Use of a gene-targeted phage display random epitope library to map an antigenic determinant on the bluetongue virus outer capsid protein VP5.
We describe the use of a gene-targeted random epitope library for the mapping of antigenic determinants. A DNA clone encoding the target antigen was digested randomly with DNase I to generate a population of DNA fragments of different sizes and sequences. After size fractionation, small DNA fragments (100-200 bp) were isolated and cloned into the phage expression vector fUSE2 to form an expression library displaying random polypeptide sequences as fusion proteins at the N terminus of the phage gene III protein. This library, termed a gene-targeted random epitope library to distinguish it from totally random synthetic epitope libraries, was then screened by affinity selection for recombinant phages which were specifically bound by the antibody of interest. Using this approach, we have mapped a monoclonal antibody (mAb)-defined epitope on the bluetongue virus outer capsid protein VP5. This epitope is not accessible on the intact virus surface, but is recognised by the immune system of sheep and cattle during virus infection. Although the example given here utilised a DNA fragment of known sequence and the library was screened for a mAb-defined epitope, the strategy described should be equally applicable to genes of unknown sequence and for screening of epitopes using polyclonal antibodies. The approach can also be extended to identify immunodominant epitope from much more complex genome-targeted random epitope library for virus, bacteria and eukaryotic organisms. Other applications of recombinant phages expressing defined immunodominant epitopes include serodiagnosis and vaccine development.
Wang
L F
LF
CSIRO Australian Animal Health Laboratory, Geelong, Victoria.
Du Plessis
D H
DH
White
J R
JR
Hyatt
A D
AD
Eaton
B T
BT
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology* / Q000648:ultrastructure; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D001820:Bluetongue virus / Q000235:genetics / Q000276:immunology* / Q000648:ultrastructure; D002213:Capsid / Q000235:genetics / Q000276:immunology* / Q000648:ultrastructure; D036022:Capsid Proteins; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D005822:Genetic Vectors / Q000235:genetics; D017979:Gold Colloid; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D036022:Capsid Proteins; D000939:Epitopes; D017979:Gold Colloid; D010446:Peptide Fragments