Biochemistry (Moscow)

, Volume 73, Issue 10, pp 1108–1113 | Cite as

Highly efficient expression of Escherichia coli heat-labile enterotoxin B subunit in plants using potato virus X-based vector

  • N. V. Ravin
  • V. V. Kuprianov
  • L. A. Zamchuk
  • A. V. Kochetov
  • Yu. L. Dorokhov
  • J. G. Atabekov
  • K. G. Skryabin


A synthetic gene of the B-subunit of Escherichia coli heat-labile toxin, optimized for expression in plants, was designed and synthesized. The recombinant viral vector was constructed on the basis of potato virus X containing the LTB gene instead of the removed triple block of transport genes and the coat protein gene, which provides for LTB expression in plants. The vector is introduced into the plant cells during cell infiltration by agrobacteria incorporating a binary vector, the T-DNA region of which contains a cDNA copy of the recombinant viral genome. Under conditions of posttranscriptional gene silencing inhibition, the LTB yield in Nicotiana benthamiana plants is 1–2% of total soluble protein; in this case, LTB synthesized in plants forms pentameric complexes analogous to those found in the native toxin. The designed viral system of LTB transient expression can be used to obtain in plants a vaccine against enteropathogenic Escherichia coli.

Key words

plant as “biofactories” LTB vaccine viral vector 



green fluorescent protein


heat-labile toxin of Escherichia coli


B-subunit of Escherichia coli heat-labile toxin


pectin methyl esterase


post-transcriptional gene silencing


potato virus X


RNA-dependent RNA polymerase


35S RNA of cauliflower mosaic virus


synthetic gene of LTB


subgenomic RNA promoter


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  1. 1.
    Clements, J. D., and Finkelstein, R. A. (1979) Infect. Immun., 24, 760–769.PubMedGoogle Scholar
  2. 2.
    Clements, J. D., Yancey, R. J., and Finkelstein, R. A. (1980) Infect. Immun., 29, 91–97.PubMedGoogle Scholar
  3. 3.
    Clements, J. D., and Finkelstein, R. A. (1978) Infect. Immun., 21, 1036–1039.PubMedGoogle Scholar
  4. 4.
    Spangler, B. D. (1992) Microbiol. Rev., 56, 622–647.PubMedGoogle Scholar
  5. 5.
    Dickinson, B. L., and Clements, J. D. (1995) Infect. Immun., 63, 1617–1623.PubMedGoogle Scholar
  6. 6.
    Dickinson, B. L., and Clements, J. D. (1996) in Mucosal Vaccines (Kiyono, H., Ogra, P. L., and McGhee, eds.) Academic Press, N. Y., pp. 73–87.CrossRefGoogle Scholar
  7. 7.
    Mason, H. S., Haq, T. A., Clements, J. D., and Arntzen, C. J. (1998) Vaccine, 16, 1336–1343.PubMedCrossRefGoogle Scholar
  8. 8.
    Tacket, C. O., Mason, H. S., Lasonsky, G., Clements, J. D., Levine, M. M., and Arntzen, C. J. (1998) Nature Med., 4, 607–609.PubMedCrossRefGoogle Scholar
  9. 9.
    Haq, T. A., Mason, H. S., Clements, J. D., and Arntzen, C. J. (1995) Science, 268, 714–716.PubMedCrossRefGoogle Scholar
  10. 10.
    Stoger, E., Sack, M., Fischer, R., and Christou, P. (2002) Curr. Opin. Biotechnol., 13, 161–166.PubMedCrossRefGoogle Scholar
  11. 11.
    Streatfield, S. J., Major, J. M., Barker, D. K., Brooks, C., Woodard, S. L., Horn, M., Nikolov, Z. L., Hood, E. E., Jilka, J. M., and Howard, J. A. (2002) In vitro Cell. Dev. Biol. Plant, 38, 11–17.CrossRefGoogle Scholar
  12. 12.
    Streatfield, S. J., Lane, J. R., Brooks, C. A., Barker, D. K., Poage, M. L., Mayor, J. M., Lamphear, B. J., Drees, C. F., Jilka, J. M., Hood, E. E., and Howard, J. A. (2003) Vaccine, 21, 812–815.PubMedCrossRefGoogle Scholar
  13. 13.
    Chikwamba, R., McMurray, J., Shou, H., Frame, B., Peggs, S. E., Scott, P., Mason, H., and Wang, K. (2002) Molec. Breeding, 10, 253–265.CrossRefGoogle Scholar
  14. 14.
    Bock, R. (2001) J. Mol. Biol., 312, 425–438.PubMedCrossRefGoogle Scholar
  15. 15.
    Kang, T. J., Loc, N. H., Jang, M. O., Jang, Y. S., Kim, Y. S., Seo, J. E., and Yang, M. S. (2003) Transgenic Res., 12, 683–691.PubMedCrossRefGoogle Scholar
  16. 16.
    Marillonnet, S., Thoeringer, C., Kandzia, R., Klimyuk, V., and Gleba, Y. (2005) Nat. Biotechnol., 23, 718–723.PubMedCrossRefGoogle Scholar
  17. 17.
    Yusibov, V., Hooper, D., Spitsin, S., Fleysh, N., Kean, R., Mikheeva, T., Deka, D., Karasev, A., Cox, S., Randall, J., and Koprowski, H. (2002) Vaccine, 20, 3155–3164.PubMedCrossRefGoogle Scholar
  18. 18.
    Santi, L., Giritch, A., Roy, C. J., Marillonnet, S., Klimyuk, V., Gleba, Y., Webb, R., Arntzen, C. J., and Mason, H. S. (2006) Proc. Natl. Acad. Sci. USA, 103, 861–866.PubMedCrossRefGoogle Scholar
  19. 19.
    Giritch, A., Marillonnet, S., Engler, C., van Eldik, G., Botterman, J., Klimyuk, V., and Gleba, Y. (2006) Proc. Natl. Acad. Sci. USA, 103, 14701–14706.PubMedCrossRefGoogle Scholar
  20. 20.
    Canizares, M. C., Nicholson, L., and Lomonossoff, G. P. (2005) Immunol. Cell. Biol., 83, 263–270.PubMedCrossRefGoogle Scholar
  21. 21.
    Dorokhov, Y. L., Skurat, E. V., Frolova, O. Y., Gasanova, T. V., Ivanov, P. A., Ravin, N. V., Skryabin, K. G., Makinen, K. M., Klimyuk, V. I., Gleba, Y. Y., and Atabekov, J. G. (2006) FEBS Lett., 580, 3329–3334.PubMedCrossRefGoogle Scholar
  22. 22.
    Komarova, T. V., Skulachev, M. V., Zvereva, A. S., Shwarts, A. M., Dorokhov, Yu. L., and Atabekov, J. G. (2006) Biochemistry (Moscow), 71, 846–850.CrossRefGoogle Scholar
  23. 23.
    Kochetov, A. V., Ischenko, I. V., Vorobiev, D. G., Kel, A. E., Babenko, V. N., Kisselev, L. L., and Kolchanov, N. A. (1998) FEBS Lett., 440, 351–355.PubMedCrossRefGoogle Scholar
  24. 24.
    Kochetov, A. V., Ponomarenko, M. P., Frolov, A. S., Kisselev, L. L., and Kolchanov, N. A. (1999) Bioinformatics, 15, 704–712.PubMedCrossRefGoogle Scholar
  25. 25.
    Likhoshvai, V. A., and Matushkin, Y. G. (2002) FEBS Lett., 516, 87–92.PubMedCrossRefGoogle Scholar
  26. 26.
    Hirst, T. R. (1995) in Handbook of Natural Toxins, Vol. 8, Bacterial Toxins and Virulence Factors in Disease (Moss, J., Ihlewski, B., Vaughan, M., and Tu, A. T., eds.) Marcel Dekker, N. Y., pp. 123–184.Google Scholar
  27. 27.
    Schonberger, O., Hirst, T. R., and Pines, O. (1991) Mol. Microbiol., 5, 2663–2671.PubMedCrossRefGoogle Scholar
  28. 28.
    MacDiarmid, R. (2005) Annu. Rev. Phytopathol., 43, 523–544.PubMedCrossRefGoogle Scholar
  29. 29.
    Dorokhov, Y. L., Frolova, O. Y., Skurat, E. V., Ivanov, P. A., Gasanova, T. V., Sheveleva, A. A., Ravin, N. V., Makinen, K. M., Klimyuk, V. I., Skryabin, K. G., Gleba, Y. Y., and Atabekov, J. G. (2006) FEBS Lett., 580, 3872–3878.PubMedCrossRefGoogle Scholar
  30. 30.
    Lauterslager, T. G., Florack, D. E., van der Wal, T. J., Molthoff, J. W., Langeveld, J. P., Bosch, D., Boersma, W. J., and Hilgers, L. A. (2001) Vaccine, 19, 2749–2755.PubMedCrossRefGoogle Scholar
  31. 31.
    Chikwamba, R. K., Cunnick, J., Hathaway, D., McMurray, J., Mason, H., and Wang, K. (2002) Transgenic Res., 11, 479–493.PubMedCrossRefGoogle Scholar
  32. 32.
    Wagner, B., Hufnagl, K., Radauer, C., Wagner, S., Baier, K., Scheiner, O., Wiedermann, U., and Breiteneder, H. (2004) J. Immunol. Meth., 287, 203–215.CrossRefGoogle Scholar
  33. 33.
    Nashar, T. O., Webb, H. M., Eaglestone, S., Williams, N. A., and Hirst, T. R. (1996) Proc. Natl. Acad. Sci. USA, 93, 226–230.PubMedCrossRefGoogle Scholar

Copyright information

© MAIK Nauka 2008

Authors and Affiliations

  • N. V. Ravin
    • 1
  • V. V. Kuprianov
    • 1
  • L. A. Zamchuk
    • 1
  • A. V. Kochetov
    • 2
  • Yu. L. Dorokhov
    • 3
  • J. G. Atabekov
    • 3
  • K. G. Skryabin
    • 1
  1. 1.Center “Bioengineering”Russian Academy of SciencesMoscowRussia
  2. 2.Institute of Cytology and GeneticsSiberian Branch of the Russian Academy of SciencesNovosibirskRussia
  3. 3.Faculty of BiologyLomonosov Moscow State UniversityMoscowRussia

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