A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

Abstract

Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (V H ) and light (V L ) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (V L -V H ) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. The genus Potyvirus, one of the largest groups of plant viruses, includes 158 species, many of which are economically important, such as Potato virus Y (PVY) and Plum pox virus (1, 2). Most potyviruses infect various host plants and are spread worldwide by aphid vectors and mechanical inoculation (3). Potyviruses have a single-stranded, positive-sense RNA genome of approximately 10 kb in length that encodes a polyprotein translated from a large open reading frame (ORF) (3) and a trans-frame movement-related protein (P3N-PIPO) encoded from the 5=-terminal portion of P3 and the PIPO cistrons (4, 5). During proteolytic processing, the large polyprotein can be separated into 10 mature proteins that are involved in virus replication, movement, and virion assembly (3). Potyviral capsid proteins (CPs) not only form the viral particle but also participate in cell-to-cell and systemic movement (6, 7). The variable N-and C-terminal regions of the CPs exposed on the virion surface are required for long-distance transport, are involved in aphid transmission (DAG motif), and may also assist in cell-to-cell spreading (7-10), whereas the conserved core regions of CPs are necessary for virion assembly and the cell-to-cell movement of potyviruses (6, 7).In Taiwan, the viral disease caused by potyviruses is one of the limiting factors for production of calla lilies. At least five potyvirus...