en Genomics & Proteomics and Medical Biotechnology Genomics & Proteomics and Medical Biotechnology Original Article Original Article <p style="text-align: justify;">The type IV pili (T4P) is a major virulence factor of <em>Pseudomonas aeruginosa</em> (<em>P. aeruginosa</em>) that is associated with primary adhesion, biofilm formation and twitching motility. This study focuses on the introduction of a novel biologically active subunit vaccine derived from the disulfide loop (DSL) of <em>P. aeruginosa</em> pilin. We investigated the expression of the novel PilA in-frame with pET26a vector, which contains three domains, that each domain contains three tandem repeats. The flexible (GGGGS) and (GGGGS)3 linkers were linked between the three tandem repeats and each <em>pilA</em> domain, respectively. &nbsp;The recombinant construct (pET26b/<em>pilA</em>) was transformed and expressed in <em>Escherichia coli</em> BL21 (DE3). The reactivity of specific antiserum against PilA was assessed by ELISA method. The biological activities of this candidate vaccine were evaluated by western blotting, opsonophagocytosis and twitching inhibition assays. The pET26b/<em>pilA</em> plasmid was confirmed by enzymatic digestion. The purified PilA protein was confirmed by immunoblot analysis. The checkerboard titration showed that the optimal dilution of the antibody to react with antigen was 1:8. The results of opsonophagocytosis assay revealed that the antibodies raised against PilA promoted phagocytosis of the PAO1 and 6266E strains, to some extent (17.5% and 16.3%, respectively), so that the twitching inhibition test confirmed this result. &nbsp;Taken together, these are preliminary results based on a first chimerical structure failure in order to induce antibodies that promote the opsonization and eradication of the pathogen. Therefore, the biological activity of the PilA protein showed that it should be introduced with other proteins or target antigens against <em>P. aeruginosa</em> in the future studies.</p> Pseudomonas aeruginosa, type IV pili, disulfide loop, vaccine, pET26b 96 108 http://ijmcmed.org/browse.php?a_code=A-10-1074-1&slc_lang=en&sid=1 Sobhan Faezi Sobhan.faezi@gmail.com 10031947532846009341 10031947532846009341 No Departments of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran. Ahmad Reza Bahrmand Padideh79@yahoo.com 10031947532846009342 10031947532846009342 Yes Departments of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran. Mehdi Mahdavi mahdavivac@gmail.com 10031947532846009343 10031947532846009343 No Departments of Immunology, Pasteur Institute of Iran, Tehran, Iran. Seyed Davar Siadat siadat@pasteur.ac.ir 10031947532846009344 10031947532846009344 No Departments of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran. Iraj Nikokar nikokariraj@yahoo.com 10031947532846009345 10031947532846009345 No Laboratory of Microbiology and Immunology of Infectious Diseases, Para Medicine Faculty, Guilan University of Medical Sciences, Rasht, Iran. Soroush Sardari ssardari@hotmail.com 10031947532846009346 10031947532846009346 No Biotechnology Research Center, Drug Design and Bioinformatics Group, Pasteur Institute of Iran, Tehran, Iran.