Location: Aquatic Animal Health ResearchTitle: Construction, characterization, expression and immune responses of flagellar proteins of channel catfish, important pathogen Edwardsiella ictaluri Author
Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 3/3/2010
Publication Date: 5/24/2010
Citation: Yeh, H., Klesius, P.H. 2010. Construction, characterization, expression and immune responses of flagellar proteins of channel catfish, important pathogen Edwardsiella ictaluri [abstract]. American Society for Microbiology. p. 64.
Technical Abstract: Background: Edwardsiella ictaluri causes enteric septicemia of catfish, which is the leading disease in channel catfish (Ictalurus punctatus)and is responsible for $50 - 60 million economic losses to catfish producers annually in the southeastern U.S. Bacterial flagella are complex polymeric structures consisting of more than 40 subunit proteins. Besides for their movement, bacterial flagella have involved in adhesion, quorum sensing, biofilm formation etc. In this study, we constructed, characterized and expressed E. ictaluri flagellar proteins in a bacterial expression system, and tested their antigenicity. Method: Genomic DNA of E. ictaluri was isolated, and flagellar genes were amplified by PCR. The Champion pET Directional TOPO Expression kit (Invitrogen) was used to construct and express the recombinant flagellar proteins. The expressed proteins were identified by SDS-PAGE and confirmed by MALDI-TOF. Each recombinant protein was overexpressed in 1-liter LB broth and purified by a nickel-chelating resin. The purified proteins were cleaved with a protease to remove the tags. The recombinant proteins with and without 6x His tag were tested for their antigenicity by using sera from channel catfish experimentally infected E. ictaluri. Results: Putative recombinant E. ictaluri flagellar proteins were cloned, expressed and confirmed by MALDI-TOF. Each recombinant protein was reacted to the immune sera from channel catfish experimentally infected E. ictaluri. Conclusion: Further study of these flagellar recombinant proteins for their immunogenicity may hold important insights in E. ictaluri pathogenesis in channel catfish.