Brucellosis is a well-known infectious disease in most parts of the world, especially in developing countries, common between humans and animals. Brucellosis is diagnosed by serological tests based on lipopolysaccharides (LPSs), which are bacterial cell wall antigens, and due to the similarities between LPSs antigens of some gram-negative bacterias, false-positive responses are inevitable. Alternatively, Outer membrane proteins (Omps), as antigenic conserved membrane proteins, can be used to diagnose brucellosis instead of LPS antigens. In this study, by using bioinformatics tools, linear B-cell epitopes were selected from Omp22, Omp25, and Omp31 antigens and fused with the rigid KP linker (K = Lysine, P=Proline). Designed gene cassette was cloned into pET-28a (+) vector and expressed recombinant protein was purified using Ni-NTA chromatography column and was confirmed with Poly-Histidine-HRP antibody. Finally, recombinant protein's seroreactivity with serum samples from 37 patients and 27 healthy individuals was evaluated by western blotting and enzyme-linked immunosorbent assay (ELISA) methods. Western blotting results showed high reactivity of the recombinant protein with serum samples of Brucella infected patients. ELISA results were analyzed using the receiver operating curve (ROC). Optical density cut-off point, accuracy, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Youden index J for recombinant protein were > 0.809, 84.37%,83.78%,88.89%,88.57%, 79.31% and 0.72 respectively. Western blotting and ELISA results showed that our recombinant protein has good sensitivity and specificity for the diagnosis of brucellosis.
Keywords: B-cell epitopes; Brucellosis; ELISA; Outer membrane protein; Recombinant protein.
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