Computational analysis and gene cloning: design and preparation of a multi subunit vaccine consisting of EspA, Stx2B and Intimin antigens against enterohaemrrhagic <em>Escherichia coli</em>

16-26Escherichia coli O157:H7 is an intestinal pathogen that made diarrhoea, haemolytic uremia syndrome (HUS) and hemorrhagic colitis (HC) in patients. Roles of EspA and Intimin at the beginning of bacteria colonization in intestine are critical. Destruction of protein synthesis route with shiga toxins of E. coli O157:H7 is mediated through B-subunit of toxins. In this study, in silico approaches were performed to design a suitable construct from EspA, Intimin and Stx2B and a recombinant chimeric antigen was produced. Bioinformatics analyses such as physicochemical data, mRNA folding, 3D structures of chimera and various immunoinformatic data, such as linear and conformational B-cell epitopes, T-cell epitopes were reported according to authentic data base. The chimeric gene was prepared as synthetic construct after designing and cloning. The validation result showed that 83.9% residues lie in favoured or additional allowed region of the Ramachandran plot. Epitope prediction results proved very good distribution of conformational B-cell epitopes in the 3D structure of chimera. The identified T-cell epitopes are apt to bind MHC molecules. A good quantity of recombinant chimeric antigen was achieved in host cells. From in silico approach, an appropriate multi subunit vaccine candidate was designed and prepared for immunological examinations