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 - 2.19

STABLE SINGLE-CHAIN Fv ANTIBODY FRAGMENTS (scFvs) FOR CUCUMBER MOSAIC VIRUS (CMV) DETECTION AND IMMUNOTHERAPY

VILLANI M.E.*, ROGGERO P.**, BITTI O.*, FRANCONI R.*

*) ENEA, BIOTEC GEN, CR Casaccia, Via Anguillarese 301, 00060, Roma

**) CNR, Istituto di fitovirologia Applicata, Strada delle Cacce 73 10135 Torino

PVX, scFv, immunotherapy, CMV

We isolated recombinant single-chain variable fragment (scFv) antibodies specific to the plant virus cucumber mosaic (CMV) using a phage display library composed of intrinsically stable scFvs. After four rounds of selection and enrichment (‘biopanning’) on the immobilised virus, a panel of different phage clones were obtained. High yields of fully functional soluble scFvs were produced in Escherichia coli. Two of them were analysed in detail. Periplasmic extracts or purified scFvs were used to successfully detect the virus in infected plant extracts. Affinity measurements revealed good affinities for the CMV, in the range of nanomolarity, demonstrating that these scFv fragments represent a valuable tool for inexpensive diagnosis.

Moreover, transient expression in the cytoplasm of N. benthamiana plants through a PVX-derived vector resulted in soluble scFvs. This analysis opened the way for the construction of transgenic microtomato plants. Several transgenic lines have been selected. Next step will be the challenge of the T2 generation with the virus to check effective protection against CMV.