Development of polyclonal antibodies using bacterially expressed recombinant coat protein for the detection of Onion yellow dwarf virus (OYDV) and identification of virus free onion genotypes

Abstract

Onion yellow dwarf virus (OYDV) belonging to the genus Potyvirus, family Potyviridae, is one of the widely distributed viruses of Allium species worldwide. It causes dwarfing, yellow striping, crinkling and flaccidity of the leaves of onion and garlic. To see the occurrence and incidence of OYDV on Allium crop, an attempt was made to develop antibody based diagnostic assay which would be useful for routine indexing and screening of the germplasm. The total RNA was isolated from the symptomatic leaves of onion and the gene encoding coat protein (CP) was cloned. The nucleotide sequencing analysis of the cloned RT-PCR product revealed ~ 774bp amplicon (OYDV CP) and it was further cloned in pET-28a ( +) expression vector which yielded ~ 30kDa fusion protein with Histidine tag (His6BP). The expression of fusion CP was primarily checked on SDS-PAGE and further confirmed by Western blot. The His6BP-OYDV-CP was obtained in soluble state after purification and was used to immunize New Zealand white rabbit for the production of polyclonal antibody (PAb). The produced PAb against the purified fusion protein successfully detected OYDV from onion and garlic samples at 1:2000 dilutions in indirect-enzyme linked immunosorbent assay (DAC-ELISA). Thus, this study presents first report that Histidine tag (His6BP) fusion OYDV-CP based antibody production and its successful application in identification of virus free onion and garlic genotypes.