Skip to main content
Clinical and Diagnostic Laboratory Immunology logoLink to Clinical and Diagnostic Laboratory Immunology
. 1995 May;2(3):263–267. doi: 10.1128/cdli.2.3.263-267.1995

Cloning and sequence analysis of a newly identified Brucella abortus gene and serological evaluation of the 17-kilodalton antigen that it encodes.

F Hemmen 1, V Weynants 1, T Scarcez 1, J J Letesson 1, E Saman 1
PMCID: PMC170142  PMID: 7664168

Abstract

A thus far unknown gene encoding a Brucella abortus protein has been isolated from a lambda gt11 expression library probed with sera from Brucella-infected sheep. Sequence analysis of the cloned gene revealed the presence of an open reading frame of 158 amino acids encoding a protein of 17.3 kDa (calculated molecular mass). The recombinant B. abortus protein, expressed in Escherichia coli, and the corresponding Brucella melitensis protein migrated at the same apparent molecular masses as shown by Western blotting (immunoblotting). Among a series of serum samples from B. melitensis- or B. abortus-infected sheep and cows, 51 and 39%, respectively, showed a signal at 17 kDa on Western blot analysis of total protein extract from Brucella bacteria. These figures amount to 70 and 61% for sheep and cattle, respectively, in a competitive enzyme-linked immunosorbent assay with a specific monoclonal antibody. These data indicate that the 17-kDa antigen may be useful for serological diagnosis of Brucella infection.

Full Text

The Full Text of this article is available as a PDF (281.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Cloeckaert A., de Wergifosse P., Dubray G., Limet J. N. Identification of seven surface-exposed Brucella outer membrane proteins by use of monoclonal antibodies: immunogold labeling for electron microscopy and enzyme-linked immunosorbent assay. Infect Immun. 1990 Dec;58(12):3980–3987. doi: 10.1128/iai.58.12.3980-3987.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Corbel M. J., Stuart F. A., Brewer R. A. Observations on serological cross-reactions between smooth Brucella species and organisms of other genera. Dev Biol Stand. 1984;56:341–348. [PubMed] [Google Scholar]
  3. Fekete A., Bantle J. A., Halling S. M., Stich R. W. Amplification fragment length polymorphism in Brucella strains by use of polymerase chain reaction with arbitrary primers. J Bacteriol. 1992 Dec;174(23):7778–7783. doi: 10.1128/jb.174.23.7778-7783.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fensterbank R. Le diagnostic allergique des brucelloses animales. Dev Biol Stand. 1984;56:401–405. [PubMed] [Google Scholar]
  5. Ficht T. A., Bearden S. W., Sowa B. A., Adams L. G. A 36-kilodalton Brucella abortus cell envelope protein is encoded by repeated sequences closely linked in the genomic DNA. Infect Immun. 1988 Aug;56(8):2036–2046. doi: 10.1128/iai.56.8.2036-2046.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ficht T. A., Bearden S. W., Sowa B. A., Adams L. G. DNA sequence and expression of the 36-kilodalton outer membrane protein gene of Brucella abortus. Infect Immun. 1989 Nov;57(11):3281–3291. doi: 10.1128/iai.57.11.3281-3291.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goldbaum F. A., Leoni J., Wallach J. C., Fossati C. A. Characterization of an 18-kilodalton Brucella cytoplasmic protein which appears to be a serological marker of active infection of both human and bovine brucellosis. J Clin Microbiol. 1993 Aug;31(8):2141–2145. doi: 10.1128/jcm.31.8.2141-2145.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  9. Mayfield J. E., Bricker B. J., Godfrey H., Crosby R. M., Knight D. J., Halling S. M., Balinsky D., Tabatabai L. B. The cloning, expression, and nucleotide sequence of a gene coding for an immunogenic Brucella abortus protein. Gene. 1988;63(1):1–9. doi: 10.1016/0378-1119(88)90540-9. [DOI] [PubMed] [Google Scholar]
  10. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  11. Perry M. B., Bundle D. R. Antigenic relationships of the lipopolysaccharides of Escherichia hermannii strains with those of Escherichia coli O157:H7, Brucella melitensis, and Brucella abortus. Infect Immun. 1990 May;58(5):1391–1395. doi: 10.1128/iai.58.5.1391-1395.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Schoerner C., Wartenberg K., Röllinghoff M. Differentiation of serological responses to Yersinia enterocolitica serotype O9 and Brucella species by immunoblot or enzyme-linked immunosorbent assay using whole bacteria and Yersinia outer membrane proteins. J Clin Microbiol. 1990 Jul;28(7):1570–1574. doi: 10.1128/jcm.28.7.1570-1574.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Tibor A., Weynants V., Denoel P., Lichtfouse B., De Bolle X., Saman E., Limet J. N., Letesson J. J. Molecular cloning, nucleotide sequence, and occurrence of a 16.5-kilodalton outer membrane protein of Brucella abortus with similarity to pal lipoproteins. Infect Immun. 1994 Sep;62(9):3633–3639. doi: 10.1128/iai.62.9.3633-3639.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Tsang V. C., Peralta J. M., Simons A. R. Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods Enzymol. 1983;92:377–391. doi: 10.1016/0076-6879(83)92032-3. [DOI] [PubMed] [Google Scholar]
  16. Verstreate D. R., Winter A. J. Comparison of sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and antigenic relatedness among outer membrane proteins of 49 Brucella abortus strains. Infect Immun. 1984 Oct;46(1):182–187. doi: 10.1128/iai.46.1.182-187.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Zygmunt M. S., Gilbert F. B., Dubray G. Purification, characterization, and seroactivity of a 20-kilodalton Brucella protein antigen. J Clin Microbiol. 1992 Oct;30(10):2662–2667. doi: 10.1128/jcm.30.10.2662-2667.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. van Gelder P., Bosman F., de Meuter F., van Heuverswyn H., Hérion P. Serodiagnosis of toxoplasmosis by using a recombinant form of the 54-kilodalton rhoptry antigen expressed in Escherichia coli. J Clin Microbiol. 1993 Jan;31(1):9–15. doi: 10.1128/jcm.31.1.9-15.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Clinical and Diagnostic Laboratory Immunology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES