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 chains of monoclonal antibodies (mAbs) to beet necrotic yellow vein virus (BNYVV) coat protein (cp) or the 25 kDa nonstructural protein (P25) were 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 the sequences encoding the pectate lyase B (PelB) or the phytohemagglutinin (PHA) signal peptides in the vector constructs or they were targeted to the cytoplasm by omitting a signal peptide-encoding sequence from 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 suspension cultures, but were retained in the cells. The amount of expression of scFv in the best expressing plants was at least as high as in bacterial 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-specific scFv had near-normal growth whereas the three plants expressing P25-specific scFv grew poorly and did not form roots.
Fecker
L F
LF
Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Biochemie und Pflanzenvirologie, Braunschweig, Germany.
Kaufmann
A
A
Commandeur
U
U
Commandeur
J
J
Koenig
R
R
Burgermeister
W
W
D000914:Antibodies, Viral / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D002213:Capsid / Q000276:immunology* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D010942:Plant Viruses / Q000276:immunology*; D010947:Plants, Toxic; D021382:Protein Sorting Signals / Q000378:metabolism; D012328:RNA Viruses / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014026:Nicotiana; D017361:Viral Nonstructural Proteins / Q000235:genetics
D000914:Antibodies, Viral; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C055966:immunoglobulin Fv