Optimization of gene expression and purification of enterotoxigenic Escherichia coli recombinant LTB protein and antibody production against it

Aims: It has been estimated that gastroenteritis is caused by bacteria in 30-70% of cases. Enterotoxigenic Escherichia coli or ETEC is one of the most common agents causing diarrhea. Protective immunity may be induced against this disease. Designing and producing a vaccine against this disease is one of the purposes of World Health Organization. Vaccine candidate molecules have to induce protective immunity against a broad spectrum of ETEC bacteria. Most ETEC strains can produce labile toxin; therefore labile toxin may be a proper candidate for being used as a vaccine molecule. The aim of this study was to optimize Heat-labile Toxin B Subunit expression in order to investigate its immunological properties. Materials & Methods: Optimizing of 3 parameters (IPTG concentration, time and temperature of promoter induction) was performed. Recombinant protein was purified with Ni-NTA column. Purified Heat-labile Toxin B Subunit was injected to mice subcutaneously in 4 sessions. Blood samples were taken during the interval between the injections and after last injection. Then, ELISA was performed. Results: The optimum expression occurred at 1mM IPTG concentration, after 3 hours and at 37oC. Recombinant protein was highly purified (>95%) with Ni-NTA column. Also, ELISA showed high titer of antibody production in mice. Conclusion: Expressed Heat-labile Toxin B Subunit is an immunogenic protein and can be one of the important