Protective properties of nontoxic recombinant exotoxin A [domain I-II] against pseudomonas aeruginosa infection

ABSTRACT

Antibiotic resistance and the need for long-term treatments especially for chronic infections necessitate the development of a vaccine against Pseudomonas aeruginosa infection. In this study, recombinant exotoxin A [domains I and II], [ExoA I-II] protein was expressed, purified and its immunological characteristics were evaluated in a mouse model. The genomic DNA was extracted from P. aeruginosa strain PAO1. The DNA encoding for domains I and II of exotoxin A was amplified by PCR and cloned into the pET22b expression vector. The construct was then transformed into E. coli BL21 and the protein expression was evaluated by the SDS-PAGE method. The Ni-NTA affinity chromatography was used for recombinant protein purification. Mice were then immunized subcutaneously on day 0, 21, 42 and 72 with exotoxin A [Domains I, II]. Antibody production was evaluated by the ELISA method. The immunized and control group mice were exposed to an approximate 2 x LD50 [7.5 x 10[7] CFU] of clinical strain of mucoid P. aeruginosa. Sequencing of the cloned gene showed that the sequence of ExoA one-two gene was in accordance with ExoA I-II from P. aeruginosa PAO1. SDS-PAGE analysis indicated the expression of recombinant protein with a molecular weight of 45 KDa. Vaccination with ExoA I-II produced a significant amount of specific IgG antibodies in mice. Also immunization of mice with ExoA one-two increased survival times against intra-peritoneal challenge with an approximate 7.5 x 10[7] CFU [2 x LD50] of clinical strain of P. aeruginosa. Results of this study suggested that recombinant ExoA I-II is a highly immunogenic protein which can be used as a new vaccine candidate against P. aeruginosa