Generation and characterization of functional recombinant antibody fragments against tomato yellow leaf curl virus replication-associated protein.
Tomato yellow leaf curl virus (TYLCV) is a complex of geminivirus species prevalent in the tropics and sub-tropics, which causes severe diseases in economically important crops such as tomato. Conventional strategies for disease management have shown little success and new approaches based on genetic engineering need to be considered. We generated two single-chain variable fragment antibodies (scFv-ScRep1 and scFv-ScRep2) that bound strongly to continuous epitopes within the TYLCV replication-associated protein (Rep). The TYLCV-Ir C1 gene (encoding Rep) was expressed as glutathione-S-transferase (GST) and maltose-binding protein (MBP) fusions. Purified MBP-Rep was used to immunize mice allowing the construction of naïve and pre-immunized scFv phage display libraries. Immunoassays showed that scFv-ScRep1 recognized an N-terminal epitope of Rep, whereas scFv-ScRep2 recognized a more central epitope. This is the first successful production of scFv antibodies against a geminivirus Rep, the initial step in the production of transgenic plants with resistance to TYLCV.
Safarnejad
M R
MR
Institute of Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringerweg 1, DE-52074-Aachen, Germany. rsafarnejad@molbiotech.rwth-aachen.de
Fischer
R
R
Commandeur
U
U
D000595:Amino Acid Sequence; D000818:Animals; D053839:Begomovirus / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D018506:Gene Expression Regulation, Plant; D015967:Gene Expression Regulation, Viral; D015723:Gene Library; D005821:Genetic Techniques; D018551:Solanum lycopersicum / Q000235:genetics / Q000276:immunology / Q000821:virology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000276:immunology / Q000821:virology*; D018515:Plant Leaves / Q000276:immunology / Q000821:virology; D046549:Plantibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000276:immunology / Q000821:virology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D004279:DNA, Viral; D046549:Plantibodies; D011994:Recombinant Proteins; D014760:Viral Fusion Proteins