LC-MS/MS followed by SDS-PAGE. Based on searches of bioinformatics by Mascot software and protein database. [Results] Five proteins were verified which can interact with the OmpU in vitro, including α-actin 2, Filamin A, α-actinin 4, Transgelin-2, Calponin-2. [Conclusion] GO annotation indicated that these proteins are cytoskeletal protein and regulatory protein of cytoskeletal which play an important role in adhesion of pathogens, internalization and pathogenicity. These results provided a basis for studies on future development of adhesion antagonist and exploration the molecular pathogenesis of V. mimicus.