Construction and immunogenic properties of a chimeric protein comprising CfaE, CfaB and LTB against Enterotoxigenic Escherichia coli

Biologicals. 2016 Nov;44(6):503-510. doi: 10.1016/j.biologicals.2016.09.003. Epub 2016 Oct 10.

Abstract

ETEC (Enterotoxigenic Escherichia coli) is a major cause of diarrhea in developing countries and children. ETEC has two virulence factors including colonization factors antigen (CFA) and labile enterotoxins (LTs). CFA/I consists the major pilin subunit CfaB and a minor adhesive subunit, CfaE. In this study a tripartite fusion protein containing CfaB, CfaE and LTB was designed. In silico analysis of the tertiary structure of the chimeric protein showed a protein with three main domains linked together with linkers. Linear and conformational B-cell epitopes were identified. A chimera consisting cfaB, cfaE and ltB(BET)was then synthesized with E. coli codon bias in pUC57 and sub cloned into pET32 vector. Recombinant protein was expressed and purified by affinity chromatography and confirmed by western blotting. Mice were immunized with recombinant protein and the antibody titer and specificity of the sera were analyzed by ELISA. The efficiency of the immune sera against ETEC was evaluated by binding assay and GM1-ELISA. VaxiJen analysis of the protein showed high antigenicity. Post-immune sera contained high titers of anti-BET IgG. Pretreatment of ETEC cells with sera from immunized mice decreased their ability to adhere to cells of the human colon adenocarcinoma cell line HT29.

Keywords: Chimeric protein; Colonization factors; Enterotoxigenic Escherichia coli; LT toxin.

MeSH terms

  • Animals
  • Bacterial Toxins* / chemistry
  • Bacterial Toxins* / genetics
  • Bacterial Toxins* / immunology
  • Cell Line, Tumor
  • Computer Simulation
  • Enterotoxigenic Escherichia coli* / genetics
  • Enterotoxigenic Escherichia coli* / immunology
  • Enterotoxins* / chemistry
  • Enterotoxins* / genetics
  • Enterotoxins* / immunology
  • Epitopes, B-Lymphocyte* / chemistry
  • Epitopes, B-Lymphocyte* / genetics
  • Epitopes, B-Lymphocyte* / immunology
  • Escherichia coli Proteins* / chemistry
  • Escherichia coli Proteins* / genetics
  • Escherichia coli Proteins* / immunology
  • Escherichia coli Vaccines* / chemistry
  • Escherichia coli Vaccines* / genetics
  • Escherichia coli Vaccines* / immunology
  • Fimbriae Proteins* / chemistry
  • Fimbriae Proteins* / genetics
  • Fimbriae Proteins* / immunology
  • Humans
  • Mice
  • Mice, Inbred BALB C
  • Protein Domains
  • Recombinant Fusion Proteins* / chemistry
  • Recombinant Fusion Proteins* / genetics
  • Recombinant Fusion Proteins* / immunology

Substances

  • Bacterial Toxins
  • CfaE protein, E coli
  • Enterotoxins
  • Epitopes, B-Lymphocyte
  • Escherichia coli Proteins
  • Escherichia coli Vaccines
  • Recombinant Fusion Proteins
  • heat-labile enterotoxin, E coli
  • Fimbriae Proteins