Isolation and characterization of single-chain Fv genes encoding antibodies specific for Drosophila Poxn protein.

ABSTRACT

The usefulness of intrabodies as specific inhibitors of gene function has been extensively demonstrated in cell culture assays. However, very few experiments have been conducted with intrabodies expressed in whole organisms. To evaluate the intrabody technology in Drosophila, we focused on poxn protein, since its effects can be easily studied. We purified the recombinant poxn protein. We next isolated three single-chain variable fragments (scFv) which specifically recognize poxn protein. Two scFvs, designated alpha-Poxn2 and alpha-Poxn4, react with both denatured and native Poxn with half maximal inhibition values of 100 nM and 40 nM, respectively. The alpha-Poxn5 scFv also recognizes denatured Poxn but either does not recognize native Poxn or its half maximal inhibition value for native Poxn is high.