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References

  1. Barre, F.-X., L. M. Mir, Y. Lécluse, A. Harel-Bellan. 1998. Highly efficient oligonucleotide transfer into intact yeast cells using square-wave pulse electroporation. Biotechniques 25:294-296.
    Pubmed
  2. Bharati, K. and S. Vrati. 2006. Japanese encephalitis: Development of new candidate vaccines. Expert. Rev. Anti. Infect. Ther. 4:313-324.
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  3. Burke, D. S. and T. P. Monath. 2001. Flaviviruses, pp. 991-
    1055. In D. M. Knipe and P. M. Howley (eds.). Fields virology,
    Forth Edition. Lippincott Williams & Wilkins, Philadelphia,
    Pennsylvania, USA.
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  5. Konishi, E., A. Fujii, and P. W. Mason. 2001. Generation and characterization of a mammalian cell line continuously expressing Japanese encephalitis virus subviral particles. J. Virol. 75: 22042212.
    Pubmed PMC CrossRef
  6. Konishi, E., P. W. Mason, and R. E. Shope. 1996. Enzymelinked immunosorbent assay using recombinant antigens for serodiagnosis of Japanese encephalitis. J. Med. Virol. 48: 7679.
    CrossRef
  7. Marx, F., T. S. Gritsun, B. Grubeck-Loebenstein, E. A. Gould. 2001. Diagnostic immunoassays for tick-borne encephalitis virus based on recombinant baculovirus protein expression. J. Virol. Methods 91: 75-84.
    CrossRef
  8. Mason, P. W., M. U. Zügel, A. R. Semproni, M. J. Fournier, T. L. Mason. 1990. The antigenic structure of dengue type 1 virus envelope and NS1 proteins expressed in Escherichia coli. J. Gen. Virol. 71(Pt 9): 2107-2114.
    Pubmed CrossRef
  9. Matsuura, Y., M. Miyamoto, T. Sato, C. Morita, K. Yasui. 1989. Characterization of Japanese encephalitis virus envelope protein expressed by recombinant baculoviruses. Virology 173: 674682.
    CrossRef
  10. McAda, P. C., P. W. Mason, C. S. Schmaljohn, J. M. Dalrymple, T. L. Mason, M. J. Fournier. 1987. Partial nucleotide sequence of the Japanese encephalitis virus genome. Virology 158: 348-360.
    CrossRef
  11. Possee, R. D. 1997. Baculoviruses as expression vectors. Curr. Opin. Biotechnol. 8: 569-572.
    CrossRef
  12. Russell, P. K., W. E. Brandt, and J. M Dalrymple. 1980. Chemical and antigenic structure of flaviviruses. 1980: 503-529.
  13. Seif, S. A., K. Morita, and A. Igarashi. 1996. A 27 amino acid coding region of JE virus E protein expressed in E. coli as fusion protein with glutathione-S-transferase elicit neutralizing antibody in mice. Virus Res. 43: 91-96.
    CrossRef
  14. Shapiro, D., W. E. Brandt, R. D. Cardiff, P. K. Russell. 1971. The proteins of Japanese encephalitis virus. Virology 44: 108124.
    CrossRef
  15. Sugrue, R. J., J. Fu, J. Howe, Y. C. Chan. 1997. Expression of the dengue virus structural proteins in Pichia pastoris leads to the generation of virus-like particles. J. Gen. Virol. 78(Pt 8):1861-1866.
    Pubmed
  16. Wengler, G., E. Castle, U. Leidner, T. Nowak, G. Wengler. 1985. Sequence analysis of the membrane protein V3 of the Flavivirus West Nile virus and of its gene. Virology 147: 264274.
    CrossRef
  17. Westaway, E. G. 1987. Flavivirus replication strategy. Adv. Virus Res. 33: 45-90.
    CrossRef
  18. Wright, P. J. 1982. Envelope protein of the Flavivirus Kunjin is apparently not glycosylated. J. Gen. Virol. 59(Pt 1): 29-38.
    Pubmed CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2012; 22(11): 1580-1587

Published online November 28, 2012 https://doi.org/10.4014/jmb.1205.05047

Copyright © The Korean Society for Microbiology and Biotechnology.

Protective Immunity of Pichia pastoris-Expressed Recombinant Envelope Protein of Japanese Encephalitis Virus

Woo-Taeg Kwon 1, Woo-Sik Lee 2, Pyo-Jam Park 3, Tae-Kyu Park 3 and Hyun Kang 3*

1Department of Environmental Health and Safety, Eulji University, Sungnam 461-713, Korea, 2Department of Environment and Energy Engineering, Gachon University, Sungnam 461-701, Korea, 3Department of Biotechnology, Research Institute for Biomedical and Health Science, College of Biomedical and Health Science, Konkuk University, Chungju, 380-701, Korea

Received: May 21, 2012; Accepted: July 4, 2012

Abstract

Japanese encephalitis virus (JEV) envelope (E) protein holds
great promise for use in the development of a recombinant
vaccine. Purified recombinant E (rE) protein may be
useful for numerous clinical applications; however, there
are limitations in using the Escherichia coli expression
system for producing high-quality rE protein. Therefore,
in this study, the yeast expression system was used to
generate the rE protein. For protein production using the
yeast system, the full-length JEV E gene was cloned into
Pichia pastoris. SDS-PAGE and immunoblotting analysis
demonstrated that the rE protein had a molecular mass of
58 kDa and was glycosylated. The predicted size of the
mature unmodified E protein is 53 kDa, suggesting that
post-translational modifications resulted in the higher
molecular mass. The rE protein was purified to greater
than 95% purity using combined ammonium sulfate
precipitation and a SP-Sepharose Fast Flow column. This
purified rE protein was evaluated for immunogenicity
and protective efficacy in mice. The survival rates of mice
immunized with the rE protein were significantly increased
over that of Hyphantria cunea nuclear polyhedrosis virus
E protein (HcE). Our results indicate that the rE protein
expressed in the P. pastoris expression system holds great
promise for use in the development of a subunit vaccine
against JEV.

Keywords: Japanese encephalitis virus (JEV), Envelop (E), Recombinant E (rE), Pichia pastoris, Protective immunity

References

  1. Barre, F.-X., L. M. Mir, Y. Lécluse, A. Harel-Bellan. 1998. Highly efficient oligonucleotide transfer into intact yeast cells using square-wave pulse electroporation. Biotechniques 25:294-296.
    Pubmed
  2. Bharati, K. and S. Vrati. 2006. Japanese encephalitis: Development of new candidate vaccines. Expert. Rev. Anti. Infect. Ther. 4:313-324.
    Pubmed CrossRef
  3. Burke, D. S. and T. P. Monath. 2001. Flaviviruses, pp. 991-
    1055. In D. M. Knipe and P. M. Howley (eds.). Fields virology,
    Forth Edition. Lippincott Williams & Wilkins, Philadelphia,
    Pennsylvania, USA.
  4. Kitano, T., K. Suzuki, and T. Yamaguchi. 1974. Morphological, chemical, and biological characterization of Japanese encephalitis virus virion and its hemagglutinin. J. Virol. 14: 631-639.
    Pubmed KoreaMed
  5. Konishi, E., A. Fujii, and P. W. Mason. 2001. Generation and characterization of a mammalian cell line continuously expressing Japanese encephalitis virus subviral particles. J. Virol. 75: 22042212.
    Pubmed KoreaMed CrossRef
  6. Konishi, E., P. W. Mason, and R. E. Shope. 1996. Enzymelinked immunosorbent assay using recombinant antigens for serodiagnosis of Japanese encephalitis. J. Med. Virol. 48: 7679.
    CrossRef
  7. Marx, F., T. S. Gritsun, B. Grubeck-Loebenstein, E. A. Gould. 2001. Diagnostic immunoassays for tick-borne encephalitis virus based on recombinant baculovirus protein expression. J. Virol. Methods 91: 75-84.
    CrossRef
  8. Mason, P. W., M. U. Zügel, A. R. Semproni, M. J. Fournier, T. L. Mason. 1990. The antigenic structure of dengue type 1 virus envelope and NS1 proteins expressed in Escherichia coli. J. Gen. Virol. 71(Pt 9): 2107-2114.
    Pubmed CrossRef
  9. Matsuura, Y., M. Miyamoto, T. Sato, C. Morita, K. Yasui. 1989. Characterization of Japanese encephalitis virus envelope protein expressed by recombinant baculoviruses. Virology 173: 674682.
    CrossRef
  10. McAda, P. C., P. W. Mason, C. S. Schmaljohn, J. M. Dalrymple, T. L. Mason, M. J. Fournier. 1987. Partial nucleotide sequence of the Japanese encephalitis virus genome. Virology 158: 348-360.
    CrossRef
  11. Possee, R. D. 1997. Baculoviruses as expression vectors. Curr. Opin. Biotechnol. 8: 569-572.
    CrossRef
  12. Russell, P. K., W. E. Brandt, and J. M Dalrymple. 1980. Chemical and antigenic structure of flaviviruses. 1980: 503-529.
  13. Seif, S. A., K. Morita, and A. Igarashi. 1996. A 27 amino acid coding region of JE virus E protein expressed in E. coli as fusion protein with glutathione-S-transferase elicit neutralizing antibody in mice. Virus Res. 43: 91-96.
    CrossRef
  14. Shapiro, D., W. E. Brandt, R. D. Cardiff, P. K. Russell. 1971. The proteins of Japanese encephalitis virus. Virology 44: 108124.
    CrossRef
  15. Sugrue, R. J., J. Fu, J. Howe, Y. C. Chan. 1997. Expression of the dengue virus structural proteins in Pichia pastoris leads to the generation of virus-like particles. J. Gen. Virol. 78(Pt 8):1861-1866.
    Pubmed
  16. Wengler, G., E. Castle, U. Leidner, T. Nowak, G. Wengler. 1985. Sequence analysis of the membrane protein V3 of the Flavivirus West Nile virus and of its gene. Virology 147: 264274.
    CrossRef
  17. Westaway, E. G. 1987. Flavivirus replication strategy. Adv. Virus Res. 33: 45-90.
    CrossRef
  18. Wright, P. J. 1982. Envelope protein of the Flavivirus Kunjin is apparently not glycosylated. J. Gen. Virol. 59(Pt 1): 29-38.
    Pubmed CrossRef