Production of polyclonal antibodies to the coat protein gene of Indian isolate of Apple stem grooving virus expressed through heterologous expression and its use in immunodiagnosis

Abstract

Apple stem grooving virus (ASGV) belonging to the genus Capillovirus under family Betaflexiviridae is one of the widely distributed latent viruses mainly on pome fruit trees. It infects apple causing considerable economic losses that is a threat to the apple industry. To study the occurrence and incidence of ASGV disease, sensitive antisera based diagnostic tool has been developed, which would be helpful in large scale indexing, certification and quarantine programmes. Coat protein gene of ASGV was cloned into pET-32a(+) and pHIS-Parallel expression vectors. Expression characteristics of the proteins expressed from both the systems were compared and taken for raising the antisera. Sensitivity and specificity of the antiserum against virus infection was compared by Double Antibody Sandwich (DAS-ELISA). It was observed that the antisera raised from the protein expressed from pHIS-Parallel expression system (smaller ~ 3 kDa His tag) was more sensitive as compared to the antisera raised from the protein expressed from pET-32a(+) expression system (~ 18 kDa His tag) and the commercially available antisera. The antisera could also be successfully used for western blotting of infected samples and in DAS-ELISA. Thus, this study presents the first report on production of polyclonal antiserum against recombinant coat protein of ASGV from India, for reliable detection of the virus.