Production of polyclonal antiserum against beet western yellows virus coat protein expressed in Escherichia coli

Authors
Arezu Lotfi 1
Masoud Shams-Bakhsh 1
Bagher Yakhchali 2
1 Department of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran.
2 National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
10.48311/jcp.2015.1255
Abstract
Serological methods are commonly used methods for detection of viruses. Preparation of pure viral antigens is a crucial step in production of antibodies required for serological studies. In this research the gene encoding coat protein of a Beet western yellows virus (BWYV) isolate from Iran was amplified by PCR and was ligated into a bacterial expression vector (pET26b) to obtain pET-BWYV-CP clone. Escherichia coli BL21 was transformed with pET-BWYV-CP and expression of the recombinant coat protein was induced by IPTG. The expressed recombinant coat proteins were purified and used as an antigen for rabbit immunization. The antiserum was able to detect recombinant coat protein in total protein extracts of induced E. coli BL21 cells in western blot analysis.
Keywords
Polerovirus
Expression
western blot
BWYV
Cerovska, N., Moravec, T., Plchova, H., Hoffmeisterova, H. and Folwarczna, J. 2010. Production of polyclonal antibodies to Potato virus X using recombinant coat protein. Journal of Phytopathology, 158: 66-68.
Dea, S., Wilson, L., Therrien, D. and Cornaglia, E. 2000. Competitive ELISA for detection of antibodies to porcine reproductive and respiratory Syndrome virus using recombinant E. coli–expressed nucleo capsid protein as antigen. Journal of Virological Methods, 87: 109-122.
Hertel, K., Schwinghamer, M. and Bambach, R. 2004. Virus diseases in canola and mustard. Bill Noad Agnote DPI 495, 1st edition, The State of New South Wales (NSW) Department of Primary Industries. Avaliable on: http:// www.dpi.nsw.gov.au/_data/assets/pdf file /0003/148377/virus-diseases-in-canola-and-mustard.pdf.
Hewings, D. A. and D'Arcy, J. C. 1984. Maximizing the detection capability of a Beet western yellows virus ELISA system. Journal of Virological Methods, 9: 131-142.
Howard, R. L. 2005. Cloning and expression of Phanerochaete chrysosporium cellobiohydrolase (cbhI.2). African Journal of Biotechnology, 4: 1180-1184.
Iracheta-Cardenas, M., Sandoval-Alejos, B. D., Roman-Calderon, M. E., Manjunath, K. L., Lee, R. F and Rocha-Pe˜na, M. A. 2008. Production of polyclonal antibodies to the recombinant coat protein of Citrus tristeza virus and their effectiveness for virus detection. Journal of Phytopathology, 156: 243-250.
Jakab, G., Droz, E., Brigneti, G., Baulcombe, D. and Malnoe, P. 1997. Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of Potato virus Y. Journal of General Virology, 78: 3141-3145.
Jelkmann, W. and Keim-Konrad, R. 1997. Immuno-capture polymerase chain reaction and plate-trapped ELISA for the detection of apple stem pitting virus. Journal of Phytopathology, 145: 499-503.
Komorowska, B. and Malinowski, T. 2009. Attempts to produce antiserum against Apple stem pitting virus coat protein (ASPV-CP) obtained in prokaryotic and eukaryotic expression systems. Journal of Fruit and Ornamental Plant Research, 17: 21-30
Mayo, M. A. and D’Arcy, C. J. 1999. Family Luteoviridae: a reclassification of luteoviruses. In: Smith, H. G. and Barker, H. (Eds), The Luteoviridae, CAB International, Oxford, PP: 15-22.
Nikolaeva, O. V., Karasev, A. V., Gumpf, D. J., Lee, R. F and Garnsey, S. M. 1995. Production of polyclonal antisera to the coat protein of Citrus tristeza virus expressed in Escherichia coli: application for immunodiagnosis. Phytopathology, 85: 691-694.
Noueiry, A. O., Lucas, W. J. and Gilbertson, R.L. 1994. Two proteins of a plant DNA virus coordinate nuclear and plasmodesmal transport. Cell, 76: 925-932.
Osman, T. A. M. and Buck, K. W. 1991. Detection of the movement protein of Red clover necrotic mosaic virus in a cell wall fraction from infected Nicotiana clevelandii plants. Journal of General Virology, 72: 2853-2856.
Sa´–Pereira, P., Duarte, J. and Costa–Ferreira, M. 2000. Electroelution as a simple and fast protein purification method: isolation of an extracellular xylanase from Bacillus sp. CCMI 966. Enzyme and Microbial Technology, 27: 95-99.
Sambrook, J. and Russell, M. D. W. 2001. Molecular Cloning: a Laboratory Manual. 3rd Edition. Cold Spring Harbor Laboratory Press. New York, USA.
Shams-Bakhsh, M. and Symons, R. H. 2004. Cloning and expression of the coat protein gene of Barley yellow dwarf virus-PAV in Escherichia coli. Iranian Journal of Biotechnology, 2: 84-89.
Stevens, M., Freeman, B., Liu, H. Y., Herrbach E. and Lemaire O. 2005. Beet poleroviruses: close friends or distant relatives? Molecular Plant Pathology, 6: 1-9.
Sørensen, H. P. and Mortensen, K. K. 2005. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microbial Cell Factories, 115: 113-128.
Vaira, A. M., Vecchiati, M., Masenga, V. and Accotto, G. P. 1996. A polyclonal antiserum against recombinant viral protein combines specificity and versatility. Journal of Virological Methods, 56: 209-219.
Zahedi Tabarestani, A., Shams-bakhsh, M. and Safaie, N. 2012. Comparison of the coat protein gene sequence of Iranian canola-infecting Beet western yellows virus isolates. Journal of Crop Protection, 1: 211-219.
Journal of Crop Protection
Volume 4, Issue 5
December 2015
Pages 739-746