Expression of a Functional Anti-Cucumber Mosaic Virus Single-Chain Variable Fragment Antibody in Tobacco Plants (Nacotiana tabacum)

  • Heng Chua Kek (Department of Molecular Medicine, Faculty of Medicine, University of Malaya) ;
  • Khalid Norzulaani (Institute of Biological Sciences, Faculty of Science, University of Malaya) ;
  • Othman Retina Yasmin (Institute of Biological Sciences, Faculty of Science, University of Malaya)
  • Published : 2006.03.01


As an alternative method to produce low cost reagents for immunodiagnosis and protect the plants from viral disease, a gene encoding a single chain variable fragment(scFv) recombinant antibody targeted to the coat protein of cucumber mosaic virus (CMV) was expressed in Nacotiana tabacum. The source of the scFv recombinant antibody gene was from spleen tissue of an immunized mouse. The gene was initially cloned into the pCANTAB5E phagemid and expressed in E. coli. In the following study, the antibody gene was subcloned into the plant expression vector, pCAMBIA-1301 and introduced into tobacco leaf tissue via Agrobacterium tumefacients mediated transformation. After transformation, 56 out of 58 plants were shown to carry the desired anti-CMV scFv gene by PCR analysis. Overall, only 12.5% of the 56 putative transgenic plants were found to express the antibody to a detectable level.


  1. Bird RE, Hardman KD, Jacobson JW, Johnson S, Kaufman BM, Lee T, Pope HS, Riordan GS, Whitlow M (1988) Single-chain antibody binding proteins. Science 242: 423-426
  2. Chen B, Francki RIB (1990) Cucumovirus transmission by the aphid Myzus-persicae is determined solely by the viral coat protein. J Gen Virol 71: 939-944
  3. Chua KH, Noorzulaani K, Tan CS, JA Harikhrisna, Rofina YO (2003) Synthesis of a soluble flag-tagged single chain variable fragment (scFv) antibody targeting cucumber mosaic virus (CMV) coat protein. Asia Pacific J Mol Biol Biotechnol 11: 93-100
  4. Chua KH, JA Harikrishna, Norzulaani K, Rofina YO (2005) Direct cloning approach for expression of an anti-cucumber mosaic virus single chain variable fragment in plants. Biotechnol 4: 206-210
  5. Clackson T, Hoogenboom HR, Griffiths AD, Winter G (1991) Making antibody fragments using phage display libraries. Nature 352: 624-628
  6. Doolittle SP (1916). A new infectious mosaic disease of cucumber. Phytopathology 6: 145
  7. Fiedler U, Philips J, Artsaenko O, Conrad U (1997) Optimisation of scFv antibody production in transgenic plants. Immunotechnol 3: 205-216
  8. Franconi R, Roggero P, Pirazzi P, Arias FJ, Desiderio A, Bitti O, Pashkoulov D, Mattei B, Bracci L, Masenga V, Milne RG, Benvenuto E (1999) Functional expression in bacteria and plants of an scFv antibody fragment against tospoviruses. Immunotechnol 4: 189-201
  9. Fuchs M, Prowidenti R, Slightom JL, Gonsalves D (1996) Evaluation of transgenic tomato plants expressing the coat protein gene of cucumber mosaic-virus strain WL under field conditions. Plant Dis 80: 270-275
  10. Fuchs M, McFerson JR, Tricoli DM, McMaster JR, Dang RZ, Boeshore ML, Reynolds JF, Russell PF, Quemada HD (1997) Cantaloupe line CZW-30 containing coat protein genes of cucumber mosaic virus, zucchini yellow mosaic virus, and watermelon mosaic virus-2 is resistant to these three viruses in the field. Mol Breeding 3: 279-290
  11. Gera A, Leobenstein G, Raccah B (1979) Protein coats of two strains of cucumber mosaic virus affect transmission by Aphis gossypii. Phytopathology 69: 369-399
  12. Gonda TJ, Symons RH (1979) Cucumber mosaic virus replication in cowpea protoplasts: time course of virus, coat protein and RNA synthesis. J Gen Virol 45: 723-736
  13. Harrison SC (1984) ultiple-modes of subunit association in the structures of simple spherical viruses. Trends Biochem Sci 9: 345-351
  14. Houwing CJ, Jaspars EMJ (1986) Coat protein blocks the in vitro transcription of virion RNAs of Alfafa mosaiv-virus. FEBS Lett 209: 284-288
  15. Hutson JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotny J, Margolies MN, Ridge RJ, Bruccoleri RE, Haber E, Crea R, Oppermann H (1988)' Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci USA 85: 5879-5883
  16. Jagger IC (1916) Experiments with the cucumber mosaic disease. Phytopathology 6: 148
  17. Kaper JM, Waterworth HE (1981) Cucumoviruses. In: Kurstak E (ed), Handbook of Plant Virus Infectious and Comparative Diagnosis. Elsevier/North-Holland Biomedical Press, The Netherlands, pp 257-332
  18. Lin NS, Hsieh CE, Hsu YH (1996) Capsid protein of cucumber mosaic virus accumulates in the nuclei and at the periphery of the nucleoli in infected cells. Arch Virol 141: 727-732
  19. Larrick JW, Yu L, Chen J, Jaiswal S, Wycoff K (1998) Production of antibodies in transgenic plants. Res Immunol 149: 603-608
  20. McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348: 552-554
  21. Michael, F. C., Richard M.L. and Moshe B. J. (1988) Methods for plant molecular biology: ELISA Techniques (Arthur W. and W. Herbert (ed.). Academic Press, London, pp 507-530
  22. Palukaitis P, Roossinck MJ, Dietzgen RG, Francki RIB (1992) Cucumber mosaic virus Avd Virus Res 41: 281-384
  23. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual (2nd eds) Cold Spring Harbor: Cold Spring Harbor laboratory Press
  24. Schouten A, Roosien J, De Boer JM, Wilmink A, Rosso MN, Bosch D, Stiekema WJ, Gommers FJ, Bakker J, Schots A (1997) Improving scFv antibody expression level in the plant cytosol. FEBS Lett 415: 234-241
  25. Schouten A, Roosien J, Van Engelen FA, De Jong GAM, Borst-Vrenssen AWM, Zilverentant JF, Bosch D, Stiekema WJ, Gommers FJ, Schots A, Bakker J (1996) The Cterminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both cytosol and the secretory pathway in transgenic tobacco. Plant Mol Biol 30: 781-793
  26. Skerra, A, Pluckthun, A. (1988). Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science 240: 1038-1040
  27. Tavladoraki P, Benvenuto E, Trinca S, De Martinis D, Cattaneo A, Galeffi P (1993) Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature 366: 469-472
  28. Tavladoraki P, Girotti A, Donini M, Arias FJ, Mancini C, Morea K, Chiaraluce R, Consalvi V, Benvenuto E (1999) A single-chain antibody fragment is functionally expressed in the cytoplasm of both Escherichia coli and transgenic plants. Eur J Biochem 262: 617-624
  29. Tricoli DM, Carney KJ, Russell PF, McMaster R, Groffl DW, Hadden KC, Himmel PT, Hubbard JP, Boeshore ML, Ouemada HD (1995) Field evaluation of transgenic squash containing single or multiple virus coat protein gene constructs for resistance to cucumber mosaic virus, watermelon mosaic virus 2, and zucchini yellow mosaic virus. Biotechnol 13: 1458-1465
  30. Yie Y, Wu ZX, Wang SY, Zhoa SZ, Zhang TQ, Yao GY, Tien P (1995) Rapid production and field testing of homozygous transgenic tobacco lines with virus-resistance conferred by expression of satellite RNA and coat protein of cucumber mosaic-virus. Transgen Res 4: 256-263
  31. Zimmermann S, Schillberg S, Liao YC, Fischer R (1998) Intracellular expression of TMV-specific single-chain Fv fragments leads to improved virus resistance in Nicotiana tabacum. Mol Breed 4: 369-379