Recombinant Coat Protein of Banana Bract Mosaic Virus as a Potential Antigen for Serological Detection of the Virus
Darsana Dilip *
Department of Plant Pathology, College of Horticulture, Kerala Agricultural University, Vellanikkara, Thrissur, Kerala, 680656, India.
Vimi Louis
Division of Plant Pathology, Banana Research Station, Kerala Agricultural University, Kannara, Thrissur, Kerala, 680652, India.
Pallavi Sabharwal
Department of Biochemistry, New Biological Sciences Building, Indian Institute of Science, Bangalore, 560012, India.
H. S. Savithri
Department of Biochemistry, New Biological Sciences Building, Indian Institute of Science, Bangalore, 560012, India.
P. M. Namitha
Division of Plant Pathology, Banana Research Station, Kerala Agricultural University, Kannara, Thrissur, Kerala, 680652, India.
K. Anita Cherian
Department of Plant Pathology, College of Horticulture, Kerala Agricultural University, Vellanikkara, Thrissur, Kerala, 680656, India.
*Author to whom correspondence should be addressed.
Abstract
Banana bract mosaic disease caused by Banana bract mosaic potyvirus (BBrMV) is reported to instigate heavy loss in banana and plantain across Asia. Almost all the cultivars of banana succumb to the disease resulting in malformed bunches weighing less than half of normal ones. In the current study the coat protein (CP) gene segment present at the 3’ terminal region of the viral genome amplified by RT-PCR was cloned into expression vectors, pRSET-C and pGEX-4T-2 to use it for raising polyclonal antiserum which in turn will aid in developing assays to detect the virus. Recombinant BBrMV CP (rCP) in pRSET-C when expressed was insoluble whereas, it was in the soluble fraction when expressed from pGEX-4T-2. The GST-fusion protein was purified by GSH sepharose affinity column chromatography and western blot analysis was performed using anti GST antibodies. 360 µg/ml of protein was purified from 1 l of culture. The GST tag was cleaved from the purified protein by incubation with thrombin at 25°C overnight. The rCP was characterized using ultracentrifugation, fluorescence spectroscopy and electron microscopy. The tagless monomer failed to assemble to virus like particles (VLPs) in vitro which was substantiated by fluorescence spectroscopy. This study will be first step towards deciphering structure and functions of Banana bract mosaic virus coat protein.
Keywords: Banana bract mosaic virus, recombinant coat protein, affinity column chromatography, virus like particles.