IN VITRO EXPRESSION AND PURIFICATION OF THE COAT PROTEIN GENE OF GARLIC COMMON LATENT VIRUS AND ITS APPLICATION IN ELISABASED DIAGNOSTICS
D. Pramesh, P. Singh, V.K. Baranwal
The coat protein (CP) gene of Garlic common latent virus (GarCLV) was expressed in Escherichia coli strain BL21 expression system as a 40 kDa fusion protein with histidine tag (6His) at its N and C terminals. The purified protein reacted positively in Western blot with a commercial GarCLV polyclonal antiserum and hence, used as immunogen for production of polyclonal antisera in two New Zealand white rabbits. Antisera to GarCLV (titer 1:2000) detected the virus by direct antigen-coated ELISA in GarCLV positive garlic samples. Further, the specific reactivity of the antisera was confirmed through Western blotting and immunosorbent electron microscopy. Antisera developed were successfully utilized for detection of GarCLV in 46 out of 48 different garlic accessions. The immunoreagents developed will be useful for virus indexing in garlic tissue culture programs as well as in quarantine and/or certification programs.