Antibody production against enterobacteriacea using recombinant Ferric Enterobactin protein (FepA)

Objective: Escherichia coli O۱۵۷:H۷ is a gram-negative rod-shaped bacterium. E coli O۱۵۷:H۷ is an enterohemorrhagic (EHEC) strain of the bacterium Escherichia coli and a cause of foodborne illness. Infection often leads to bloody diarrhea by producing a toxin called Shiga toxin, which damages the intestines, and occasionally leads to kidney failure, especially in young children and elderly people. A ۲۲۴۱ bp fepA gene of E.coli O۱۵۷:H۷ codes for production of a ferric enterobactin binding membrane protein that is essential for iron uptake by the bacterium. Inhibition of iron uptake can protect invasion of host by the bacterium. In this study we attempted to evaluate immunogenicity of the membrane protein, FepA. Materials and Methods: In order to produce recombinant FepA protein, the genomic, fepA gene of ۲۲۴۱ bp long was amplified by PCR from E coli O۱۵۷:H۷. The PCR product was ligated to pET۲۸a. The recombinant protein was then expressed in E. coli BL۲۱DE۳ by IPTG induction. SDS-PAGE analysis was carried out and the recombinant protein was purified by Ni-NTA affinity chromatography. The purified recombinant protein was injected to Balb/C mice in order to induce immunity. Antibody titer was determined by ELISA. Results: The recombinant protein of ۸۵ KD was produced and purified. Immunogenicity of the recombinant protein was determined by injecting Balb/C mice. The antibody produced therein could efficiently recognize and bind ferric enterobactin binding protein, thus heaving mice tolerance of ۱۰۶LD۵۰. Conclusion: With a view to the significant recognition by the antibody of ferric enterobactin binding protein, the notion of its application in restriction of enterobacteriacea propagation could be feasible.