Immunodiagnostics of cucumber mosaic virus using antisera developed against recombinant coat protein

Abstract

Cucumber mosaic virus (CMV), causing mosaic disease in banana cultivars, is the most widespread virus after Banana bunchy top virus. The entire coat protein (CP) gene of CMV was amplified by polymerase chain reaction (PCR) and engineered to be expressed in bacterial expression vector pQE30, under the control of isopropyl-thio-β-d galactopyranoside (IPTG)-inducible T7lac promoter. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed expressed CP as ∼25 kDa protein. Expression conditions for maximum recovery of soluble recombinant protein were standardised. The recombinant CP (r-CP) fused with His-tag was purified from Escherichia coli using nickel-nitrilotriacetic acid (Ni-NTA) resin and used as an antigen for the production of polyclonal antisera. This antiserum was used to develop enzyme-linked immunosorbent assay (ELISA) and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection of CMV isolates. The antiserum had a titre of 1:8000 when tested with r-CP. The reaction of polyclonal antisera was much better than that of commercial antibody. On comparing IC-RT-PCR with direct antigen coating-enzyme-linked immunosorbent assay (DAC-ELISA) using polyclonal antisera, IC-RT-PCR was found to be more sensitive, hence can be used to detect the presence of virus in tissue culture based banana plant as well as in various host plants.