Development of immunodiagnostics for the detection of Grapevine leafroll-associated virus 3 (GLRaV-3) in grapevine using in vitro expression and purification of its coat protein gene

  • Sandeep Kumar
  • Priyanka Singh
  • Richa Rai
  • Virendra Kumar Baranwal
Original Article
  • 24 Downloads

Abstract

A previously cloned coat protein (CP) gene of Grapevine leafroll-associated virus 3 (GLRaV-3) from cultivar Cabernet Souvignon was over-expressed in Escherichia coli strain BL21 expression system as ~ 43 kDa fusion protein containing polyhistidine tag (6His) at its N terminal. The protein was purified from insoluble fraction and reacted positively in western blotting with commercial anti GLRaV-3 polyclonal antiserum (Bioreba, Switzerland) and hence, used as immunogen for the production of polyclonal antisera in New Zealand white rabbits. Polyclonal antiserum specific to GLRaV-3 detected the virus by double antibody sandwich enzyme linked immunosorbent assay using commercial alkaline phosphatase (ALP) conjugated globulin fraction (Bioreba, Switzerland) in GLRaV-3 positive grapevine samples. The immunoreactivity of the antiserum was confirmed through western blotting. The purified antiserum was conjugated with ALP. The primary antiserum along with ALP conjugate successfully detected the GLRaV-3 from the infected sample at 1:8000 and 1:10,000 dilutions, respectively. To the best of our knowledge, it is the first global study wherein the CP of GLRaV-3 was cloned in pET28a(+) expression vector having many advantages over the earlier used expression vectors. The cloned CP gene was expressed, purified and subjected to the production of immunoreagents. The developed immunoreagents will be useful for certification programmes as well as for large scale virus screening to produce GLRaV-3 free grapevines. The indigenously developed immunereagents will provide a cost-effective way of managing grapevine leafroll disease in Indian sub-continent.

Keywords

CP GLRaV-3 Primary antiserum IgG ALP conjugate DAS-ELISA Quarantine certification 

Notes

Acknowledgement

Sandeep Kumar would like to acknowledge Department of Science and Technology (DST), Ministry of Science and Technology, Government of India for providing INSPIRE (Innovation in Science Pursuit for Inspired Research) fellowship during his Ph.D. programme. Financial support from Outreach Project of ICAR, New Delhi is duly acknowledged.

Supplementary material

13562_2018_451_MOESM1_ESM.docx (154 kb)
Supplementary material 1 (DOCX 153 kb)

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Copyright information

© Society for Plant Biochemistry and Biotechnology 2018

Authors and Affiliations

  • Sandeep Kumar
    • 1
    • 2
  • Priyanka Singh
    • 1
  • Richa Rai
    • 1
  • Virendra Kumar Baranwal
    • 1
  1. 1.Advanced Centre for Plant Virology, Division of Plant PathologyIndian Agricultural Research InstituteNew DelhiIndia
  2. 2.All India Coordinated Research Project on Medicinal and Aromatic Plants and BetelvineOrissa University of Agriculture and TechnologyBhubaneswarIndia

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