In silico Design, and In vitro Expression of a Fusion Protein Encoding Brucella abortus L7/L12 and SOmp2b Antigens

Background: L7/L12 is a protective antigen conserved in main Brucella pathogens and is considered as potential vaccine candidate. Outer membrane protein 2b is an immunogen conserved in all Brucella pathogens. Materials and Methods: The purpose of the current study was to in silico design a L7/L12-SOmp2b fusion protein and in vitro production of the chimera. Two possible fusion forms, L7/L12-SOmp2b and SOmp2b-L7/L12, were subjected to in silico modeling and analysis. Cloning and expression of the fusion protein has been done in the pET28a vector and Escherichia coli Bl21 (DE3), respectively. Results: Analysis and validation of the fusion proteins three-dimensional models showed that both models are in the range of native proteins. However, L7/L12-SOmp2b structure was more valid than the SOmp2b-L7/L12 model and subjected to in vitro production. The major histocompatibility complex II (MHC-II) epitope mapping using Immune Epitope DataBase indicated that the model contained good MHC-II binders. The L7/L12-Omp2b coding sequence was cloned in pET28a vector. The fusion was successfully expressed in E. coli BL21 by induction with isopropyl-β-d-thiogalactopyranoside. The rL7/L12-SOmp2b was purified with Ni-NTA column. The yield of the purified rL7/L12-SOmp2b was estimated by Bradford method to be 240 μg/ml of the culture. Western blot analysis revealed a specific reactivity with purified rL7/L12-SOmp2b produced in E. coli cells and showed the expression in the prokaryotic system. Conclusions: Our data indicates that L7/L12-SOmp2b fusion protein has a potential to induce both B- and T-cell-mediated immune responses and it can be evaluated as a new subunit vaccine candidate against brucellosis.