E Choubini , Mr Asadi Karam, A Khorshidi, M Habibi , A Ghasemi , S Bouzari ,
Volume 3, Issue 3 (11-2016)
Abstract
Introduction: Pathogenic strains of Proteus mirabilis have important roles in urinary tract infection. Proteus toxic agglutinin (Pta) is amongst the most important virulence factors of P. mirabilis. This protein has a conserved sequence present in all the strains which could be evaluated as a novel vaccine target against them. The aims of the current study were the expression, purification and characterization of a truncated Pta protein of P. mirabilis strain HI4320 as well as the bioinformatics analysis of the truncated protein. Methods: The passenger domain of pta genes in P. mirabilis was evaluated by bioinformatics studies. The selected domain (residues 207-730) was amplified by PCR and cloned into pET28a expression vector. The Pta was expressed in BL21 (DE3) host and purified by Ni-NTA resin. The analyses of the purified protein were performed by SDS-PAGE and Western blotting. Results: The bioinformatics studies predicted the appropriateness of the passenger domain of Pta protein in terms of conservation, stability and cell-surface exposure. The length of PCR fragment of truncated form of pta gene was ~1500 bp. The cloning and expression of the truncated pta gene was successfully performed using pET28a-BL21 (DE3) system. Analyses of the purified Pta by SDS-PAGE and Western blotting confirmed the purification of a ~60 kDa His-tagged polypeptide. Conclusion: The high frequency of P. mirabilis infection, especially in patients with abnormalities in their urinary tracts and also the rising of antibiotic resistance among the strains of this pathogen point to the need for effective controlling measures against them. In this regard, the passenger domain of Pta could be considered as a vaccine target. The efficacy and in-vivo immunogenicity of this purified protein is currently under study.
Sheida Hedayat, Mehri Habibi, Reza Hosseini Doust, Mohammad Reza Asadi Karam,
Volume 10, Issue 1 (6-2023)
Abstract
Introduction: Uropathogenic Escherichia coli (UPEC) is the main cause of urinary tract infections (UTIs). Increasing antibiotic resistance among UPEC isolates complicate UTI treatment in the future. Finding alternative approaches against UPECs seems necessary. Despite many efforts to develop a vaccine for UPEC, there is no yet effective vaccine against the bacteria. Methods: Designing a multi-epitope vaccine based on the main UPEC virulence factors can be an effective strategy for prevention of UTI. In the previous study, three important proteins from UPEC strains including FimH, FyuA and CNF-1 were selected to design a multi-epitope antigen which was used for production of polyclonal antibody in rabbits. In the present study, the collected sera were used for evaluating the sensitivity, specificity, cell adherence, and biofilm inhibition of the developed polyclonal antibody. Results: ELISA results showed high binding activity of the rabbit polyclonal antibody against the multi-epitope protein even in low antibody titers. In addition, the polyclonal antibody showed antigenic specificity against the multi-epitope protein and UPEC UTI89 strain. Cross-reactivity of the polyclonal antibody was observed with Klebsiella pneumonia. Bacterial adhesion was reduced significantly in the presence of antibody, compared to the controls. Conclusion: The generated polyclonal antibody significantly reduced in vitro biofilm formation of UTI89 strain while did not significantly affect the biofilm degradation. These results highlight the potential of the designed multi-epitope protein as a promising vaccine candidate for the prevention of UTI caused by UPEC.
