1887

Abstract

The extracellular portion (amino acids 1 to 844) of the equine herpesvirus type 1 (EHV-1) glycoprotein gpl4, the homologue of gB of herpes simplex virus, was expressed in and in insect cells using a recombinant baculovirus. Immunoblot analysis revealed that the recombinant expressed a fusion protein of 135K which was composed of the truncated gpl4 and the maltose-binding protein (MBP) provided by the vector and a 90K protein lacking the MBP moiety. Both proteins were sequestered within the cells in form of inclusion bodies. Infection of insect cells with the recombinant baculovirus resulted in the production of a 115K to 118K glycoprotein which was cleaved intracellularly into two subunits of 55K and 63K to 65K. The cleaved subunits were secreted into the cell culture supernatant and formed disulphide-linked dimers of 120K to 122K. The recombinant proteins produced in and in insect cells elicited EHV-1-specific antibodies in goats as demonstrated by Western blot analysis. The gpl4 expressed in insect cells induced antibodies with virus-neutralizing activity. In contrast, the truncated gpl4 expressed by failed to elicit neutralizing antibodies. The results suggest that posttranslational modification of the EHV-1 gpl4 may be important for the expression of epitopes necessary for the induction of neutralizing antibodies.

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1994-08-01
2024-03-28
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References

  1. Allen G. P., Bryans J. T. 1986; Molecular epizootology, pathogenesis, and prophylaxis of equine herpesvirus-1 infections. In Progress in Veterinary Microbiology and Immunology 2 pp. 78–144 Pandey R. Edited by Basel: Karger;
    [Google Scholar]
  2. Allen G. P., Yeargan M. R. 1987; Use of γgt11 and monoclonal antibodies to map the genes for the six major glycoproteins of equine herpesvirus 1. Journal of Virology 61:2454–2461
    [Google Scholar]
  3. Bell C. W., Boyle D. B., Whalley J. M. 1990; Transcript analysis of the equine herpesvirus glycoprotein B gene homologue and its expression by a recombinant vaccinia virus. Journal of General Virology 11:1119–1129
    [Google Scholar]
  4. Edington N., Bridges C. G., Patel J. R. 1986; Endothelial cell infection and thrombosis caused by equid herpesvirus-1: equine stroke. Archives of Virology 90:111–124
    [Google Scholar]
  5. Guo P., Goebel S., Perkus M. E., Taylor J., Norton E., Allen G., Languet B., Desmettre P., Paoletti E. 1990; Coexpression by vaccinia virus recombinants of equine herpesvirus 1 glycoproteins gpl3 and gp14 results in potentiated immunity. Journal of Virology 64:2399–2406
    [Google Scholar]
  6. Laffin J., Lehmann J. M. 1990; Detection of intracellular virus. In Flow Cytometry pp. 271–284 Darzynkiewicz Z., Crissman H. A. Edited by San Diego: Academic Press;
    [Google Scholar]
  7. Meredith D. M., Stocks J.-M., Whittaker G. R., Halliburton I. W., Snowden B. W., Killington R. A. 1989; Identification of the gB homologues of equine herpesvirus types 1 and 4 as disulphide-linked heterodimers and their characterization using monoclonal antibodies. Journal of General Virology 70:1161–1172
    [Google Scholar]
  8. Meyer H., Hübert P. H., Eichhorn W. 1987; Changes in the restriction pattern of the equine herpesvirus 1 (EHV-1) strain RacH during attenuation. Journal of Veterinary Medicine B 34:310–313
    [Google Scholar]
  9. Saiki R. K., Gelfand D.H, Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. 1988; Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491
    [Google Scholar]
  10. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual, 2nd edn. New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  11. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U.S.A 74:5463–5467
    [Google Scholar]
  12. Shimizu M., Satou K., Nishioka N. 1989; Monoclonal antibodies with neutralizing activity to equine herpesvirus 1. Archives of Virology 104:169–174
    [Google Scholar]
  13. Shine J., Dalgarno L. 1974; The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementary to nonsense triplets and ribosome binding sites. Proceedings of the National Academy of Sciences, U.S.A 71:1342–1346
    [Google Scholar]
  14. Sinclair R., Binns M. M., Chirnside E. D., Mumford J. A. 1993; Detection of antibodies against equine herpesvirus types 1 and 4 by using recombinant protein derived from an immunodominant region of glycoprotein B. Journal of Clinical Microbiology 31:265–271
    [Google Scholar]
  15. Stokes A., Allen G. P., Pullen L. A., Murray P. K. 1989; A hamster model of equine herpesvirus type 1 (EHV-1) infection: passive protection by monoclonal antibodies to EHV-1 glycoproteins 13, 14 and 17/18. Journal of General Virology 70:1173–1183
    [Google Scholar]
  16. Sullivan D. C., Allen G. P., O’Callaghan D. J. 1989; Synthesis and processing of equine herpesvirus type 1 glycoprotein 14. Virology 173:638–646
    [Google Scholar]
  17. Summers M. D., Smith G. E. 1987 A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures Texas Agricultural Experimental Station Bulletin 1555
    [Google Scholar]
  18. Whalley J. M., Robertson G. R., Scott N. A., Hudson G. C., Bell C. W., Woodworth L. M. 1989; Identification and nucleotide sequence of a gene in equine herpesvirus 1 analogous to the herpes simplex virus gene encoding the major envelope glycoprotein B. Journal of General Virology 70:383–394
    [Google Scholar]
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