IMMUNODIAGNOSIS OF CUCUMBER MOSAIC VIRUS (CMV), WITH POLYCLONAL ANTIBODIES RAISED AGAINST COAT PROTEIN (CP) GENE OF THE VIRUS
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Date
2015-10-10
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UNIVERSITY OF AGRICULTURAL SCIENCES GKVK, BENGALURU
Abstract
Cucumber Mosaic Virus (CMV) disease is one of the most common disease affecting cucumber. It has a wide host range causing severe damage in many economically important agricultural as well as ornamental crops, resulting in remarkable yield loss. CMV is difficult to diagnose due to the periodic change in symptoms, hence methods of early detection and identification of virus which can be employed in both laboratory and field, play a critical role in virus disease management. Cloning and expression of CMV CP gene is an impm1ant strategy for obtaining large scale recombinant protein that can be used in antibody production and further these antibodies with higher specificity, play a critical role in carrying out serological tests. In this study CMV CP gene (-657 bp) was isolated using RT-PCR with gene specific primers which was cloned into pRSET B expression vector and expressed in E. coli DH5a cells. Coat protein of -25 KDa was isolated and purified by using Ni-NT A column. Dot immunobinding assay (DIBA) and plate enzyme linked immuno sorbent assay (ELISA) were standardized for expressed CMV CP and CMV infected plants using the antibodies raised against the whole virus, with optimum titre values at 1: 100 dilution of antigen and 1: 1000 dilution of primary and secondary antibody.
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