Recombinant Production and Immunogenicity Assessment of Pseudomonas aeruginosa Exotoxin A and PilA Fragments in a Murine Model

Naimi Shamel, Nasrin; Fathi Najafi, Mohsen; Moghbeli, Majid; Dolatabadi, Samaneh

doi:10.22092/ari.2026.370684.3903

Recombinant Production and Immunogenicity Assessment of Pseudomonas aeruginosa Exotoxin A and PilA Fragments in a Murine Model

Articles in Press

Document Type : Original Articles

Authors

¹ Department of microbiology, Damghan Branch, Islamic Azad University, Damghan, Iran.

² Razi Vaccine and Serum Research Institute, Agriculture Research, Education and Extension Organization (AREEO), Mashhad, Iran.

³ Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran.

⁴ Department of Microbiology, Neyshabur Branch, Islamic Azad University, Neyshabur, Iran.

10.22092/ari.2026.370684.3903

Abstract

Introduction: Exotoxin A and pili are key virulence factors of Pseudomonas aeruginosa, and their neutralization is a promising strategy to prevent or attenuate infections caused by this pathogen.
Objective: To evaluate the immunogenicity of recombinant exotoxin A and pilin A (PilA) fragments as conserved antigens against different P. aeruginosa serotypes.
Materials and Methods: Genomic DNA was extracted from P. aeruginosa using the phenol–chloroform method, and exoA and pilA genes were amplified by PCR with specific primers. Purified PCR products were cloned into the pGEM vector and transformed into Escherichia coli DH5α. After confirmation by restriction digestion with BamHI and XhoI, the inserts were subcloned into the pET 26b expression vector and introduced into E. coli BL21 (DE3). Recombinant proteins were expressed, purified, and analyzed by SDS PAGE, which showed bands of approximately 21–23 kDa for EXO and PILI. Expression of pET 26b/PilA in E. coli BL21 (DE3) yielded about 12% (v/v) protein after 5 h of IPTG induction. Purified proteins were injected into BALB/c mice with experimental burn injuries. Humoral immune responses were assessed by antigen specific ELISA, and protein expression and antigenicity were confirmed by dot blot and Western blot.
Results: Immunization with recombinant exotoxin A and PilA induced robust antigen specific antibody responses in mice, indicating strong immunogenicity.
Conclusion: Recombinant exotoxin A and PilA produced in this study showed promising potential as vaccine candidates against P. aeruginosa. Immunization with these conserved components may offer an effective strategy for preventing infections caused by multiple P. aeruginosa serotypes.

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