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Functional expression in bacteria and plants of an scFv antibody fragment against tospoviruses

TitleFunctional expression in bacteria and plants of an scFv antibody fragment against tospoviruses
Publication TypeArticolo su Rivista peer-reviewed
Year of Publication1999
AuthorsFranconi, Rosella, Roggero P., Pirazzi P., Arias F.J., Desiderio Angiola, Bitti O., Pashkoulov D., Mattei B., Bracci L., Masenga V., Milne R.G., and Benvenuto Eugenio
Pagination189 - 201
Date Published1999
ISBN Number13802933 (ISSN)
KeywordsAmino Acid Sequence, Antibodies, antibody production, antibody specificity, antiviral activity, article, Base Sequence, Diagnostic, Escherichia coli, Immunoglobulin Fragments, immunoglobulin variable region, Molecular Sequence Data, Monoclonal, nonhuman, Phage display, plantibody, Plants, priority journal, protein engineering, PVX vector, Recombinant Proteins, Secretory pathway, serodiagnosis, Tobacco, Tospovirus, Toxic, Viral, virus infection

Background: Recombinant antibodies expressed in plants (’plantibodies’), directed against crucial antigens and addressed to the right cell compartment, may be able to protect against viral diseases. Moreover, antibody fragments produced in bacteria or plants may provide low cost reagents for immunodiagnosis. Objectives: In an attempt to develop genetic immunisation against tomato spotted wilt tospovirus (TSWV), we engineered an scFv fragment starting from a monoclonal antibody (mAb) able to recognise an epitope of the glycoprotein G1 conserved among a large number of tospoviruses. After establishing functional expression in bacteria, we aimed to drive expression of this molecule in the secretory pathway of plants. Study design: An antibody phage display expression system was used to isolate the correct VH and VL binding regions from the hybridoma secreting the original mAb. To assess functional expression in plant, we first used an epichromosomal expression vector derived from potato virus X (PVX). In this vector the scFv gene was cloned to produce a cytosolic or a secretory protein. For secretion, the signal sequence derived from the polygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris was used. Subsequently, the gene encoding the secretory scFv, was used to transform Nicotiana benthamiana plants. Results: High expression levels of fully active molecule were obtained in Escherichia coli. The engineered molecule retained the binding specificity and dissociation rate constant (k(off)) of the cognate monoclonal antibody. Both PVX-infected and transformed plants expressed fully functional scFv molecules in the secretory pathway. Conclusion: This engineered scFv may be valuable for inexpensive diagnosis, for studying the role of the glycoproteins in virus transmission and, possibly, for a ’plantibody’-mediated resistance to tospoviruses.


Cited By :37Export Date: 16 July 2015CODEN: IOTEECorrespondence Address: Benvenuto, E.; ENEA, Dipartimento Innovazione, Divisione Biotecnol. e Agricoltura, PO Box 2400, Rome, Italy; email: benvenutoe@casaccia.enea.itChemicals/CAS: Antibodies, Monoclonal; Antibodies, Viral; Immunoglobulin Fragments; immunoglobulin Fv; Immunoglobulin Variable Region; Recombinant ProteinsReferences: Cattaneo, A., Biocca, S., (1997) Intracellular Antibodies: Development and Applications, , Berlin: Springer;Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman, B.M., Lee, S.M., Lee, T., Whitlow, M., (1988) Science, 242, pp. 423-426; Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, M.S., Novotny, J., Margolies, M.N., Ridge, R.J., Oppermann, H., (1988) Proc Natl Acad Sci USA, 85, pp. 5879-5883; Tavladoraki, P., Benvenuto, E., Trinca, S., De Martinis, D., Cattaneo, A., Galeffi, P., (1993) Nature, 366, pp. 469-472; Marasco, W.A., Haseltine, W.A., Chen, S., (1993) Proc Natl Acad Sci USA, 90, pp. 7889-7893; Hartl, F.U., (1996) Nature, 381, pp. 571-580; German, T.L., Ullman, D.E., Moyer, J.W., (1992) Annu Rev Phytopathol, 30, pp. 315-348; Goldbach, R., Peters, D., (1996) The Bunyaviridae, pp. 129-157. , Elliot RM, editor. New York: Plenum Press; Prins, M., Goldbach, R., (1998) Trends Microbiol, 6, pp. 31-35; Kormelink, R., De Haan, P., Meurs, C., Peters, D., Golbach, R., (1992) J Gen Virol, 73, pp. 2795-2804; Adkins, S., Choi, T.-J., Israel, A.B., Bandla, M.D., Richmond, K.E., Schultz, K.T., Sherwood, J.L., German, T.L., (1996) Phytopathology, 86, pp. 849-855; Adam, G., Yeh, S.D., Reddy, D.V.R., Green, S.K., (1993) Arch Virol, 130, pp. 237-250; Feldhoff, A., Kikkert, M., Kormelink, R., Krczal, G., Goldbach, R., Peters, D., (1997) Arch Virol, 142, pp. 781-793; Kitajima, E.W., De Avila, A.C., Resende, R., De, O., Goldbach, R.W., Peters, D., (1992) J Submicrosc Cytol Pathol, 24, pp. 1-14; Resende, R., De, O., De Haan, P., De Avila, A.C., Kitajima, E.W., Kormelink, R., Goldbach, R., Peters, D., (1991) J Gen Virol, 72, pp. 2375-2383; Wijkamp, I., (1995) Virus-vector Relationships in the Transmission of Tospoviruses, pp. 1-143. , Ph.D. Thesis, University of Wageningen; Adam, G., Roggero, P., Malavasi, F., Milne, R.G., Papa, G., (1995) OEPP/EPPO Bull, 25, pp. 247-257; Roggero, P., Ogliara, P., Dellavalle, G., Lisa, V., Malavasi, F., Adam, G., (1996) Acta Hort, 431, pp. 167-175; Roggero, P., Ogliara, P., Ramasso, E., Arzone, A., Tavella, L., Alma, A., (1996) Acta Hort, 431, pp. 333-340; Adam, G., Peters, D., Goldbach, R., (1996) Acta Hort, 431, pp. 135-158; Toubart, P., Desiderio, A., Salvi, G., Cervone, F., Daroda, L., De Lorenzo, G., Bergmann, C., Albersheim, P., (1992) Plant J, 2, pp. 367-373; Chapman, S., Kavanagh, T., Baulcombe, D., (1992) Plant J, 2, pp. 549-557; Evan, G.I., Lewis, G.K., Ramsay, G., Bishop, J.M., (1985) Mol Cell Biol, 5, pp. 3610-3616; Roggero, P., Adam, G., Milne, R.G., Lisa, V., (1996) Eur J Plant Pathol, 102, pp. 563-568; Bradbury, A., Ruberti, F., Werge, T., Amati, V., Di Luzio, A., Gonfloni, S., Hoogenboom, H., Cattaneo, A., (1995) Antibody Engineering 2, pp. 295-361. , C.A.K. Borrebaeck. Oxford: Oxford University Press; Hoogenboom, H.R., Griffiths, A.D., Johnson, K.S., Chiswell, D.J., Hudson, P., Winter, G., (1991) Nucl Acids Res, 19, pp. 4133-4137; Tavladoraki, P., Franconi, R., Benvenuto, E., (1995) Antibody Engineering, pp. 363-376. , C.A.K. Borrebaeck. Oxford: Oxford University Press; Sambrook, J., Fritsch, E.F., Maniatis, T., (1989) Molecular Cloning: A Laboratory Manual, 2. , Cold Spring Harbor: Cold Spring Harbor Laboratory Press; Tavazza, R., Ordas, R.J., Tavazza, M., Ancora, G., Benvenuto, E., (1988) J Plant Physiol, 133, pp. 640-644; Bradford, M.M., (1976) Anal Biochem, 72, pp. 248-254; De Wilde, C., De Neve, M., De Rycke, R., Bruyns, A.-M., De Jaeger, G., Van Montagu, M., Depicker, A., Engler, G., (1996) Plant Sci, 114, pp. 233-241; Kabat, E.A., Wu, T.T., Perry, H.M., Gottesmann, K.S., Foeller, C., (1991) Sequences of Proteins of Immunological Interest. 5th Ed., , US Department of Health and Human Services, US Government Printing Office; Malmqvist, M., (1993) Nature, 361, pp. 186-187; Ruberti, F., Cattaneo, A., Bradbury, A., (1994) J Immunol Meth, 173, pp. 33-39; Winter, G., Griffiths, A.D., Hawkins, R.E., Hoogenboom, H.R., (1994) Annu Rev Immunol, 12, pp. 433-455; Jiang, W., Bonnert, T.P., Venugopal, K., Gould, E.A., (1994) Virology, 200, pp. 21-28; Knappick, A., Krebber, C., Plückthun, A., (1993) Bio/technology, 11, pp. 77-83; Knappick, A., Plückthun, A., (1995) Protein Eng, 8, pp. 81-89; Steipe, B., Schiller, B., Plückthun, A., Steinbacher, S., (1994) J Mol Biol, 240, pp. 188-192; Kerschbaumer, R.J., Hirschl, S., Kaufmann, A., Ibl, M., Koenig, R., Himmler, G., (1997) Anal Biochem, 249, pp. 219-227; Neri, D., Montigiani, S., Kirkham, P.M., (1996) TIBTECH, 14, pp. 465-470; Scholthof, H.B., Scholthof, K.-B.G., Jackson, A.O., (1996) Annu Rev Phytopathol, 34, pp. 299-323; Glockshuber, R., Schmidt, T., Plückthun, A., (1992) Biochemistry, 31, pp. 1270-1279

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