Potential uses of in vitro expressed and purified recombinant Prunus necrotic ringspot virus coat protein gene

Abstract

Prunus necrotic ring spot virus (PNRSV), an ilarvirus, has a wide host range especially on important commercial crops like almond, apple, apricot, hop, peach, and rose. PNRSV coat protein gene was cloned in expression vector pGEX-2Tk (Amersham Pharmecia, USA) using E. Coli BL 21 strain competent cells. Expression conditions were standardized for maximum recovery of soluble recombinant protein. The in vitro expressed protein was purified and used as an antigen for raising antisera. Both intramuscular and sub-cutaneous routes were used separately for antisera raising. A part of the raised and purified antisera was used in enzyme conjugate preparation. It was then formulated in the form of an ELISA-based diagnostic kit for PNRSV detection. It was also compared with the commercially available kit.

Keywords:

• Antiserum
• diagnostic kit
• ELISA
• in vitro expression
• PNRSV
• protein purification

Acknowledgements

The authors are thankful to the Director, IHBT, Palampur, India for providing the necessary facilities and the Department of Biotechnology, Government of India, for financial support (grant No. BT/PR/1790/AGR/08/126/99). S.K. thanks the Council of Scientific and Industrial Research, New Delhi for awarding a Senior Research Fellowship.

Notes

IHBT Communication # 586.

This work has been protected by a patent in order to permit an industrial development.