SELECTION OF SPECIFIC SINGLE CHAIN VARIABLE FRAGMENTS (SCFV) AGAINST POLYMYXA BETAE FROM PHAGE DISPLAY LIBRARIES

Open access

Abstract

Sugar beet is one of the most important industrial crops in Iran. For the last two decades it has been mainly affected by a destructive virus, beet necrotic yellow vein virus (BNYVV). The Polymyxa betae is the only natural transmitting agent of the disease among the plants. Developing accurate diagnostic methods may have a major impact on the rising of resistant germplasms. In the present study, specific monoclonal recombinant antibodies in the form of single chain variable fragments (scFv) were obtained from naïve phage display libraries. The fungus specific glutathione-S-transferase (GST) protein was chosen as an antigen for developing antibodies and diagnostic purposes. To generate specific scFv, screening of Tomlinson phage display libraries was performed by applying both recombinant and native fungal GST. Using the recombinant GST in the panning process resulted in the isolation of an antibody only bound to recombinant GST but it failed to detect native GST in the infected plants. Alternatively, the process of panning was carried out by applying native fungal GST trapped to immunotubes through specific polyclonal antibody intermediate. The recent approach resulted in the selection of a specific scFv binding to native GST which was able to detect the presence of the fungi within infected plants. To the best of our knowledge, this is the first report on the generation of recombinant antibodies against Polymyxa betae, fungal vector of sugar beet rhizomania disease.

Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A., Struhl K. (eds.). 1995. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, NJ: 3.1.1-3.1.21.

Cervera M., Esteban O., Gil M., Gorris M.T., Martínez M.C., Peña L. 2010. Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance. Transgenic Res. 19 (6): 1001-1015.

Fischer R., Emans N., Schillberg S. 2001. Achieving plant disease resistance by antibody expression. Can. J. Plant Pathol. 23 (3): 236-245.

Franconi R., Roggero P., Pirazzi P., Arias F.J., Desiderio A., Bitti O., Pashkoulov D., Mattei B., Bracci L., Masenga V., Milne R.G., Benvenuto E. 1999. Functional expression in bacteria and plants of an scFv antibody fragment against tospoviruses. Immunotechnology 4 (3-4): 189-201.

Griep R.A., Van Twisk C., Schots A. 1999. Selection of Beet necrotic yellow vein virus-specific monoclonal antibodies from a semisynthetic combinatorial antibody library. Eur. J. Plant. Pathol. 105 (2): 147-156.

Griffiths A.D., Williams S.C., Hartley O., Tomlinson I.M., Waterhouse P., Crosby W.L., Kontermann R.E., Jones P.T., Low N.M., Allison T.J., Prospero T.D., Hoogenboom H.R., Nissim A., Cox J.P.L., Harrison J.L., Zaccolo M., Gherardi E., Winter G. 1994. Isolation of high affinity antibodies directly from large synthetic repertoires. EMBO J. 13 (14): 3245-3260.

Hoogenboom H.R., Griffiths A.D., Johnson K.S., Chiswell D.J., Hudson P., Winter G. 1991. Multi-subunit proteins on the surface of filamentous phage: Methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res. 19 (15): 4133-4137.

Hu Z.Q., Liu J.L., Li H.P., Xing S., Xue S., Zhang J.B., Wang J.H., Nölke G., Liao Y.C. 2012. Generation of a highly reactive chicken-derived single-chain variable fragment against Fusariumverticillioides by phage display. Int. J. Mol. Sci. 13 (6): 7038-7056.

Jahromi Z.M., Salmanian A.H., Rastgoo N., Arbabi M. 2009. Isolation of BNYVV coat protein specific single chain Fv from a mouse phage library antibody. Hybridoma (Larchmt) 28 (5): 305-313.

Keskin B. 1964. Polymyxa betae n. sp., ein parasit in den Wurzeln von Beta vulgaris Tournefort, besonders während der Jugendentwicklung der Zuckerrübe. Arch. Mikrobiol. 49 (4): 348-374.

Kirakosyan A., Kaufman P.B. 2009. Recent advances in plant biotechnology. p. 35-63. In “Molecular Farming of Antibodies in Plants” (R. Fischer, S. Schillberg, R.M. Twyman, eds.). Springer, USA, 428 pp.

Krishnaswamy S., Kabir M.E., Rahman M.M., Miyamoto M., Furuichi Y., Komiyama T. 2011. Isolation and characterization of recombinant single chain fragment variable anti-idiotypic antibody specific to Aspergillus fumigates membrane protein. J. Immunol. Methods 366 (1): 60-68.

Li H.P., Zhang J.B., Shi R.P., Huang T., Fischer R., Liao Y.C. 2008. Engineering Fusarium head blight resistance in wheat by expression of a fusion protein containing a Fusarium-specific antibody and an antifungal peptide. Mol. Plant. Microbe. In. 21: 1242-1248.

Liu F., Sukhacheva E., Erokhina T., Schubert J. 1999. Detection of potyviral nuclear inclusion b proteins by monoclonal antibodies raised to synthetic peptides. Eur. J. Plant Pathol. 105 (4): 389-395.

Marks J.D., Hoogenboom H.R., Bonnert T.P., McCafferty J., Griffiths A.D., Winter G. 1991. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222 (3): 581-597.

Mowat W. 1985. Simplified enzyme immunoassay for plant virus detection and identification. Report of the Scottish Crop Research Institute for 1984, 188 pp.

Mutasa-Gottgens E.S., Chwarszczynska D.M., Halsey K., Asher M.J.C. 2000. Specific Polyclonal antibodies for the obligate plant parasite Polymyxa - a targeted recombinant DNA approach. Plant Pathol. 49 (2): 276-287.

Öztetik E. 2008. A tale of plant glutathione S-transferases: since 1970. Bot. Rev. 74 (3): 419-437.

Peschen D., Li H.P., Fischer R., Kreuzaler F., Liao Y.C. 2004. Fusion proteins comprising a Fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen. Nat. Biotechnol. 22 (6): 732-738.

Prins M., Lohuis D., Schots A., Goldbach R. 2005. Phage displayselected single-chain antibodies confer high levels of resistance against Tomato spotted wilt virus. J. Gen. Virol. 86 (7): 2107-2113.

Richard-Molard M. 2002. Rhizomania: interactions varietes x lieux et consequences. Proceedings of the 65th IIRB Congress, Brussels: 239-245.

Rodi D.J., Makowski L., Kay B.K. 2001. One from column A and two from column B: the benefits of phage display in molecular- recognition studies. Curr. Opin. Chem. Biol. J. 6 (1): 92-96.

Safarnejad M.R., Fischer R., Commandeur U. 2008. Generation and characterization of functional recombinant antibody fragments against Tomato yellow leaf curl virus replication- associated protein. Commun. Agric. Appl. Biol. Sci. 73 (2): 311-323.

Safarnejad M.R., Fischer R., Commandeur U. 2009. Recombinant- antibody-mediated resistance against Tomato yellow leaf curl virus in Nicotiana benthamiana. Arch. Virol. 154 (3): 457-467.

Safarnejad M.R., Salehi Jouzani G.R., Tabatabaie M., Twyman R.M., Schillberg S. 2011. Antibody-mediated resistance against plant pathogens. Biotechnol. Adv. 29 (6): 961-971.

Safarpour H., Safarnejad M.R., Basirat M., Hasanzadeh F., Kamyab F. 2012. Development of a specific serological kit for detection of Polymyxa betae, transmitting agent of sugar beet Rhizomania disease. J. Food Agric. Environ. 10 (3-4): 729-732.

Schillberg S., Zimmermann S., Zhang M.Y., Fischer R. 2001. Antibody- based resistance to plant pathogens. Transgenic Res. 10 (1): 1-12.

Treuheit M.J., Kosky A.A., Brems D.N. 2001. Inverse relationship of protein concentration and aggregation. Pharmaceut. Res. 19 (4): 511-516.

Villani M.E., Roggero P., Bitti O., Benvenuto E., Franconi R. 2005. Immunomodulation of cucumber mosaic virus infection by intra bodies selected in vitro from a stable single framework phage display library. Plant Mol. Biol. 58 (3): 305-16.

Yajima W., Rahman M.H., Das D., Suresh M.R., Kav N.N. 2008. Detection of Sclerotinia sclerotiorum using a monomeric and dimeric single-chain fragment variable (scFv) antibody. J. Agric. Food Chem. 56 (20): 9455-9463.

Journal of Plant Protection Research

The Journal of Polish Society of Plant Protection, Committee of Plant Protection; Polish Academy of Sciences, Institute of Plant Protection – National Research Institute

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CiteScore 2016: 0.84

SCImago Journal Rank (SJR) 2016: 0.332
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