BIOINFORMATICS DESIGN AND PRODUCTION OF RECOMBINANT CHIMERIC ANTIGENS OF E.COLI O157:H7 AND BRUCELLA ABORTUS AND EVALUATION OF ITS STRUCTURE

Background and Aim:Diarrhea remains a major health problem for human societies and Livestock breeding systems. Intestinal bacteria which can produce intestinal poisons, plays an important role in the development of these diseases. Escherichia coli hemorrhage’s shiga Toxin (EHEC), is known to cause intestinal infections in developing countries. Also, Brucella spp. are facultative intracellular Gram-negative bacteria and an important etiological agent that cause zoonotic diseases.Methods:EspB 150-312 and Omp3161-201 were attached together with suitable linker. These multi genes were synthesized with codon optimization for E.coli host and were fused together by the application of three repeats of five hydrophobic amino acids as linkers. The structure of the synthetic construct gene, its mRNA and deduced protein and their stabilities were analyzed by bioinformatic software. Furthermore, the immunogenicity of this multimeric recombinant protein consisting of three different domains was predicted.Results:The chimeric gene was synthesised and subcloned into pET28a by related company , then we expressed in E.coli BL21 (DE3) and purified succesfully with Ni- NTA agarose. Expression of recombinant chimeric protein was confirmed by Western blot. Concentration of recombinant protein was assayed by Bradford. The produced recombinant protein was purified by mixed mix chromatography.Conclusion:Results from circular dichroism,with some difference, these structures can be detected in the second structure of the protein.

Keywords:

E.coli O157:H7 , EspB , Brucella Abortus , Omp31