تخلیص و ارزیابی خصوصیات ایمونولوژیک دومينهاي I وII اگزوتوكسينA سودوموناس آئروژينوزا

Title

Purification and Immunological Characterization Evaluation of Recombinant Exotoxin A (Domains I, II) Protein isolated from Pseudomonas aeruginosa

English Abstract

Background & Objectives: Pseudomonas aeruginosa (P.aeruginosa) is a commonly isolated pathogen from nosocomial infections worldwide this bacterium has evolved several antibiotic resistance mechanisms and antibiotics have failed in treatment of P. aeruginosa infections. Hence, immunoprophlaxy and immunotherapy may be an effective method for control and treatment of P. aeruginosa infections. In the present study, the immunological properties of exotoxin A (domain I,II) was evaluated Methods: The DNA encoding for domains I and II of Exotoxin A was amplified by PCR and cloned in PET22b expression vector. The construct was then transformed in to E. coli BL21 and the protein expression was evaluated by SDS-PAGE method. The Ni-NTA affinity chromatography was used for recombinant protein purification. The identity of recombinant protein was confirmed by western blotting using anti exotoxin A antibody (Sigma). The reactivity of recombinant exotoxin A with sera from P.aeruginosa infected patients was used for evaluation of immunogenicity. Results: The electrophoresis of PCR products on 1% agarose showed 1722 bp band that was in accordance with ExoA (domain I- II) gene size. Sequencing of cloned gene showed that the sequence of ExoA I-II gene was in accordance with Exo A from P.aeruginosa PAO1. SDS-PAGE analysis indicated expression of recombinant protein with a molecular weight of approximately 45kDa. Western blotting with anti-exotoxin A antibody (Sigma) verified the identity of recombinantly expressed protein. Immunologic analysis using P.aeruginosa infected patients sera showed that the exoA I-II is highly immunogenic protein that identified by patients sera. Conclusion: Results of this study showed that recombinant exotoxinA (domainI, II) can be tested as a sub-unit vaccine candidate in P. aeruginosa infections.

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