Production of Polyclonal Antibodies Specifi c to the Recombinant Coat Protein of Blackeye cowpea mosaic virus and Its Use in Disease Detection

Abstract

The coat protein gene of Blackeye cowpea mosaic virus (BICMV-CP) was amplifi ed by reverse transcription polymerase chain reaction and cloned into the expression vector pQE-80L. This plasmid was transformed into Escherichia coli DH5α competent cells. The BICMV-CP gene was expressed as a fusion protein containing a fragment of 6xHis-tag. Bacterial cells were disrupted by repeated freezethawing three times and the BICMV-CP fusion proteins were purifi ed under denaturing conditions by affi nity chromatography with Ni-NTA Agarose. A sample of 500 μg of purifi ed protein was mixed with Freund’s complete adjuvant at a ratio of 1:1 (volume to volume). Initially, the emulsion was subcutaneously injected into a New Zealand White rabbit, followed at weekly intervals by three additional immunizations with 500 μg of the purifi ed protein mixed with Freund’s incomplete adjuvant. Bleeding was done every week during weeks 5-12 and titers of the antisera ranging from 800-51,200 were obtained. Up to a dilution of 1:320 of the BICMV-infected yardlong bean sap could be detected by indirect enzyme-linked immunosorbent assay. The produced antiserum reacted specifi cally with the BICMV-infected plant without any cross reaction with other virus species tested. However, a weakly positive reaction with Bean common mosaic virus could be observed.