Expression of a hybrid protein composed of several antigensfrom uropathogenic Escherichia coli in Lactococcus lactis andconfirmation of its expression on the surface of the bacteria

Background and Aim : Uropathogenic Escherichia coli (UPEC) are the most frequent pathogencausing urinary tract infections (UTIs). Nowadays, emerging of antibiotic resistance among UPECisolates is considered as an important challenge, making the development of an efficacious vaccineagainst these infections more urgent. Lactic acid bacteria (LAB) such as Lactococcus lactis byinduction the humoral response especially induction of mucosal antibody against the appliedantigen can be used as the carrier in the vaccine combinations. Therefore, in the current study, weexpressed a hybrid protein composed of virulence factors of UPEC including FimH adhesin oftype ۱ pili, cytotoxic necrotizing factor type ۱ (CNF۱) and iron scavenger receptor FyuA inlactococcus system and then confirmed its expression on the surface of bacteria by differentmethods.Methods : In this study, a multi-epitope composed of proteins FimH, FyuA and CNF-۱ wasdesigned. The synthesized gene was ligated into the pT۱NX plasmid and then recombinantplasmids were transformed into the Lactococcus lactis MG۱۳۶۳ strain. The recombinant plasmidswere screened using PCR, enzymatic digestion, and sequencing. The expression of the hybrid inLactococcus lactis was evaluated by SDS-PAGE and Western blot. Immunofluorescencemicroscopy and whole-cell ELISA were used to confirm cell surface display of the multi-epitopeon L. lactis.Results : After synthesis of the hybrid gene, its transformation into the Lactococcus lactis wereperformed. After culture on M۱۷ medium, the recombinant clones containing the gene wereselected. Analysis of protein expression by SDS-PAGE and western blot showed the expressionof this protein with a size of ۶۶ KD. Evaluation of protein expression on the surface of Lactococcuslactis by immunofluorescent showed strong fluorescent on the surface of recombinant bacteria.The results of the ELISA method based on the whole bacterial cell showed that the recombinantbacteria expressing the protein showed a higher absorption rate at OD۴۵۰nm compared to theLactococcus lactis carrying the vector alone.Conclusion : Despite the expression of the hybrid protein on surface of Lactococcus lactis, it isable to induce the immune response against the designed protein. Thus, we are going to evaluatethe immunogenicity of the induced responses in animal model.

کلیدواژه ها:

Lactococcus lactis ، Uropathogenic Escherichia coli ، urinary tract infections ، immunofluorescent ، ELISA