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United States Department of Agriculture

Agricultural Research Service

Research Project: Pre-Harvest Interventions For Application During Poultry Production To Reduce Food-Borne Bacterial Pathogens

Location: Poultry Microbiological Safety Research

Title: Construction, Expression, and Characterization of Flagellar Proteins for the Food-borne Pathogen Campylobacter jejuni

Authors
item YEH, HUNG-YUEH
item HIETT, KELLI
item LINE, JOHN
item OAKLEY, BRIAN
item SEAL, BRUCE

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 19, 2012
Publication Date: N/A

Technical Abstract: Background: Campylobacter jejuni, a Gram-negative bacterium, is the leading etiologic agent of human acute bacterial gastroenteritis worldwide. The source of this bacterium for human infection has been implicated as consumption and handling of poultry where Campylobacter jejuni is a commensal in the gut. Although this microorganism has been extensively studied, there are many unanswered questions regarding pathogenesis and host immune responses unanswered. To provide a framework for studying host immune responses during Campylobacter infection, our goal is to produce Campylobacter protein arrays that contain the proteins involved in bacterial cell motility. We first targeted bacterial flagella, which are complex polymeric structures, consisting of more than 35 subunit proteins. Bacterial flagella are involved not only in motility, but also adhesion, quorum sensing, biofilm formation and other virulence activities. In this study, we constructed, characterized and expressed Campylobacter jejuni flagellar proteins in a bacterial expression system. Methods: Genomic DNA of the Campylobacter jejuni D1-39 was isolated, and flagellar genes were amplified by PCR. The Expresso™ Rhamnose Cloning and Expression kit (Lucigen Corp.) was used to construct and express the recombinant flagellar proteins. The expressed proteins were identified by SDS-PAGE and confirmed by anti-His tag monoclonal antibody. Each recombinant protein was over-expressed in 0.5-liter LB broth and purified by a cobalt-chelating resin. The recombinant proteins were tested for their antigenicity by using sera from experimentally infected Campylobacter jejuni. Results: In this study, 35 flagellar genes were PCR amplified, sequenced and cloned from the Campylobacter jejuni D1-39 genome. Twenty-two flagellar genes were successfully expressed in the E. cloni® host cells. All recombinant proteins had the expected sizes on Coomassie-stained SDS-PAGE, and were reactive to a mouse anti-His tag antibody. We also observed the optical density of cultures from other clones for which proteins were not detected on the SDS-PAGE decreased or leveled off after addition of rhamnose, suggesting that the proteins are toxic to the E. cloni® host. Conclusion: Further study of these flagellar recombinant proteins for their immunogenicity and vaccine potential may hold important insights for Campylobacter jejuni commensalism in chickens and pathogenesis in humans.

Last Modified: 9/10/2014