1887

Abstract

A panel of 11 monoclonal antibodies (MAbs) raised against pseudorabies virus (PRV) was used to map epitopes on the virus glycoprotein I (gl). We employed three approaches to map epitopes on gl. By a competition binding assay, six groups of MAbs were defined as reacting with distinct antigenic domains on gl. To identify regions along the gl polypeptide chain encompassing the domains recognized by these MAbs, DNA fragments derived from the gI-coding region were cloned into pEX expression plasmids. The antigenic reactivity of the fusion proteins expressed in was analysed by immunoblotting. Five antigenic domains were mapped within the first 238 amino acids of gl: domains A, B and D were mapped between amino acids 52 and 123 and domains C and E between amino acids 78 and 238. One MAb, representing domain F, did not react with the expressed fusion proteins. To assess the precise location and amino acid sequences of the epitopes, overlapping nonapeptides covering the amino acid sequence 52 to 238 were synthesized. The antibody-binding activity of these peptides was tested by an ELISA (Pepscan-method). Three antigenic domains were mapped: domain A was localized to amino acids 64 to 73 and 75 to 84, domain B to amino acids 52 to 67 and domain D to amino acids 68 to 82. Four MAbs representing antigenic domains C, E and F did not react in the Pepscan. Finally, sera from pigs infected experimentally with PRV reacted with the fusion protein of plasmid ps1 (amino acids 52 to 238).

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-71-4-881
1990-04-01
2022-10-07
Loading full text...

Full text loading...

/deliver/fulltext/jgv/71/4/JV0710040881.html?itemId=/content/journal/jgv/10.1099/0022-1317-71-4-881&mimeType=html&fmt=ahah

References

  1. Chou P. Y., Fasman G. D. 1978; Prediction of the secondary structure of proteins from their amino acid sequence. Advances in Enzymology 47:45–148
    [Google Scholar]
  2. Davis L. G., Dibner M. D., Battey J. F. 1986 Basic Methods in Molecular Biology New York: Elsevier;
    [Google Scholar]
  3. Eloit M., Fargeaud D., Vannier P., Toma B. 1989; Development of an ELISA to differentiate between animals either vaccinated or infected by Aujeszky’s disease virus. Veterinary Record 128:91–94
    [Google Scholar]
  4. Getzoff E. D., Geysen H. M., Rodda S. J., Alexander H., Tainer J. A., Lerner R. A. 1987; Mechanisms of antibody binding to a protein. Science 235:1191–1196
    [Google Scholar]
  5. Geysen H. M., Meloen R. H., Barteling S. J. 1984; Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proceedings of the National Academy of Sciences, U.S.A 81:3998–4002
    [Google Scholar]
  6. Geysen H. M., Barteling S. J., Meloen R. H. 1985; Small peptides induce antibodies with a sequence and structural requirement for binding antigen comparable to antibodies raised against the mature protein. Proceedings of the National Academy of Sciences, U.S.A 82:178–182
    [Google Scholar]
  7. Geysen H. M., Tainer J. A., Rodda S. J., Mason T. J., Alexander H., Getzoff E. D., Lerner R. A. 1987; Chemistry of antibody binding to a protein. Science 235:1184–1190
    [Google Scholar]
  8. Gielkens A. L. J., Moormann R. J. M., Van Oirschot J. T., Berns A. J. M. 1989; Vaccine efficacy and innocuity of strain 783 of Aujeszky’s disease virus. In Vaccination and Control of Aujeszky’s Disease, CEC Seminar pp. 27–35 Van Oirschot J. T. Edited by Dordrecht: Kluwer Academic Publishers;
    [Google Scholar]
  9. Hampl H., Ben-Porat T., Ehrlicher L., Habermehl K. O., Kaplan A. S. 1984; Characterization of the envelope proteins of pseudorabies virus. Journal of Virology 52:583–590
    [Google Scholar]
  10. Kit S., Sheppard M., Ichmura H., Kit M. 1987; Second generation pseudorabies virus vaccine with deletions in thymidine kinase and glycoprotein genes. American Journal of Veterinary Research 48:780–793
    [Google Scholar]
  11. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 227680–685
    [Google Scholar]
  12. Lenstra J. A., Kusters J. G., Koch G., van der Zeijst B. A. M. 1989; Antigenicity of the peplomer protein of infectious bronchitis virus. Molecular Immunology 26:7–15
    [Google Scholar]
  13. Luytjes W., Geerts D., Posthumus W., Meloen R., Spaan W. 1989; Amino acid sequence of a conserved neutralizing epitope of murine coronaviruses. Journal of Virology 63:1408–1412
    [Google Scholar]
  14. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: A Laboratory Manual New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  15. Marchioli C. C., Yancey R. J., Wardley R. C., Thomsen D. R., Post L. E. 1987; A vaccine strain of pseudorabies virus with deletions in the thymidine kinase and glycoprotein X genes. American Journal of Veterinary Research 48:1577–1583
    [Google Scholar]
  16. Mehra V., Sweetser D., Young R. A. 1986; Efficient mapping of protein antigenic determinants. Proceedings of the National Academy of Sciences, U.S.A 83:7013–7017
    [Google Scholar]
  17. Mettenleiter T. C., Lukacs N., Rziha H. J. 1985a; Pseudorabies virus avirulent strains fail to express a major glycoprotein. Journal of Virology 56:307–311
    [Google Scholar]
  18. Mettenleiter T. C., Lukacs N., Rziha H. J. 1985b; Mapping of the structural gene of pseudorabies virus glycoprotein A and identification of two non-glycosylated precursor polypeptides. Journal of Virology 53:52–57
    [Google Scholar]
  19. Middeldorp J. M., Meloen R. H. 1988; Epitope-mapping on the Epstein-Barr virus major capsid protein using systematic synthesis of overlapping oligopeptides. Journal of Virological Methods 21:147–159
    [Google Scholar]
  20. Nunberg J. H., Rodgers G., Gilbert J. H., Snead R. M. 1984; Method to map antigenic determinants recognized by monoclonal antibodies: localization of a determinant of virus neutralization on the feline leukemia virus envelope protein gp70. Proceedings of the National Academy of Sciences, U.S.A 81:3675–3679
    [Google Scholar]
  21. Petrovskis E. A., Timmins J. G., Post L. E. 1986; Use of the Agtl 1 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoprotein. Journal of Virology 60:185–193
    [Google Scholar]
  22. Quint W., Gielkens A. L. J., Van Oirschot J. T., Berns A., Cuypers H. T. 1987; Construction and characterization of deletion mutants of pseudorabies virus: a new generation of ‘live’ vaccines. Journal of General Virology 68:523–534
    [Google Scholar]
  23. Stanley K. K. 1983; Solubilization and immuno-detection of β-galactosidase hybrid proteins carrying foreign antigenic determinants. Nucleic Acids Research 12:4017–4092
    [Google Scholar]
  24. Stanley K. K., Luzio J. P. 1984; Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins. EMBO Journal 3:1429–1434
    [Google Scholar]
  25. Van Oirschot J. T.editor 1989; The antibody response to glycoprotein I and the control of Aujeszky’s disease. In Vaccination and Control of Aujeszky’s Disease. CEC Seminar pp. 129–138 Dordrecht: Kluwer Academic Publishers;
    [Google Scholar]
  26. Van Oirschot J. T., Gielkens A. L. J. 1984; Intranasal vaccination of pigs against pseudorabies. 2. Absence of vaccinal virus latency and failure to prevent latency of virulent virus. American Journal of Veterinary Research 45:2069–2103
    [Google Scholar]
  27. Van Oirschot J. T., Rziha H. J., Moonen P. J. L. M., Pol J. M. A., van Zaane D. 1986; Differentiation of serum antibodies from infected pigs vaccinated or infected with Aujeszky’s disease virus by a competitive enzyme immunoassay. Journal of General Virology 67:1179–1182
    [Google Scholar]
  28. Van Oirschot J. T., Houwers D. I., Rziha H. J., Moonen P. J. L. M. 1988; Development of an ELISA for detection of antibodies to glycoprotein I of Aujeszky’s disease virus: a method for the serological differentiation between infected and vaccinated pigs. Journal of Virological Methods 22:191–206
    [Google Scholar]
  29. Wilson M. B., Nabcane P. K. 1978; Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In Immunofluorescence and Related Staining Techniques pp. 215–224 Knapp W., Holibar K., Wick G. Edited by Amsterdam: Elsevier/North-Holland;
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-71-4-881
Loading
/content/journal/jgv/10.1099/0022-1317-71-4-881
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error