Rotavirus VP7 epitope mapping using fragments of VP7 displayed on phages.
cDNA copies of the complete porcine rotavirus CRW-8 VP7 gene were randomly digested to fragments of about 30-60 or 30-500 base pairs by DNase1 in the presence of Mn(2+). The fragments were cloned and expressed in a filamentous phage fd-tet-derived vector to create specific-gene-related peptide libraries. Polyclonal antibodies were then used to pan the SGRP libraries for antibody-binding phages. Analysis of the phage isolates revealed that the majority (86%) of them only had a single insert. However, phages displaying composite inserts containing the VP7 antigenic regions A, B, and C, originally defined by neutralising monoclonal antibody escape mutants, were also isolated. Inserts containing A or C region peptide were found to contain extra sequences from the C region, while the B region epitope was linear and had additional sequence from either upstream or downstream. In addition a dominant and possibly non-neutralising VP7 epitope was identified around amino acids 263-270. One of the recreated antigenic epitopes has also been fused to the outer membrane protein A (OmpA) of Escherichia coli and shown to maintain its antigenicity. The results in this study may have significant implication for recreation of conformational epitopes and vaccine development.
Huang
J A
JA
Department of Microbiology and Immunology, University of Melbourne, Parkville, Australia.
Wang
L
L
Firth
S
S
Phelps
A
A
Reeves
P
P
Holmes
I
I
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral; D001435:Bacteriophages / Q000235:genetics*; D002213:Capsid / Q000276:immunology*; D036022:Capsid Proteins; D018604:Epitope Mapping; D015723:Gene Library; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D012401:Rotavirus / Q000276:immunology*; D013552:Swine
D000956:Antigens, Viral; D036022:Capsid Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C050779:VP7 protein, Rotavirus