Generation and characterization of a recombinant antibody fragment that binds to the coat protein of grapevine leafroll-associated virus 3.
Pathogen-specific recombinant antibodies have been used to characterize pathogen infections and to engineer resistance in crops. We selected a single-chain antibody fragment (scFvLR3cp-1) specific for the coat protein of grapevine leafroll-associated virus 3 (GLRaV-3), one of the agents of grapevine leafroll (GLR) disease, from a phage display library. The antibody binds specifically to the entire length of GLRaV-3 particles and has a high binding affinity value (K(D)) of 42 nM. The amino acid motif AQEPPRQ located at the N terminus of the GLRaV-3 coat protein was identified as the antibody-binding epitope by PEPSCAN analysis. To evaluate scFv stability in the reducing environment of the plant cell cytosol, transient expression assays were performed using Nicotiana benthamiana as a model plant. Capture ELISA demonstrated that the scFv fragment was produced and retained its antigen-binding capacity in the plant cytosol. Further functional assays showed that scFvLR3cp-1 binds with high specificity to at least four members of the family Closteroviridae. Therefore, the GLRaV-3-specific scFv fragment could be an ideal candidate for mediating broad-spectrum virus resistance if produced in transgenic grapevine plants.
Orecchia
Martin
M
Institute for Molecular Biotechnology (Biology VII), RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany.
Nölke
Greta
G
Saldarelli
Pasquale
P
Dell'Orco
Mariangela
M
Uhde-Holzem
Kerstin
K
Sack
Markus
M
Martelli
Giovanni
G
Fischer
Rainer
R
Schillberg
Stefan
S
D000914:Antibodies, Viral / Q000276:immunology*; D000915:Antibody Affinity; D036022:Capsid Proteins / Q000737:chemistry / Q000276:immunology*; D029381:Closteroviridae / Q000276:immunology*; D003429:Cross Reactions; D000939:Epitopes / Q000276:immunology / Q000302:isolation & purification; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000276:immunology*; D019151:Peptide Library; D010935:Plant Diseases / Q000821:virology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D014026:Nicotiana / Q000378:metabolism*; D027843:Vitis / Q000821:virology*
D000914:Antibodies, Viral; D036022:Capsid Proteins; D000939:Epitopes; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; C055966:immunoglobulin Fv