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.
Ogunniyi
Abiodun D
AD
Department of Molecular Biosciences, Adelaide University, Adelaide, SA 5005, Australia. david.ogunniyi@adelaide.edu.au
Kotlarski
Ieva
I
Morona
Renato
R
Manning
Paul A
PA
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