U104184.pdf (9.17 MB)
Engineering recombinant antibodies for virus resistance
thesisposted on 2014-12-15, 10:33 authored by Sharifah Zabidah. Haron
An important step in the development of plant protection strategies using anti-viral recombinant antibodies will be to devise generic systems for the reliable expression of the antibodies to the required levels in the desired cellular compartment. Currently, expression levels of recombinant antibodies are apparently antibody dependent, highly variable and in the cytoplasmic compartment in particular, often very low.;An aim of the work reported here was to investigate the possibility that the fusion of recombinant antibodies to a protein known to be highly expressed in plants would increase the level of the fusion protein over that of the separately expressed recombinant antibody. The potato virus-S virus coat protein accumulates at high levels in the plant and was therefore selected as a fusion partner for two combinant antibodies; a model scFv, AS32scFv, known to be functional and to accumulate, although at low levels, in the cytosol of Nicotiana tabacum; and an scFv antibody to coat protein of potato virus-V, scFv4.5. These gene fusions were constructed and expressed in a protoplast transient expression system and an Agrobacterium rhizogenes hairy root system. The hairy root system, as determined by the se of GUS reporter gene expression, proved to be convenient and reliable. Evidence was obtained with ELISA detection methods which indicates that fusion of scFv to viral coat protein increases scFv accumulation in the cytoplasm although difficulties with the detection of the unfused scFv on western blots prevented thorough investigation.;Other anti-viral scFv's were made available for study but attempts to express them in a bacterial system for characterisation purposes were unsuccessful. The Pichia yeast expression system was therefore chosen as a possible alternative means of producing these scFv's, in sufficient quantities for characterisation as scFvs and scFv fusions prior to the labour intensive and time consuming process of plant transformation. Although after considerable effort the system allowed the production of significant amounts of one of the antibodies studied it did not prove possible to detect expression of the two other anti-viral antibody genes investigated.
Date of award1997-01-01
Awarding institutionUniversity of Leicester