EXPRESSION OF SINGLE-CHAIN ANTIBODY FRAGMENTS (SCFV) SPECIFIC FOR BEET NECROTIC YELLOW VEIN VIRUS COAT PROTEIN OR 25 KDA PROTEIN IN ESCHERICHIA-COLI AND NICOTIANA-BENTHAMIANA

The coding sequences for the variable regions of heavy and light chain s of monoclonal antibodies (mAbs) to beet necrotic yellow vein virus ( BNYVV) coat protein (cp) or the 25 kDa nonstructural protein (P25) wer e cloned into the pCOCK vector and expressed as single-chain antibody fragments (scFv) in Escherichia coli. For expression in higher plants the scFv were targeted either to the secretory pathway by including th e sequences encoding the pectate lyase B (PelB) or the phytohemaggluti nin (PHA) signal peptides in the vector constructs or they were target ed to the cytoplasm by omitting a signal peptide-encoding sequence fro m the constructs. The scFv were detected mainly in plants in which the PHA signal peptide had been used for targeting demonstrating for the first time the usefulness of this peptide for enabling scFv expression in plants. The scFv were not secreted into the culture fluids of susp ension cultures, but were retained in the cells. The amount of express ion of scFv in the best expressing plants was at least as high as in b acterial culture supernatants. In a dot blot immunoassay, 0.4 ng BNYVV cp or 0.8 ng P25 were detected by the respective scFv either from E. coli or from plants. The majority of the 21 plants expressing cp-speci fic scFv had near-normal growth whereas the three plants expressing P2 5-specific scFv grew poorly and did not form roots.