Herstellung und Charakterisierung von rekombinanten Antikörpern und scFv Fusionsproteinen zur Generierung Phytophthora infestans-resistanter Kartoffelpflanzen

Potatoes are grown on approximately 19 million hectares worldwide. Following maize, rice and wheat potato is the world’s fourth most important crop in terms of production volume. Phytophthora infestans cause huge losses in cultivated potato worldwide. The objective of this work was to generate P. infestans specific single chain antibody fragments (scFv) and genetically fuse them to antifungal peptides (AFP) in order to establish transgenic potato plants that are resistant against P. infestans by expressing the AFP-scFv fusion protein. To generate suitable scFvs two strategies were used. On the one hand spleen cells of mice that have been hyperimmunized with cell wall fragments of P. infestans were fused with myeloma cells. This approach yielded twelve P. infestans specific monoclonal antibodies (mAb). On the other hand scFv libraries constructed from splenic lymphocytes of chicken and mice hyperimmunized with P. infestans cell wall fragments were subjected to phage display panning resulting in the isolation of four scFvs. The mAbs, their corresponding scFvs and the scFvs selected by phage display were analyzed by immunoblot and concerning their reactivity towards P. infestans as well as members of the asco-, basidio- and oomycetes. Immunofluorescence microscopy was applied to localize the antibody-antigen complexes on the pathogen. Focusing on the aim of this thesis scFvPi102.2 derived from mAbPi102.2 was the most promising scFv. Cross reactivity tests revealed that the IgG1 was specific for oomycetes. Both, mAbPi102.2 and scFvPi102.2, demonstrated highest reactivity towards P. infestans in comparison to the other IgG1 antibodies or scFvs. The corresponding epitope could be detected on the surface of sporangia, germ tube and appressoria of the intact pathogen. As mAbPi102.2 had no inhibitory effect on the growth of P. infestans, the scFvPi102.2 was genetically fused to several AFPs. The reactivity of the bacterially in E. coli and transiently in N. tabacum produced AFP-scFv fusion proteins was confirmed by ELISA. Growth inhibition of Phytophthora nicotianae could be observed on tobacco leaves transiently transformed with AFP-scFv fusions. The transgenes D4E1 scFvPi102.2 und GR7 scFvPi102.2 were stably integrated into the genome of the S. tuberosum varieties Gala and Pirol. Leaves of three transgenic potato lines remained 100 % disease free when inoculated with P. infestans in comparison to the completely infected wild type. The results of the present study demonstrate that the development of AFP-scFv fusions and the expression of these fusion proteins in transgenic potato plants is a promising possibility to control P. infestans.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2009

Author dc:creator

• Schleker, Sylvia

Contributors dc:contributor

• Fischer, Rainer

• info:eu-repo/classification/ddc/570
• Antikörper
• Pilze
• Gentechnologie
• Transgene Pflanzen
• Phytophthora infestans
• Phagen-Display-Bibliothek
• Kartoffel
• Resistenz
• Biowissenschaften, Biologie
• Antikörper-vermittelte Resistenz
• scFv
• antifungale Peptide
• antimikrobielle Peptide
• Plantibodies
• antibody
• resistance
• antimicrobial peptides
• potato
• transgenic plant

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-113571%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A51260