Vaccine efficacy of 45 kDa outer membrane protein of escherichia coli O157:H7 in Mice Models

This thesis report is submitted in partial fulfillment of the requirement for the degree of M.Sc in Biotechnology, 2017.Cataloged from PDF version of thesis.Includes bibliographical references (pages 71-78).Escherichia coli O157:H7 is an extremely pathogenic organism that produces a potent Shiga toxin which can cause a number of brutal diseases like Hemorrhagic Colitis (bloody diarrhoea), renal failure causing Hemolytic Uremic Syndrome (HUS), and fatal neurological defects. Cattle and other ruminant animals are the main reservoirs of Shiga-toxin producing E. coli (STEC) organisms. CD-17 is the bovine isolate of this strain, whose antigenic structural parts had been employed in this study to be used as a vaccine against these virulent strains. In previous studies, despite undergoing several trials and strategies to produce an effective vaccine against STEC, no successful treatment could be launched. However in recent years, a 45kDa antigen was found to be present in normal healthy human serum against E. coli O157:H7. The main objective of this study was to determine the vaccine efficacy of the 45kDa outer membrane protein (OMP) of E. coli O157:H7 (CD-17 isolate) in mice models (in vivo) to develop and establish an efficient vaccine that can be used against these pathogens in the target regions of the world. For this, the outer membrane protein (OMP) of E. coli O157:H7 (CD-17 isolate) was extracted by the TSE (Tris-Sucrose-EDTA) extraction method, followed by its SDS-PAGE analysis and Western Blotting. The distinct band of 45kDa that appeared in the membrane after Western Blot was sonicated into a suspension and injected into only one set of experimental mice, keeping another set as control. After immunization, another western blotting was done, which confirmed the presence of 45kDa protein bands in the immunized set of mice and their babies (First Generation F-1, who were not injected 45kDa). When all the three sets of mice (experimental/immunized, F-1 and control/non-immunized) were challenged with the lethal dose of 1010 cells/ml of E.coli O157:H7 pathogen, it was observed that within a time period of 11 days, 85.7% of control mice have died whereas even after 60 days, 100% of the immunized mice were as healthy as before. Moreover, 100% of the non-injected babies (F-1) that were produced from mating of the immunized mice had also survived brilliantly like their parents. Therefore, this study demonstrates that the 45kDa protein vaccine will serve as an efficient treatment against the highly virulent E.coli O157:H7 strain, and will also sustain its immunogenicity in their next generation. This vaccine targets the developed countries where this lethal strain of E.coli O157:H7 still imposes a huge life-threat to the community leading to death. The world-wide problem of traveler’s diarrhoea of foreigners can also be expectantly solved by this vaccine. However, further characterization of the 45kDa surface protein is necessary to confirm other functions of this protein.Afsana Tasnim OshinM. Biotechnolog