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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR ANALYSIS OF VIRULENCE DETERMINANTS OF SELECT BACTERIA IN FISH DISEASES

Location: Aquatic Animal Health Research

Title: Development and use of Edwardsiella ictaluri type three secretion system protein array

Authors
item Yeh, Hung-Yueh
item Klesius, Phillip

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: February 20, 2009
Publication Date: May 17, 2009
Citation: Yeh, H., Klesius, P.H. 2009. Development and use of Edwardsiella ictaluri type three secretion system protein array. American Society for Microbiology Annual Meeting. p. 614.

Technical Abstract: Background: Edwardsiella ictaluri is the etiological agent of enteric septicemia of catfish, which is among the most common disease of channel catfish (Ictalurus punctatus) and is responsible for $50 - 60 million economic losses to catfish producers annually in the Southeastern U.S. The type three secretion system (TTSS) in Gram-negative bacteria is one of the most common modes for the toxins to be specifically exported into the host cell. In this study, we developed, characterized and used E. ictaluri TTSS protein arrays. Method: Genomic DNA of E. ictaluri was isolated, and TTSS genes were amplified by PCR. The Expression of Escherichia coli System with Gateway™ Technology (Invitrogen) 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 broth with appropriate amounts of L-arabinose and antibiotic, 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 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. Sera from channel catfish experimentally infected E. ictaluri were collected. Antibody reactions to the recombinant proteins were carried out. Results: Forty recombinant putative E. ictaluri TTSS proteins were constructed, expressed and confirmed by MALDI-TOF. Each recombinant protein was reacted with antibody to the 6x His tag at the amino terminus as well as sera from channel catfish experimentally infected E. ictaluri. Conclusion: Further study of these recombinant proteins for their antibody reactivities may hold important insights in E. ictaluri pathogenesis in catfish.

Last Modified: 4/24/2014
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