Construction, expression, purification and characterization of secretin domain of PilQ and triple PilA-related disulfide loop peptides fusion protein from Pseudomonas aeruginosa

Abstract

<strong><em>Objective(s)</em></strong>: Infection with <em>Pseudomonas aeruginosa</em> has been a long-standing obstacle for clinical therapy due to the complexity of the genetics and pathogenesis, as well for widespread resistance to antibiotics, thus attaching great importance to explore effective vaccines for prevention and treatment. This paper focuses on the introduction of novel <em>Pseudomonas aeruginosa</em> type IV pili (T4P)-based fusion protein containing the secretin domain of PilQ and tandem PilA-related peptides.<br /> <strong><em>Materials and Methods: </em></strong>We surveyed the expression of the PilQ_380-705<em>-</em>PilA fusion protein in-frame with pET26b vector in which a rigid linker was used between two polypeptides and flexible linkers were inserted between the three tandem repeats and each <em>pilA</em> domain<strong>s</strong>. The transformants were expressed in <em>Escherichia coli</em> BL21. The reactivity of specific antisera to the fusion protein was assessed by ELISA. The biological activities of this candidate vaccine were evaluated by western blotting, opsonophagocytosis, and twitching inhibition assays.<br /> <strong><em>Results:</em></strong> The fusion protein was purified in high yield by osmotic shock method using HisTrap affinity column. The protein was confirmed by immunoblot analysis. The checkerboard titration showed that the optimal dilution of the antibody to react with antigen is 1:128. Results of opsonophagocytosis assay revealed that the antibodies elevated to the fusion protein promoted phagocytosis of the PAO1 and 6266E strains, so that the twitching immobilization test confirmed these results.<br /> <strong><em>Conclusion:</em></strong> Due to excellent killing activity mediated by opsonic antibodies and efficient immobilization of the strains, it seems that PilQ_380-705-PilA fusion protein could be a reliable candidate vaccine against <em>P. aeruginosa</em> infection.