1- Departments of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran. 2- Departments of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran. , Padideh79@yahoo.com 3- Departments of Immunology, Pasteur Institute of Iran, Tehran, Iran. 4- Laboratory of Microbiology and Immunology of Infectious Diseases, Paramedicine Faculty, Guilan University of Medical Sciences, Guilan, Iran. 5- Biotechnology Research Center, Drug Design and Bioinformatics Group, Pasteur Institute of Iran, Tehran, Iran. 6- Departments of Microbiology and Surgery, College of Medicine, University of Cincinnati, and Shriners Burns Institute, Cincinnati, Ohio, USA.
Abstract: (8422 Views)
Pseudomonas aeruginosa as an opportunistic pathogen is a significant cause of acute and chronic infections in patients with compromised defenses. This bacterium is motile via a single polar flagellum made of polymerized flagellin subunits differentiated into two major serotypes: A and B. flagellin plays an important role as a virulence factor in the adhesion, colonization and invasion of P. aeruginosa into host epithelial cells. To develop a functional vaccine that can be used in practical application to prevent and treat infection, type B-flagellin was produced as recombinant protein. In this work, the fliC gene was introduced into a pET28a vector and expressed in Escherichia coli BL21 (DE3). The expressed recombinant protein was purified by a modified method without sonication using a HisTrap affinity column. The functional activities of produced flagellin were confirmed by ELISA, western blot analysis, motility inhibition assay and opsonophagocytosis test. The purification process of the type B-flagellin was lead to a high yield. The produced recombinant type B-flagellin showed high biological activity in all of these standard assays. In conclusions, this report provides the new protocol to efficiently obtain the type B-flagellin with high biological activity and immunogenicity. This immunogen can be introduced as an adjuvant or vaccine in the future study.
Faezi S, Bahrmand A R, Mahdavi M, Siadat S D, Nikokar I, Sardari S et al . High Yield Overexpression, Refolding, Purification and Characterization of Pseudomonas aeruginosa Type B-Flagellin: An Improved Method Without Sonication. Int J Mol Cell Med 2016; 5 (1) :37-48 URL: http://ijmcmed.org/article-1-437-en.html