Development of a polyclonal antibody against the coat protein of Prunus necrotic ring spot virus

Document Type : Research paper

Authors

Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Zanjan, Iran.

Abstract

The Prunus necrotic ring spot virus (PNRSV) poses a significant threat to the global stone fruit industry. In this study, samples suspected of PNRSV infection were gathered from Iran’s primary stone fruit-growing areas. The coat protein (CP) gene of the isolates was amplified, and the nucleic acid and amino acid sequences of the PNRSVs were determined. Phylogenetic analysis revealed that these isolates belonged to the PV-96-II, a prominent group of PNRSVs found worldwide. An isolate from this clade was chosen to express the CP gene in Escherichia coli. The CP gene of this isolate was cloned into the pET28 vector for expression, and the resulting coat protein was purified as a native protein using Ni-NTA agarose. The purified protein served as a recombinant antigen to generate anti-PNRSV-CP antiserum in rabbits. Purified anti-PNRSV-CP IgG and conjugated IgG showed specificity and sensitivity, successfully detecting expressed CP and PNRSV isolates in infected stone fruit trees through various serological and sero-molecular techniques such as plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA), Double Antibody Sandwich-ELISA (DAS-ELISA), and western blotting. These antibodies can be valuable in virus and plant screening studies, as well as serological and sero-molecular tests. This study represents the first documentation of a polyclonal antibody preparation against the CP of an Iranian PNRSV isolate, offering potential assistance in controlling and preventing this economically significant virus in the future.

Keywords

References

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.
Afrashteh M., Koolivand D., Hajizadeh M., and Masoudi N. (2023). Preparation of recombinant polyclonal antibody against Apple chlorotic leaf spot virus and its efficiency. Genetic Engineering and Biosafety Journal, 12: 1-12. (In Persian)
Amiri Sadeghan A., Shams-Bakhsh M., and Yakhchali B. (2013). Expression of Citrus tristeza virus coat protein gene in Escherichia coli. Journal of Crop Protection, 2: 387-393.
Antony A. C., Louis V., Dilip D., and Cherian K. (2021). Purification of recombinant Cucumber mosaic virus (banana isolate) coat protein by sucrose density gradient ultra-centrifugation. Journal of Tropical Agriculture, 58: 12-25.
Aparicio F., Vilar M., Perez-Payá E., and Pallás V. (2003). The coat protein of prunus necrotic ringspot virus specifically binds to and regulates the conformation of its genomic RNA. Virology, 313: 213-223.
Barbieri M. R., Carvalho M. G. D., Zambolim E. M., and Zerbini F. (2004). Expression in Escherichia coli of the capsid protein of Watermelon mosaic virus and production of specific antiserum. Fitopatologia Brasileira, 29: 215-219.
Bragard C., Duncan G. H., Wesley S., Naidu R., and Mayo M. (2000). Virus-like particles assemble in plants and bacteria expressing the coat protein gene of Indian peanut clump virus. Journal of General Virology, 81: 267-272.
Carvalho S. L. D., Silva F. N. D., Zanardo L. G., Almeida Á. M., Zerbini F. M., and Carvalho C. (2013). Production of polyclonal antiserum against Cowpea mild mottle virus coat protein and its application in virus detection. Fitopatologia Brasileira, 38: 49-54.
Cerovska N., Filigarova M., and Pečenková 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.
Chen C. C., Hsiang T., Chiang F. L., and Chang C. (2002). Molecular characterization of Tuberose mild mosaic virus and preparation of its antiserum to the coat protein expressed in bacteria. Phytopathology, 96: 1296-1304.
Chung C., Niemela S. L., and Miller R. H. (1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proceedings of the National Academy of Sciences, 86: 2172-2175.
Dhir S., Lakshmi V., and Hallan V. (2021). Development of immunodiagnostics for Apple stem pitting virus and Apple mosaic virus infecting apple in India. Indian Phytopathology, 74: 189-199.
Fajardo T. V., Barros D. R., Nickel O., Kuhn G. B., and Zerbini F. (2007). Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies. Fitopatologia Brasileira, 32: 496-500.
Fiore N., Fajardo T. V., Prodan S., Herranz M. C., Aparicio F., et al. (2008). Genetic diversity of the movement and coat protein genes of South American isolates of Prunus necrotic ringspot virus. Archives of Virology, 153: 909-919.
Folwarczna J., Plchova H., Moravec T., Hoffmeisterova H., Dědič P., and Čeřovská N. (2008). Production of polyclonal antibodies to a recombinant coat protein of potato virus Y. Folia Microbiologica, 53: 438-442.
Glasa M., Betinova E., Kúdela O., and Šubr Z. (2002). Biological and molecular characterisation of Prunus necrotic ringspot virus isolates and possible approaches to their phylogenetic typing. Biotechnology & Biotechnological Equipment, 140: 279-283.
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 Virologica Methods, 160: 29-37.
Hamdayanty H., Hidayat S. H., Damayanti T. (2016). Expression of recombinant Sugarcane streak mosaic virus coat protein gene in Escherichia coli. HAYATI Journal of Biosciences, 23(3): 111-116.
Hammond R. W., and Crosslin J. M. (1995). The complete nucleotide sequence of RNA 3 of a peach isolate of Prunus necrotic ringspot virus. Virology, 208: 349-353.
Jacob T., and Usha R. (2002). Expression of Cardamom mosaic virus coat protein in Escherichia coli and its assembly into filamentous aggregates. Virus Research, 86: 133-141.
King, A. M. Q., Lefkowitz E. J., Adams M. J., and Carstens E. B. (2011). Virus taxonomy: ninth report of the International Committee on Taxonomy of Viruses. Elsevier, pp. 1327.
Koolivand D., Bashir N. S., Behjatnia S. A., and Joozani R. J. (2016). Production of polyclonal antibody against Grapevine fanleaf virus movement protein expressed in Escherichia coli. Plant Patthology Journal, 32: 452-463.
Koolivand D., Bashir N. S., Behjatnia S. A., and Joozani R. J. (2014). Detection of Grapevine fanleaf virus by immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) with recombinant antibody. Archives of Phytopathology and Protection, 47: 2070-2077.
Koolivand D., Sokhandan Bashir N., and Rostami A. (2017). Preparation of polyclonal antibody against recombinant coat protein of Cucumber mosaic virus isolate B13. Journal of Crop Protection, 6: 25-34.
Korimbocus J., Preston S., Danks C., Barker I., Coates D., and Boonham N. J. (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.
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. In book: Serological Diagnosis of Certain Human, Animal and Plant Diseases, IntechOpen. DOI: 10.5772/38038.
Liu W., He R., Xue Z. J. P., and Letters P. (2001.) Expression of the n-terminal segment of qbrn-2 in E. coli and tips on preparation of a recombinant protein. Protein & Peptide Letters, 8: 27-32.
Lotfi A., Shams-Bakhsh M., and Yakhchali B. (2015). Production of polyclonal antiserum against beet western yellows virus coat protein expressed in Escherichia coli. Journal of Crop Protection, 4: 739-746.
Mansour K., Kominek B., Kominkova M., and Brozova J. (2023). Characterization of Prunus Necrotic ringspot virus and Cherry virus A Infecting Myrobalan Rootstock. Viruses. 15: 1723-1735.
Masoudi N., Assareh M. H., Rouhibakhsh A., Madani R., Naderpour M., Emami T., Amini S. M., and Koolivand D. (2019). Rapid detection of Potato virus S using antibody-coated gold nanoparticles. Iranian Journal of Plant Pathology, 55: 105-114.
Masoudi N., Rouhibakhsh A., Asareh M. H., Naderpour M., and Koolivand D. (2018). Identification of dominant isolate of Potato virus S in Iran and heterologous expression of its coat protein. Genetic Engineering and Biosafety Journal, 7: 153-162.
Milne J., and Walter G. (2003). The coincidence of thrips and dispersed pollen in PNRSV‐infected stonefruit orchards–a precondition for thrips‐mediated transmission via infected pollen. Annals of Applied Biology, 142: 291-298.
Mohammadloo M., Koolivand D., Hajizadeh M., and Eini O. (2023). Designing and evaluation of ELISA kit based on preparing antibodies against Apple stem grooving virus coat protein gene expressed in Escherichia coli. Agricultural Biotechnology Journal, 15: 1-24.
Mutasa‐Gottgens E., Chwarszczynska D., Halsey K., and Asher M. (2000). Specific polyclonal antibodies for the obligate plant parasite Polymyxa—a targeted recombinant DNA approach. Journal of Plant Pathology, 49: 276-287.
Noorani M. S., Bag M. S., Khan J. A., and Pravej A. (2023). Whole genome characterization and diagnostics of prunus necrotic ringspot virus (PNRSV) infecting apricot in India. Scientific Reports, 13: 4393.
Oliver J., Freer J., Andersen R., Cox K., Robinson T., and Fuchs M. (2009). Genetic diversity of Prunus necrotic ringspot virus isolates within a cherry orchard in New York. Journal of Plant Disease, 93: 599-606.
 Pallas V., Aparicio F., Herranz M., Amari K., Sanchez-Pina M., Myrta A., and Sanchez-Navarro J. (2012). Ilarviruses of Prunus spp.: A continued concern for fruit trees. Phytopathology, 102: 1108-1120.
Pallás V., Aparicio F., Herranz M., Sánchez-Navarro J., and Scott S. (2013). The molecular biology of ilarviruses. Advances in Virus Research, 87: 139-141.
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.
Saini M., and Vrati S. (2003). High-level synthesis of Johnson grass mosaic virus coat protein in Escherichia coli and its auto-assembly to form virus-like particles. Protein Expression and Purification, 28: 86-92.
Sharma P., Sharma S., Singh J., Saha S., and Baranwal V. (2016). Incidence of Lettuce mosaic virus in lettuce and its detection by polyclonal antibodies produced against recombinant coat protein expressed in Escherichia coli. Journal of Virologica Methods, 230: 53-58.
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.
Sokhandan-Bashir N., Kashiha Z., Koolivand D., and Eini O. (2017). Detection and phylogenetic analysis of Prunus necrotic ringspot virus isolates from stone fruits in Iran. Journal of Plant Pathology, 99: 723-729.
Sokhandan- Bashir, N., Koolivand D. and Behjatnia S. A. A. (2015). Preparation of polyclonal antibodies to Grapevine fanleaf virus coat protein expressed in Escherichia coli. Biotechnology, 14: 173-180.
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.

Files

History

  • Receive Date: 07 October 2023
  • Revise Date: 08 April 2024
  • Accept Date: 09 April 2024

Share

How to cite

Statistics