Page Banner

United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR ANALYSIS OF VIRULENCE DETERMINANTS OF SELECT BACTERIA IN FISH DISEASES Title: Construction, characterization and use of Edwardsiella ictaluri flagella-type three secretion system protein arrays

Authors
item Yeh, Hung-Yueh
item Klesius, Phillip

Submitted to: European Association of Fish Pathologists
Publication Type: Abstract Only
Publication Acceptance Date: June 18, 2009
Publication Date: September 14, 2009
Citation: Yeh, H., Klesius, P.H. 2009. Construction, characterization and use of Edwardsiella ictaluri flagella-type three secretion system protein arrays. 14th European Association of Fish Pathologists. International Conference Prague, Czech Republic, September 14-19, 2009. p. 42.

Technical Abstract: Enteric septicemia of catfish, caused by Edwardsiella ictaluri, is the leading disease in channel catfish (Ictalurus punctatus) that is responsible for $50 - 60 million economic losses to catfish producers annually in the Southeastern U.S. The flagella and type three secretion system (TTSS) in Gram-negative bacteria are one of the most common modes for the toxins to be specifically exported into the host cell. In this study, we constructed, characterized and used E. ictaluri TTSS-flagella protein arrays. E. ictaluri genomic DNA was isolated, and TTSS-flagella genes were PCR amplified. The expression of Escherichia coli System with Gateway™ Technology (Invitogen) was used to construct and express the recombinant proteins. The expressed proteins were identified by SDS-PAGE and MALDI-TOF. Each recombinant protein was overexpressed in 1-liter LB-antibiotic broth in the presence of L-arabinose, and purified by a nickel-chelating resin. The purified proteins were cleaved with a protease to remove the 6x His tags. The recombinant proteins with and without 6x His tag were fabricated on aminosilan coated glass slides using a BioOdyssey Calligrapher MiniArrayer according to the manufacturer’s instruction. After fabrication, the protein arrays were examined for quality. The recombinant proteins were probed for antigenicity using sera from experimentally infected E. ictaluri channel catfish. Forty recombinant putative E. ictaluri TTSS-flagella proteins were cloned, expressed and confirmed by MALDI-TOF. Each recombinant protein was probed with the immune sera to the 6x His tag at the amino terminus, but not all recombinant proteins reacted to the immune sera from channel catfish experimentally infected E. ictaluri. Further study of these recombinant proteins for their antibody reactivities may hold important insights in E. ictaluri pathogenesis in catfish.

Last Modified: 10/23/2014
Footer Content Back to Top of Page