Production of Recombinant Coat Protein for the Generation of Specific Polyclonal Antibody Against Tomato leaf curl New Delhi virus – [Thailand:Kanchanaburi:Cucumber:2012]

Abstract

Tomato leaf curl New Delhi virus (ToLCNDV) causes curling leaf disease of cucumber, an economic plant in Thailand. Serological detection of this disease requires good quality antibody. The objective of this research is to produce anti–ToLCNDV antibody against recombinant coat protein (rCP) antigen of ToLCNDV–[TH:KR:Cu:12] isolated from curling leaf cucumber sample in Kanchanaburi province. The CP gene was cloned and ligated into a pQE–80L expression vector. After inducing gene expression, the rCP inclusion body form was obtained and required denaturing condition for protein extraction and purification by Ni–NTA column chromatography. Based on sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis, the rCPs showed the molecular weight of 31 kDa which was further used as an antigen for rabbit immunization. The antiserum had been collected started from the 5^th week and continued weekly for 14 weeks. The highest antibody titre appeared in 15^th week after the first immunization, at 102,400. Determination of antibody specificity was performed by indirect plate trapped antigen enzyme– linked immunosobent assay. The result showed that the antibody was specific to native ToLCNDV however it cross–reacted with Tomato leaf curl virus (TLCV) and African cassava mosaic virus (ACMV) which contained amino acid similarity at 76.95% and 76.95%, respectively. Since no report of the infection of TLCV and ACMV in Cucurbitaceae therefore the produced antiserum could be used for preliminary screening of ToLCNDV infection in cucurbits and ACMV in cassava. Nevertheless we do not recommend the use of this antiserum for the detection of TLCV in tomato due to the host range property of tomato for ToLCNDV.