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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, purification and antigenicity of recombinant Campylobacter jejuni flagellar proteins

Authors
item Yeh, Hung-Yueh
item Hiett, Kelli
item Line, John
item Oakley, Brian
item Seal, Bruce

Submitted to: Microbiological Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 23, 2012
Publication Date: May 6, 2013
Citation: Yeh, H., Hiett, K.L., Line, J.E., Oakley, B., Seal, B.S. 2013. Construction, expression, purification and antigenicity of recombinant Campylobacter jejuni flagellar proteins. Microbiological Research. 168(4):192-198.

Interpretive Summary: Campylobacter jejuni is an important zoonotic pathogen, which causes human acute bacterial gastroenteritis worldwide. The source of this bacterium for human infection has been linked to consumption and handling of poultry meat where this organism is a commensal in the gut. Vaccination of chickens is one of powerful means to reduce this bacterium in broiler chicken gut and minimize contamination in poultry. Because the genomes of many Campylobacter jejuni isolates have been sequenced, we wanted to use genomic approaches to develop flagellar vaccines for broiler chickens. Twelve recombinant Campylobacter jejuni flagellar proteins were purified, which were confirmed by SDS-PAGE analysis and a His tag detection kit. The FlgE1, FlgG, FlgK, FliE, FlgH/FliH and FlaA recombinant proteins were further confirmed by mass spectrophotometer. The purified recombinant proteins were tested whether they were immunogenic using antibodies from several sources. BacTrace anti-Campylobacter species antibody reacted to the FlaA recombinant protein, but not others. Rabbit anti-MOMP1 peptide antibody reacted strongly to FliE and weakly to FlaA, but not others. Rabbit anti-MOMP2 peptide antibody reacted strongly to the FlaA, FliG, FliE, FlhF, FlgG, FlgE1 and FliD recombinant proteins, less to FlgK and FlgH/FliH, and did not react to the FliY, FliS and FliH recombinant proteins. These antibody studies suggest that these recombinant flagellar proteins have potential for use as vaccines. This study is related to the goal of ARS National Program 108-Food Safety, Component 1D-Intervention and Control Strategies.

Technical Abstract: Campylobacter jejuni, a flagellated, spiral-rod Gram-negative bacterium, is the leading etiologic agent of human acute bacterial gastroenteritis worldwide. The source of this microorganism for human infection has been implicated as consumption and handling of poultry meat where this microorganism is a commensal in the gut. Because the genomes of many Campylobacter jejuni isolates have been sequenced, our ultimate goal is to develop protein arrays for exploring this microorganism and host interactions. In this communication, we report cloning, expression and purification of Campylobacter jejuni flagellar proteins in a bacterial expression system. Thirty-five flagellar genes were amplified by PCR and ligated in pRham vector in E. cloni 10G cells. Among them, 21 flagellar genes were successfully expressed, and 12 recombinant proteins were purified, which were confirmed by SDS-PAGE analysis and a His tag detection kit. The FlgE1, FlgG, FlgK, FliE, FlgH/FliH and FlaA recombinant proteins were further confirmed by LC-ESI-MS/MS. The purified recombinant proteins were tested whether they were immunogenic using antibodies from several sources. BacTrace anti-Campylobacter species antibody reacted to the FlaA recombinant protein, but not others. Rabbit anti-MOMP1 peptide (NH2-YRYDTGNFDKNF-COOH) antibody reacted strongly to FliE and weakly to FlaA, but not others. Rabbit anti-MOMP2 peptide (NH2-TGSRLNGDTGRN-COOH) antibody reacted strongly to the FlaA, FliG, FliE, FlhF, FlgG, FlgE1 and FliD recombinant proteins, less to FlgK and FlgH/FliH, and did not react to the FliY, FliS and FliH recombinant proteins. These antibody studies suggest that these recombinant flagellar proteins have potential for novel targets for vaccine development. It is also anticipated that these recombinant proteins provide us a very useful tool for investigating host immune response to Campylobacter jejuni.

Last Modified: 12/21/2014