Publikationsserver der RWTH Aachen University
Towards an antibody mediated resistance against Geminiviruses
Abstract
dc:descriptionIn this study, single chains (scFvs) against key proteins of the Tomato leaf curl virus-India were generated using phage display and/or hybridoma technologies. Purified AV1 (coat protein) and AC1 (replicase enhancer) viral proteins were used for mice immunization and screening of phage display libraries and hybridoma clones. The binding properties of the isolated scFvs were determined by western blot, ELISA and BIACORE. The missing nucleotide found in the hybridoma derived scFv-SAV against AV1 was restored and the resulting scFv (scFv-RW-AV) showed better binding properties than scFv-SAV. Four scFvs against AV1 were derived from the naïve Tomlinson I library. Two of these scFvs carried a stop codon. The stop codon was mutated in vitro to either code for glutamine (Q) or glutamic acid (E). The Q-mutated scFvs showed higher expression level but less binding activity than the E-mutated scFvs. Additionally, two scFvs against AC1 were selected. All scFvs were cloned and expressed in tobacco leaves as N-terminal fusion with the GFP for cytosolic expression in tobacco leaves. Additionally, two scFv-GFP fusions were targeted to the nucleus by a nuclear localization signal (NLS) introduced at the N-terminus of the scFvs. Both scFv-GFP fusion proteins were exclusively localized in the nucleus. The AV1 proteinwas also expressed in tobacco leaves as N-terminal fusion with DsRed. The AV1-DsRed fusion was completely localized in the nucleus. The binding activity of the AV1-specific scFvs was tested by the co-expression of the scFv- GFP fusion and AV1-DsRed. Only the hybridoma derived scFv showed clear binding and the protein complex was targeted to the nucleus by the AV1 NLS. The data presented in this study demonstrate the successful expression of specific and functional scFvs against the viral proteins in the reducing environment of the plant cytosol. Such a scFv is a potential candidate to interfere with the viral life cycle and thus abolishing or delaying the viral infection.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2006
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Zakri, Mohammed Adel
- Contributors dc:contributor
-
- Fischer, Rainer
Subjects
dc:subject × 7Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- eng
Identifiers
dc:identifier.*- OAI identifier oai:identifier
- oai:publications.rwth-aachen.de:61209