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Disease Detection and Losses

Production of Polyclonal Antisera to the Coat Protein of Citrus Tristeza Virus Expressed in Escherichia coli: Application for Immunodiagnosis. Olga V. Nikolaeva, Department of Plant Pathology, University of California, Riverside 92521-0122, University of Florida, Citrus Research and Education Center, Lake Alfred 33850-2299; Alexander V. Karasev(2), David J. Gumpf(3), Richard F. Lee(4), and Stephen M. Garnsey(5). (2)(3)Department of Plant Pathology, University of California, Riverside 92521-0122; (2)(4)University of Florida, Citrus Research and Education Center, Lake Alfred 33850-2299; (5)USDA-ARS Horticultural Research Laboratory, 2120 Camden Road, Orlando, FL 32803. Phytopathology 85:691-694. Accepted for publication 1 March 1995. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1995. DOI: 10.1094/Phyto-85-691.

Using specific primers based on the sequence of the Florida isolate T36 of citrus tristeza virus (CTV), the coat protein (CP) gene was amplified by RT-PCR (reverse transcription-polymerase chain reaction) from the severe California isolate SY568 of CTV. The RT-PCR product was cloned, sequenced, and subcloned into an expression vector pMALc2. The CTV CP was expressed as a fusion product containing a fragment of the Escherichia coli maltose-binding protein (MBP). This MBP-CP fusion protein reacted with CTV-specific antisera in immunoblotting and enzyme-linked immunosorbent assay (ELISA). After cell disruption, the MBP-CP fusion protein was purified to near homogeneity by amylose resin affinity column chromatography giving a yield of 1 mg of fusion protein per 10 ml of E. coli culture. Antisera obtained from rabbits after injection with MBP-CP protein were specific to CTV, with a titer of about 10⁵ in an indirect ELISA, and were suitable in ELISA for trapping. These polyclonal antisera reacted with a wide range of CTV isolates from different geographic sources, and of different biological properties.