PRODUCTION OF POLYCLONAL ANTISERA TO THE COAT PROTEIN OF CITRUS TRISTEZA VIRUS EXPRESSED IN ESCHERICHIA-COLI - APPLICATION FOR IMMUNODIAGNOSIS

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 pMAL-c2. 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 amy lose 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 10(5) 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.