Expression of Recombinant Chimeric EspA-intimin Protein in Nicotiana tobaccum for Oral Vaccine Development

One of the important pathogens which cause hemorrhagic colitis and hemolytic uremic syndrome in humans is enterohemorrhagic Escherichia coli (EHEC) O157:H7. Cattle are the most important reservoir of this bacterium. EspA and Intimin are two protein factors for bacteria colonization on intestinal epithelium and cause attaching/effacing lesion. The LEE pathogenicity islands code these proteins. EspA is part of type III secretion systems which delivers Tir to the host cell and integrate to membrane. Intimin encoded by eae gene and fused to Tir. In this research we supposed that chimeric recombinant form of EI gene containing EspA and Intimin were fused with a linker as an edible candidate vaccine would reduce colonization of E. coli O157:H7 in animal model. We constructed a synthetic gene EspA (E120) and intimin (Int282) fused by (EAAAK)4 sequence. The synthetic gene (EI) was codon optimized and subcloned into plant expression vector (PBI121) under CaMV35S promoter and then transferred to tobacco plant by agrobacterium mediated protocol. The presence of inserted gene in plant genome was documented by PCR and RT-PCR methods. The amount of EI protein in transgenic tobacco leaves were estimated 0.1% of the total soluble protein (TSP) by ELISA method.