Expression, purification, and immunization of a chimeric protein containing immunogenic regions of flagellin and intimin proteins against E. coli O157: H7

Enterohemorrhagic Escherichia coli (EHEC) and serotype O157: H7 is one of the most important diseases causing diarrhea. Shiga-like toxin secreted by the bacteria destroys epithelial cells and, in acute cases, causes hemolytic uremic syndrome (HUS). Antibiotic therapy is not effective against this pathogen, because it increases the production of Shiga toxin. Designing chimeric immunogens can be one of the most effective strategies against bacteria. This study aimed to evaluate the immunogenicity of recombinant chimeric antigen-containing immunogenic regions of flagellin and intimin proteins against E. coli O157: H7.

Immunogenic regions of flagellin and intimin proteinswere selected for bioinformatics evaluations. Plasmid H7-Int pET28a- was transferred to E. coli BL21(DE3). Expression of the recombinant chimeric protein was induced by IPTG and evaluated by SDS-PAGE. The recombinant protein was purified using a Ni-NTA affinity chromatography column and confirmed by Western blotting. Immunogenicity of the protein was performed in mice with pure protein and antibody titers were determined by ELISA. Bacterial shedding and mortality in mice were examined.

Expression of recombinant protein in E.coli led to the production of a protein with a molecular weight of 70 kDa. The total yield of purified protein was 80 mg/L. Immunization of mice with recombinant protein induced humoral immunity. Bacterial shedding in immunized mice’s feces was significantly reduced (102 CFU / ml). 100% and 80% of the immunized mice survived challenged with 10LD50 and 100LD50 of bacteria respectively.

The results showed that the chimeric protein provides good protective immunity in mice and can be considered an immunogenic candidate against E. coli O157: H7.