1Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Zanjan-Iran.
2Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Iran.
Cucumber mosaic virus (CMV) is one of widely-spread viruses of plants with the broadest host range encompassing over 1200 species. One major limiting factor for detection of the virus is unavailability of the virus-specific antibodies especially in developing countries. Recombinant DNA technology facilitates antibody preparation without requiring special equipment. In this study, coat protein (CP) gene cDNA of CMV was subcloned from pTZ57CMVCP into pET21a expression vector and transformed into Escherichia coli strain Rosetta. Expression of CMV CP was examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and its identity was confirmed by western blotting, dot blot immunobinding assay (DIBA) and enzyme- linked immunosorbent assay (ELISA) using anti- CMV antibody. The expressed protein was purified using T7•Tag affinity purification kit and used as antigen for raising polyclonal antibodies in two mice. The purified anti-CMV CP IgG and the conjugated IgG performed favourably in terms of specificity and sensitivity to detect both expressed CP (antigen) and CMV isolates in infected cucurbit plants using plate trapped antigen (PTA)- ELISA, double-antibody sandwich (DAS)-ELISA and western blotting. The prepared antibodies can be applied in serological and sero-molecular tests in studies on the virus and in screening of plants for the infection. This is the first report of preparation of antibodies against CP of an indigenous isolate of CMV.
Abou-Jawdah, Y., Sobh, H., Cordahi, N., Kawtharani, H., Nemer, G., Maxwell, D. P. and Nakhla, M. K. 2004. Immunodiagnosis of Prune dwarf virus using antiserum produced to its recombinant coat protein. Journal of Virological Methods, 121: 31-38.
Amer, M., El-Hammady, M., Mazyad, H., Shalaby, A. and Abo-Elabbas, F. 2004. Cloning, expression and nucleotide sequence of coat protein gene of an Egyptian isolate of Potato virus Y strain NTN Infecting Potato Plants. Egyptian Journal Of Virology, 1: 39:50.
Barbieri, M. R., Carvalho, M. G. d., Zambolim, E. M. and Zerbini, F. M. 2004. Expression in Escherichia coli of the capsid protein of Watermelon mosaic virus and production of specific antiserum. Fitopatologia Brasileira, 29: 215-219.
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248-254.
Cerovska, N., Filigarova, M. and Pecenkova, T. 2006. Production of polyclonal antibodies to a recombinant Potato mop-top virus Non-structural triple gene block protein 1. Journal of Phytopathology, 154: 422-427.
Cerovska, N., Moravec, T., Plchova, H., Hoffmeisterova, H. and Dedic, P. 2012. Production of polyclonal antibodies to the recombinant Potato virus M (PVM) Non‐structural triple gene block protein 1 and coat protein. Journal of Phytopathology, 160: 251-254.
Dijkstra, J. and de Jager, C. P. 1998. Practical Plant Virology: Protocols and Exercises. Springer-Verlag, Berlin, Germany.
Gulati-Sakhuja, A., Sears, J. L., Nunez, A. and Liu, H. Y. 2009. Production of polyclonal antibodies against Pelargonium zonate spot virus coat protein expressed in Escherichia coli and application for immunodiagnosis. Journal of Virological Methods, 160: 29-37.
Hartley, J. L. 2006. Cloning technologies for protein expression and purification. Current Opinion in Biotechnology, 17: 359-366.
Jain, R., Pandey, A. N., Krishnareddy, M. and Mandal, B. 2005. Immunodiagnosis of groundnut and Watermelon bud necrosis viruses using polyclonal antiserum to recombinant nucleocapsid protein of Groundnut bud necrosis virus. Journal of Virological Methods, 130: 162-164.
King, A. M., Adams, M. J. and Lefkowitz, E. J. 2011. Virus taxonomy: classification and nomenclature of viruses: Ninth Report of the International Committee on Taxonomy of Viruses: Elsevier, New Yourk, USA.
Komorowska, B., Malinowski, T. and Michalczuk, L. 2010. Evaluation of several RT-PCR primer pairs for the detection of Apple stem pitting virus. Journal of Virological Methods, 168: 242-247.
Koolivand, D., Sokhandan-Bashir, N., Behjatnia. S. A. A. and Jafari-Joozani, R. 2014. Detection of Grapevine fanleaf virus by immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) with recombinant antibody. Archives of Phytopathology and Plant Protection, 47: 2070-2077.
Koolivand, D., Sokhandan-Bashir, N., Behjatnia. S. A. A. and Jafari-Joozani, R. 2016. Production of polyclonal antibody against Grapevine fanleaf virus movement protein expressed in Escherichia coli. The Plant Pathology Journal, 32: 452-459.
Korimbocus, J., Preston, S., Danks, C., Barker, I., Coates, D. and Boonham, N. 2002. Production of monoclonal antibodies to Sugarcane yellow leaf virus using recombinant readthrough protein. Journal of Phytopathology, 150: 488-494.
Kumari, S., Makkouk, K., Katul, L. and Vetten, H. 2001. Polyclonal antibodies to the bacterially expressed coat protein of Faba bean necrotic yellows virus. Journal of Phytopathology, 149: 543-550.
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.
Lee, S. C. and Chang, Y. C. 2008. Performances and application of antisera produced by recombinant capsid proteins of Cymbidium mosaic virus and Odontoglossum ringspot virus. European Journal of Plant Pathology, 122: 297-306.
Lima, J. A. A., Nascimento, A. K. Q., Radaelli, P. and Purcifull, D. E. 2012. Serology applied to plant virology. Serological diagnosis of certain human, animal and plant diseases. Rijeka Croácia. InTech: 71-94.
Lima, J., Sittolin, I., Lima, R., Freire Filho, F. and Lima, J. 2005. Diagnose e estratégias de controle de doenças ocasionadas por vírus. Feijão caupi: avanços tecnológicos. Brasília: Embrapa Informação Tecnológica, pp: 404-459.
Mutasa-Gottgena, E. S., Chwarszczynska, D. M., Halsey, K. and Asher, M. J. C. 2000. Specific polyclonal antibodies for the obligate plant parasite Polymyxa a targeted recombinant DNA approach. Plant Pathology, 49: 276-278.
Nickel, O., Targon, M. L., Fajardo, T. V., Machado, M. A. and Trivilin, A. P. 2004. Polyclonal antibodies to the coat protein of Apple stem grooving virus expressed in Escherichia coli: production and use in immunodiagnosis. Fitopatologia Brasileira, 29: 558-562.
Palukaitis, P. and Garcia-Arenal, F. 2003. Cucumoviruses. Advances in Virus Research, 62: 241-323.
Plchova, H., Moravec, T., Dedic, P. and Cerovska, N. 2011. Expression of recombinant Potato leafroll virus structural and non-structural proteins for antibody production. Journal of Phytopathology, 159: 130-132.
Raikhy, G., Hallan, V., Kulshrestha, S. and Zaidi, A. 2007. Polyclonal antibodies to the coat protein of Carnation etched ring virus expressed in bacterial system: production and use in immunodiagnosis. Journal of Phytopathology, 155: 616-622.
Roossinck, M. J. 2001. Cucumber mosaic virus, a model for RNA virus evolution. Molecular Plant Pathology, 2: 59-63.
Rostami, A., Sokhandan-Bashir, N., Pirniakan, P. and Masoudi, N. 2014. Expression of Cucumber mosaic virus coat protein and its assembly into virus like particles. Biotechnology and Health Scinces, 1: 1-11.
Sokhandan-Bashir, N., Kalhor, M. R. and Zarghani, S. N. 2006. Detection, differentiation and phylogenetic analysis of Cucumber mosaic virus isolates from cucurbits in the northwest region of Iran. Virus Genes, 32: 277-288.
Surendran, S., Mathai, A. and Radhakrishna, V. V. 2015. Western blotting, In: Kurien, B. T. and Scofield, R. H. (Eds.), Methods in Molecular Biology, Springer, New York, Vol. 1312, pp. 105-108.
Wetzel, T., Candresse, T., Macquaire, G., Ravelonandro, M. and Dunez, J. 1992. A highly sensitive immunocapture polymerase chain reaction method for Plum pox potyvirus detection. Journal of Virological Methods, 39: 27-37.
Xu, Z., Hong, N., Xing, B. and Wang, G. 2006. Partial molecular characterization of a Chinese isolate of Grapevine leafroll-associated virus 2 and production of antisera to recombinant viral proteins. Journal of Plant Pathology, 88: 89-94.