Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 2.13

Plant expression of anti-TSWV nucleoprotein recombinant antibodies

Donini M., Desiderio a.

ENEA C.R. Casaccia, UTS Biotecnologie, Protezione della Salute e degli Ecosistemi, 00060 Rome, Italy

transgenic plants, scFv expression, immunotherapy

Immunomodulation of host-pathogen interactions by expressing recombinant antibodies in plants represents a novel approach in viral disease resistance (De Jaeger, G. et al. (2000) Plant Mol. Biol. 43, 419-28). There are several examples in literature of partially protected transgenic plants expressing single chain antibody fragments (scFv) against structural viral proteins (Tavladoraki, P. et al. (1993) Nature 366, 469-72; Xiao, W.X., et al.(2000) Mol. Breeding 6, 421-431).

In this work two scFv fragments (NUV1 and NUV4) obtained from TSWV-nucleoprotein specific mAbs were expressed in E. coli. The N-terminal pelB leader sequence directed expression in the bacterial periplasmic space where recombinant antibodies accumulated at high levels. ScFvs were then transiently expressed in the apoplast of N. benthamiana plants. Transient plant expression system was mediated by the recombinant PVX viral vector. The scFv NUV1 and scFv NUV4 were cloned in the pPVX vector harbouring a secretory plant signal derived from the polygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris and protein expression was assayed by Western blot. The scFv showed to accumulate in the intercellular space at levels of 0,05% of total soluble protein content.

Recombinant antibodies were then cloned in pPBI vector with and without the PGIP leader sequence to respectively target the scFvs to the apoplast (NUV-PGIP) and to the cytoplasm (NUV-intra). Transgenic N. benthamiana plants were obtained by A. tumefaciens infection. Transgenic F0 plants were screened by RT-PCR and the expression of eleven independent lines were assayed. Western blot analysis revealed low expression levels: 0,01% of total soluble protein for lines 1NUV1-intra and 2NUV1-PGIP. with Plant extracts from cytoplasmic expressing lines showed no specific binding activity to the TSWV nucleoprotein in ELISA. This result could be explained by the very low levels of scFv expression observed. Further characterisation of the transgenic lines is therefore required. The selected scFvs could be a promising tool for the immunomodulation of TSWV plant infection.