ABSTRACT
OBJECTIVES: Enterotoxigenic Escherichia coli (ETEC), a major cause of diarrhea in children under 5, is an important agent for traveler's diarrhea. Heat-labile enterotoxin (LT) and colonization factors (CFs) are two main virulence mechanisms in ETEC. CS6 is one of the most prevalent CFs consisting of two structural subunits viz., CssA, CssB, necessary for attachment to the intestinal cells. METHODS: In the present research, a chimeric trivalent protein composed of CssB, CssA and LTB was constructed. The chimeric gene was synthesized with codon bias of E. coli for enhanced expression of the protein. Recombinant proteins were expressed and purified. Mice were immunized with the recombinant protein. The antibody titer and specificity of the immune sera were analyzed by ELISA and Western blotting. Efficiency of the immune sera against ETEC was evaluated. RESULTS: Antibody induction was followed by immunization of mice with the chimeric protein. Pretreatment of the ETEC cells with immunized animal antisera remarkably decreased their adhesion to Caco-2 cells. DISCUSSION: The results indicate efficacy of the recombinant chimeric protein as an effective immunogen, which induces strong humoral response as well as protection against ETEC adherence and toxicity. .
Subject(s)
Animals , Female , Mice , Antigens, Bacterial/genetics , Chimerin Proteins/immunology , Enterotoxigenic Escherichia coli/genetics , Escherichia coli Infections/microbiology , Escherichia coli Proteins/genetics , Blotting, Western , Chimerin Proteins/chemistry , Enterotoxigenic Escherichia coli/immunology , Escherichia coli Infections/immunology , Mice, Inbred BALB CABSTRACT
Fusion of two genes at DNA level produces a single protein, known as a chimeric protein. Immunotoxins are chimeric proteins composed of specific cell targeting and cell killing moieties. Bacterial or plant toxins are commonly used as the killing moieties of the chimeric immunotoxins. In this investigation, the catalytic domain of Shiga-like toxin [A1] was fused to human granulocyte macrophage colony stimulating factor [hGM-CSF] gene and the fused gene was then expressed using an expression vector containing arabinose promoter. The protein thus obtained could be recognized by two different ELISA system designed for detection of hGM-CSF and Shiga-toxin and reconfirmed by Western-blot. The recognition of the chimeric protein by specific antibodies could be indicative of the proper form of the protein, which justifies further steps to be taken to evaluate the potential effects of the chimeric protein