Enterotoxigenic Escherichia coli (ETEC) is the main cause of diarrhea in under 5 years old children living in developing countries. The prevalence of the disease is about 400,000,000 annually among which 400,000 to 800,000 lead to deaths. Vaccination against this disease is one of the main objectives of World Health Organization (WHO).

 After colonization of intestine by ETEC, heat labile and heat stable toxin are released which lead to diarrhea. Colonization factor/I antigen (CFA/I) and the heat labile enterotoxin (LT) are the most important virulent factors in ETEC infection. The critical role of these two proteins in ETEC pathogenesis, propose them as good candidates for vaccine development. Considering the role of mucosal immunity in defense against ETEC infection, we can use the edible transgenic plants amongst the different expression and delivery systems, to induce mucosal as well as systemic immunity.

 An optimized synthetic gene, with plant codon preferences, which encodes CfaB-LTB fusion protein was designed and chemically synthesized. The gene was introduced into canola plant by insertion into a plant expression vector harboring seed specific promoter.

Southern blot and RT-PCR analysis confirmed stable integration and efficient transcription of synthetic construct. CFaB-LTB fusion protein was determined to comprise approximately 0.3% of total soluble protein in transgenic canola seeds. We also observed a specific anti CfaB-LTB IgG and IgA antibody responses in mice fed with transgenic seeds, which inhibited binding of both ETEC to Caco-2 cells and of LT to the coated GM1 ganglioside. Our results further showed no mortality in immunized mice challenged with bacteria when compared to the control group. In conclusion, the constructed CfaB-LTB fusion protein derived from transgenic canola seeds, is immunogenic and also protective when orally administered to mice, and may be used as an oral ETEC candidate vaccine.