Recombinant OMP25 protein production from endemic Brucella Abortus against brucellosis

Brucellosis is a zoonotic disease that affects numerous organs in humans and causes disease with specific and/or nonspecific symptoms, has increasing incidence recently as an bacterial infection agent originating from laboratory, causes serious economical losses due to factors such as miscarriages and the decrease in milk yield in animals, and moreover used in bioterrorism attacks. To prevent this disease, it is needed to develop effective vaccines with new technological approaches because of the lack of any vaccine that can be applied in humans and the vaccine used in animals is live vaccine with decreased virulence. In this thesis, the immunogenic outer membrane protein Omp25, which is located in the outer membrane of Brucella abortus strain, was obtained by using recombinant DNA technology. In the study, the Omp25 gene region of the endemic Brucella abortus type 3 strain provided by Erzurum Provincial Directorate of Food, Agriculture and Livestock was amplified with suitable primers and then cloned into the pET102 / D-TOPO plasmid. DNA sequencing analysis confirmed that the vector was successfully establised. Also, phylogenic analysis showed the gene of interest has a near genetic relation with other Brucella strains. The plasmids carrying the Omp25 gene were transformed into E.coli BL21 (DE3) strain and expression was performed. rOmp25 protein was purified from total protein contents by Ni-NTA affinity chromatography method in denaturing conditions. The total protein concentration of the final pure product was calculated as 0.50 mg/mL with BCA method. The necessary validations were made at all these stages and the final product was compared with the commercially purchased rOmp25.