Optimization of Expression and Characterization of Polytopic Protein as a New Vaccine Candidate against Pseudomonas aeruginosa Infections

Introduction and Objectives: Nowadays, P. aeruginosa, the highly regarded opportunistic pathogen, is the leading cause of morbidity and mortality in patients with compromised defenses and chronic diseases such as cystic fibrosis. Due to the emergence of highly resistant strains, at present, aggressive antibiotic therapy is the only choice for management of P. aeruginosa infections. Therefore, the development of novel alternative therapeutics including an effective vaccine, is necessary. Several P. aeruginosa antigens have been tested for vaccine development, including OprF (major outer membrane protein), ExoA (Exotoxin A), and LecB (fucose-binding lectin) which plays an important role in pathogenesis of P. aeruginosa. The aim of the study was to design a multi-epitope vaccine based on OprF, ExoA and LecB proteins and optimize the expression of polytopic construct contains these genes from P. aeruginosa as a vaccine candidate against P. aeruginosa infections. Materials and Methods: In order to design the polytopic construct, we predicted the most probable immunogenic epitopes of OprF, ExoA and LecB using bioinformatics methods. The chimeric gene was introduced into a pET28a vector and expressed in Escherichia coli BL21 and its expression was analyzed by SDS-PAGE and western blotting. Finally, in order to optimize the expression of the recombinant protein, cell density, induction time, growth temperature, IPTG (Isopro- pyl β-D-1-thiogalactopyranoside) concentration were studied. Results: Expression of recombinant fusion protein by E. coli using pET22b vector resulted in production of chimeric protein in high concentration. Optimum condition for recombinant protein expression was determined at OD 600 of 0.8, 0.5mM IPTG, six hours incubation time at 30 ºC and BL21 host. Conclusion: These results suggest that recombinant chimeric protein can be produced in the laboratory and expression can be optimized. Moreover, by purification of recombinant protein and evaluation of its immunogenicity in mice, it can be used as a vaccine candidate against the P. aeruginosa.

Keywords:

P. aeruginosa , OprF , ExoA and LecB , Polytopic vaccine