title: Production of Polyclonal Antibody againstGrapevine fanleaf virusMovement Protein Expressed inEscherichia coli.
abstract: The genomic region ofGrapevine fanleaf virus(GFLV) encoding the movement protein (MP) was cloned into pET21a and transformed intoEscherichia colistrain BL21 (DE3) to express the protein. Induction was made with a wide range of isopropyl-β-D-thiogalactopyranoside (IPTG) concentrations (1, 1.5, and 2 mM) each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs) and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection.
authors.affiliation: Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Zanjan 45371-38111, Iran.
authors.forename: Davoud
authors.initials: D
authors.lastname: Koolivand
authors.affiliation: Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz 51666-16471, Iran.
authors.forename: Nemat Sokhandan
authors.initials: NS
authors.lastname: Bashir
authors.affiliation: Plant Virology Research Center, College of Agriculture, Shiraz University, Shiraz 71949-84471, Iran.
authors.forename: Seyed Aliakbar
authors.initials: SA
authors.lastname: Behjatnia
authors.affiliation: Department of Veterinary Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz 51666-16471, Iran.
authors.forename: Raziallah Jafari
authors.initials: RJ
authors.lastname: Joozani
keywords: ELISA; Grapevine fanleaf virus; antibody; expression; recombinant