Cloning and expression of the immunogenic moiety of Pseudomonas aeruginosa exotoxin A

Abstract

Introduction: Pseudomonas aeruginosa, as an opportunistic microorganism, is a major cause of nosocomial infections worldwide. Exotoxin A (ETA) is an extracellular enzyme that is produced by most clinical strains of P. aeruginosa. Although the pathogenesis of the diseases due to Pseudomonas are complex, clinical and experimental data linking ETA with the morbid and lethal consequences of Pseudomonas infection are accumulating.Materials and methods: An immunogenic 490–bp DNA segment including translocation domain plus 1b domain of the ETA from P. aeruginosa strain PAO1 were reproduced by PCR. The PCR product was cloned in E.coli DH5α and expressed in E.coli BL21 using recombinant pET28a vector. The cloned polypeptide was found to have an electrophoretic mobility in sodium dodecyle sulfate- polyacrylamide gels (SDS -PAGE) of 18kDa.Results: PCR and colony PCR results approved the cloning of immunogenic moiety of exotoxin A. Analysis of the location of cloned polypeptide by SDS- PAGE electrophoresis revealed that it was exported by E.coli into the bacterial periplasmic space.Discussion and conclusion: Since the whole toxin is not necessary for enhancing the immune responses and this recombinant polypeptide has antigenic qualities, so it may serve as a useful vaccine to prevent Pseudomonas infections.

Keywords

• Pseudomonas aeruginosa PAO1
• Cloning
• Exotoxin A
• Immunogenic moiet