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

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 3.18

TRANSIENT EXPRESSION OF CLASSICAL SWINE FEVER VIRUS A1-EPITOPE IN NICOTIANA BENTHAMIANA PLANTS INFECTED WITH RECOMBINANT POTATO VIRUS X

G. MARCONI*, A. PORCEDDU**, P. BARONE*, E. ALBERTINI*, D. RUTILI***, F. VERONESI*

*) Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Università degli Studi di Perugia, Borgo XX Giugno 74, 06121 Perugia

**) IRMGPF-CNR, Via Madonna Alta 130, 06128 Perugia

***) Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Via Salvemini 1, 06121 Perugia

PVX, transient expression, CSFV

Plant viruses have been used as vector for peptide and proteins production. The high expression levels and the possibility of exposing on the coat surface foreign peptide allow their use as vaccines.The vector used in the present study is Potato Virus X (PVX), a filamentous plant-virus infecting many members of the Solanacee family including potato, tomato and pepper.

The Classical Swine Fever Virus (CSFV) is an economically important infectious disease occurring in pigs causing direct and indirect losses. In Europe it is estimated that more than 2 milion of pigs die per year. The etiological agent of CSFV is a virus of the genus Pestivirus (Flaviviridae family). Previous studies have shown that the glicoprotein E2 is highly immunogenic with four antigenics domains A to D. The domain A is divided in subdomains A1, A2 and A3. Subdomains A1 and A2 are conserved in more than 90 CSFV strains, but only subdomain A1 is neutralizing..

The PVX cDNA was modified through the fusion to the N-terminal portion of CP of a 16 amino acid sequence, called FMDV 2A and subdomain A1 the CSFV to produce the 35SPVXA12ACP construct. The FMDV 2A sequence allow to obtain a proteolityc cleavage with an efficiency of about 50-70%.

Nicotiana benthamiana plants were infected with virus carring plasmid 35SPVXA12ACP or wild-type (WT) PVX by abrading the surface of two leaves with carborundum and inoculating each leaf with 25 mg of plasmid DNA. Upon sistemic infection (10 to 15 days after inoculation) the correct expression of construct was verified through RT-PCR. The recombinant protein was analized through Maldi-Tof method. Sera from rabbit immunized with 35SPVXA12ACP showed IgG specific for E2 peptide, while no reactivity was found in the sera of control animals.