Antikörperfragmente und synthetische Antigene für die Diagnose von Viruserkrankungen der Pflanzen
The detection of Beet necrotic yellow vein virus (BNYVV) in stored sugar beets by means of monoclonal antibodies or antibody single chain fragments (scFv) often poses problems, because the immunodominant C-terminal epitope of the viral coat protein is readily lost due to proteolysis. Recombinant bacteria which produce scFv specific for protease-stable BNYVV epitopes were selected from two naïve phage display libraries. Fusion proteins of the scFv with a human IgG kappa chain (CL) or with a modified bacterial alkaline phosphatase (AP/S) allow the ELISA detection of BNYVV even in stored sugar beets with a sensitivity comparable to that achieved with polyclonal antibodies. The production of synthetic antigens of BNYVV and Beet soil borne virus (BSBV) for use as diagnostic standards were investigated in two different systems. After N-terminal fusion of different BNYVV epitopes to the coat protein (CP) of Potato virus X (PVX) all recombinant (r)PVX elicited PVX wild-type-specific symptoms on N. benthamiana. Detection of presented BNYVV epitopes with specific monoclonal antibodies was successful in ELISA and Western Blots as well as in immunogold electron microscopy. The stability of the foreign epitope sequences introduced into the PVX CP was studied by means of serial passages on systemically infected N. benthamiana. After five passages mutations and deletions were found in the inserted foreign epitope sequence, especially amino acids with positive charge were affected. Comparison of isoelectric points (pI) and charge of the rCP showed, that the initial high pIs of the rCPs were changed by the observed mutations close to that of wild-type CP. In addition to rPVX particles, “virus-like particles” (VLPs) from the Ty1 yeast retrotransposons were examined for the production of synthetic antigens. Cytoplasmatic expression of VLP constructs was successful in Escherichia coli and in Pichia pastoris. The particle morphology of VLPs produced in E. coli or P. pastoris corresponded with wild-type Ty1-VLPs from Saccharomyces cerevisiae. Apart from BNYVV epitopes also a novel, through peptide phage display identified BSBV epitope was successfully expressed in E. coli. This on VLPs presented BSBV epitope as well as all BNYVV epitopes were detected in ELISA and Western Blots with specific monoclonal antibodies.
info:eu-repo/classification/ddc/570, Biowissenschaften, Biologie
Networked Digital Library of Theses and Dissertations
Publikationsserver der RWTH Aachen University