USE OF A GENE-TARGETED PHAGE DISPLAY RANDOM EPITOPE LIBRARY TO MAP ANANTIGENIC 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 ant igen was digested randomly with DNase I to generate a population of DN A fragments of different sizes and sequences. After size fractionation , small DNA fragments (100-200 bp) were isolated and cloned into the p hage expression vector fUSE2 to form an expression library displaying random polypeptide sequences as fusion proteins at the N terminus of t he phage gene III protein. This library, termed a gene-targeted random epitope library to distinguish it from totally random synthetic epito pe libraries, was then screened by affinity selection for recombinant phages which were specifically bound by the antibody of interest. Usin g this approach, we have mapped a monoclonal antibody (mAb)-defined ep itope on the bluetongue virus outer capsid protein VP5. This epitope i s not accessible on the intact virus surface, but is recognised by the immune system of sheep and cattle during virus infection. Although th e example given here utilised a DNA fragment of known sequence and the library was screened for a mAb-defined epitope, the strategy describe d should be equally applicable to genes of unknown sequence and for sc reening 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 eukary otic organisms. Other applications of recombinant phages expressing de fined immunodominant epitopes include serodiagnosis and vaccine develo pment.