Epitope analysis of the FanC subunit protein of the K99 (F5) fimbriae of enterotoxigenic Escherichia coli using a recombinant fusion technique.
We have used a recombinant approach to characterise the B- and T-cell epitopes of FanC, the major subunit polypeptide of K99 (F5) fimbriae of enterotoxigenic Escherichia coli strains. This involved the fusion of FanC and its carboxy-terminal truncated derivatives to a reporter, the E. coli alkaline phosphatase (PhoA), generating stable, recombinant fusions. The B-cell epitopes of FanC were characterised by Western blotting of FanC::PhoA fusion proteins with a polyclonal mouse antiserum directed against K99 fimbrial antigen, and with a panel of monoclonal antibodies generated to the K99 antigen. An attempt to characterise the T-cell epitopes of the fimbrial subunit was made by standard in vitro T-cell proliferation assay. Our results suggest that the B-cell epitopes of FanC are likely to be continuous, with a potentially immunodominant epitope at the carboxy-terminus. However, T-cell proliferation assays with the FanC::PhoA fusion proteins did not indicate any immunodominant T-cell epitope(s). We hypothesise that fusion of FanC peptides to PhoA had resulted in altered folding of the peptides for antibody and T-cell recognition, highlighting the potential problems and drawbacks of the recombinant fusion technique in defining the epitopes of certain proteins.
Department of Molecular Biosciences, Adelaide University, Adelaide, SA 5005, Australia. david.ogunniyi@adelaide.edu.au
Abiodun D
AD
Ogunniyi
Ieva
I
Kotlarski
Renato
R
Morona
Paul A
PA
Manning
D000469:Alkaline Phosphatase; D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry* / Q000235:genetics; D001402:B-Lymphocytes / Q000276:immunology; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000235:genetics; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D029968:Escherichia coli Proteins; D005260:Female; D010861:Fimbriae, Bacterial / Q000737:chemistry / Q000276:immunology; D016056:Immunodominant Epitopes / Q000737:chemistry / Q000235:genetics; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D017510:Protein Folding; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology
D000954:Antigens, Surface; D001427:Bacterial Toxins; D000939:Epitopes; D029968:Escherichia coli Proteins; D016056:Immunodominant Epitopes; C031429:K99 antigen; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D018844:Cyclin-Dependent Kinases; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli