Author/Authors :
-، - نويسنده Department of Bacteriology, Faculty of Medical sciences, Tarbiat Modares University, Tehran, IR Iran Tanomand, Asghar , -، - نويسنده Department of Bacteriology, Faculty of Medical sciences, Tarbiat Modares University, Tehran, IR Iran Najar Peerayeh, Shahin , -، - نويسنده Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, IR Iran Farajnia, Safar , -، - نويسنده Department of Immunology and Immunology Research Center, Faculty of Medical Sciences, Tabriz University of Medical Sciences, Tabriz, IR Iran Majidi, Jafar
Abstract :
Background: Antibiotic resistance and the need for long-term treatments especially for chronic infections necessitate the developmentof a vaccine against Pseudomonas aeruginosa infection.Objectives: In this study, recombinant exotoxin A (domains I and II), (ExoA I-II) protein was expressed, purifid and its immunologicalcharacteristics were evaluated in a mouse model.Materials and Methods: The genomic DNA was extracted from P. aeruginosa strain PAO1. The DNA encoding for domains I and II ofexotoxin A was amplifid by PCR and cloned into the pET22b expression vector. The construct was then transformed into E. coli BL21 andthe protein expression was evaluated by the SDS-PAGE method. The Ni-NTA affity chromatography was used for recombinant proteinpurifiation. Mice were then immunized subcutaneously on day 0, 21, 42 and 72 with exotoxin A (Domains I, II). Antibody production wasevaluated by the ELISA method. The immunized and control group mice were exposed to an approximate 2 × LD50 (7.5 × 107 CFU) of clinicalstrain of mucoid P. aeruginosa.Results: Sequencing of the cloned gene showed that the sequence of ExoA I-II gene was in accordance with ExoA I-II from P. aeruginosaPAO1. SDS-PAGE analysis indicated the expression of recombinant protein with a molecular weight of 45 KDa. Vaccination with ExoA I-IIproduced a signifiant amount of specifi IgG antibodies in mice. Also immunization of mice with ExoA I-II increased survival timesagainst intra-peritoneal challenge with an approximate 7.5 × 107 CFU (2 × LD50) of clinical strain of P. aeruginosa.Conclusions: Results of this study suggested that recombinant ExoA I-II is a highly immunogenic protein which can be used as a newvaccine candidate against P. aeruginosa.
