Development of polyclonal antibodies against nucleocapsid protein of watermelon silver mottle virus and their application to diagnostic

Abstract

Summary. -Watermelon silver mottle virus (WSMoV) is an emerging disease of cucurbit crops in South China. Production of high-quality antibodies is necessary for the development of serological methods for detection of this virus. The nucleocapsid protein (NP) gene of WSMoV was amplified from WSMoV-infected watermelon leaves by RT-PCR and cloned into vector pET-28a (+) for prokaryotic expression. After identification via enzyme digestion and sequencing, the recombinant clone was transformed into Escherichia coli Rosetta (DE3) for protein expression. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the molecular weight of the WSMoV NP fusion protein was 34.1 kDa. The fusion protein was purified and used as antigen for the preparation of polyclonal antisera in rabbits. Results of indirect ELISA and western blot analysis showed that the antisera reacted specifically with WSMoV NP. In addition, sensitivity and specificity of the antisera were examined on a number of infected field samples by indirect ELISA. These findings will facilitate further immunological and serological studies of WSMoV.Keywords: watermelon silver mottle virus; polyclonal antibody; western blot; prokaryotic expression Acta virologica 58: 167 -172, 2014 doi:10.4149/av_2014_02_167 * Corresponding author. E-mail: raoxq@hotmail.com; fax: 086-020-85281107. Abbreviations: CaCV = capsicum chlorosis virus; CMV = cucumber mosaic virus; HCRV = hippeastrum chlorotic ringspot virus; IPTG = Isopropyl-β-D-thiogalactopyranoside; NP = nucleocapsid protein; sodium WSMoV = watermelon silver mottle virus

Development and Application of Droplet Digital PCR Assay for the Detection of Watermelon Silver Mottle Virus and Melon Yellow Spot Virus

“…Currently, many detection methods are used to identify WSMoV or MYSV. They include electron microscopy [4], reverse-transcriptase polymerase chain reaction (RT-PCR) [10], enzyme-linked immunosorbent assay (ELISA), Western blotting [11], and doubleantibody sandwich ELISA (DAS-ELISA) [12]. However, these methods have certain limitations [13].…”