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

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.

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.

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.

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.