An LTB-entrapped protein in PLGA nanoparticles preserves against enterotoxin of enterotoxigenic Escherichia coli

Objective(s): Enterotoxigenic Escherichia coli (ETEC) is known as the most common bacterial causes of diarrheal diseases related to morbidity and mortality. Heat-labile enterotoxin (LT) is a part of major virulence factors in ETEC pathogenesis. Antigen entrapment into nanoparticles (NPs) can protect them and enhance their immunogenicity.Materials and Methods: In the present study, recombinant LTB protein was expressed in E. coli BL۲۱ (DE۳) and purified by an Ni-NTA agarose column. The protein was entrapped in PLGA polymer by the double emulsion method. NPs were characterized physicochemically and the protein release from the NPs was evaluated. ELISA assay was performed for investigation of raised antibody against the recombinant protein in mice. The anti-toxicity and anti-adherence attributes of the immune sera against ETEC were also evaluated.Results: It showed the successful cloning of a ۳۱۳ bp DNA fragment encoding LTB protein in the pET۲۸a vector. Over-expression in BL۲۱ (DE۳) led to the formation of corresponding ۱۵.۵ kDa protein bands in the SDS-PAGE gel. Western blotting by using anti-CTX confirmed the purified LTB. Protein-entrapped NPs had a spherical shape with the size of ۲۳۸ nm mean diameter and ۸۵% entrapment efficiency.  Immunological analyses showed the production of a high titer of specific IgG antibody in immunized animals. The neutralizing antibody in the sera of immunized animals was approved by GM۱ binding and Ileal loop assays.Conclusion: The results indicate the efficacy of the entrapped LTB protein as an effective immunogen which induces the humoral responses.

Keywords:

Enterotoxigenic Escherichia coli , Heat-labile enterotoxin Immunization , Nanoparticle , PLGA