Skip to main content
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1992 Oct;30(10):2662–2667. doi: 10.1128/jcm.30.10.2662-2667.1992

Purification, characterization, and seroactivity of a 20-kilodalton Brucella protein antigen.

M S Zygmunt 1, F B Gilbert 1, G Dubray 1
PMCID: PMC270495  PMID: 1400966

Abstract

An internal protein was purified from cell extracts of Brucella melitensis B115 by a combination of preparative isoelectric focusing and high-performance size exclusion chromatography. The protein has an apparent molecular mass of 230 kDa as determined by size exclusion chromatography. The protein was resolved to a single band of 20 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native protein had an isoelectric point of 4.9. The N-terminal sequence of the 20-kDa protein was determined. The 20-kDa protein has been identified as antigen A-2 with a previously described anti-antigen A-2 serum (B. Stemshorn, K. Nielsen, and B. Samagh, Can. J. Comp. Med. 45:77-81, 1981). Antigen A-2 reacted with sera from infected sheep in immunoblotting and may be useful in developing diagnostic tests for brucellosis.

Full text

PDF

Images in this article

Selected References

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

  1. Alton G. G., Corner L. A., Plackett P. Vaccination of pregnant cows with low doses of Brucella abortus strain 19 vaccine. Aust Vet J. 1980 Aug;56(8):369–372. doi: 10.1111/j.1751-0813.1980.tb09561.x. [DOI] [PubMed] [Google Scholar]
  2. Alton G. G., Corner L. A. Vaccination of heifers with a reduced dose of brucella abortus strain 19 vaccine before first mating. Aust Vet J. 1981 Dec;57(12):548–550. doi: 10.1111/j.1751-0813.1981.tb00431.x. [DOI] [PubMed] [Google Scholar]
  3. Baughn R. E., Freeman B. A. Antigenic structure of Brucella suis spheroplasts. J Bacteriol. 1966 Nov;92(5):1298–1303. doi: 10.1128/jb.92.5.1298-1303.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beck B. L., Tabatabai L. B., Mayfield J. E. A protein isolated from Brucella abortus is a Cu-Zn superoxide dismutase. Biochemistry. 1990 Jan 16;29(2):372–376. doi: 10.1021/bi00454a010. [DOI] [PubMed] [Google Scholar]
  5. Berman D. T., Wilson B. L., Moreno E., Angus R. D., Jones L. M. Characterization of Brucella abortus soluble antigen employed in immunoassay. J Clin Microbiol. 1980 Apr;11(4):355–362. doi: 10.1128/jcm.11.4.355-362.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bricker B. J., Tabatabai L. B., Judge B. A., Deyoe B. L., Mayfield J. E. Cloning, expression, and occurrence of the Brucella Cu-Zn superoxide dismutase. Infect Immun. 1990 Sep;58(9):2935–2939. doi: 10.1128/iai.58.9.2935-2939.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Diaz R., Jones L. M., Leong D., Wilson J. B. Surface antigens of smooth brucellae. J Bacteriol. 1968 Oct;96(4):893–901. doi: 10.1128/jb.96.4.893-901.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Diaz R., Jones L. M., Wilson J. B. Antigenic relationship of Brucella ovis and Brucella melitensis. J Bacteriol. 1967 Apr;93(4):1262–1268. doi: 10.1128/jb.93.4.1262-1268.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hinsdill R. D., Berman D. T. Antigens of Brucella abortus. I. Chemical and immunoelectrophoretic characterization. J Bacteriol. 1967 Feb;93(2):544–549. doi: 10.1128/jb.93.2.544-549.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Lee C., Levin A., Branton D. Copper staining: a five-minute protein stain for sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem. 1987 Nov 1;166(2):308–312. doi: 10.1016/0003-2697(87)90579-3. [DOI] [PubMed] [Google Scholar]
  12. McGhee J. R., Freeman B. A. Separation of soluble Brucella antigens by gel-filtration chromatography. Infect Immun. 1970 Jul;2(1):48–53. doi: 10.1128/iai.2.1.48-53.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nielsen K., Cherwonogrodzky J. W., Duncan J. R., Bundle D. R. Enzyme-linked immunosorbent assay for differentiation of the antibody response of cattle naturally infected with Brucella abortus or vaccinated with strain 19. Am J Vet Res. 1989 Jan;50(1):5–9. [PubMed] [Google Scholar]
  14. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  15. Plommet M., Fensterbank R. Vaccination against bovine brucellosis with a low dose of strain 19 administered by the conjunctival route. III. -- Serological response and immunity in the pregnant cow. Ann Rech Vet. 1976;7(1):9–23. [PubMed] [Google Scholar]
  16. Schurig G. G., Jones L. M., Speth S. L., Berman D. T. Antibody response to antigens distinct from smooth lipopolysaccharide complex in Brucella infection. Infect Immun. 1978 Sep;21(3):994–1002. doi: 10.1128/iai.21.3.994-1002.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schurig G. G., Pringle A. T., Breese S. S., Jr Localization of brucella antigens that elicit a humoral immune response in Brucella abortus-infected cells. Infect Immun. 1981 Dec;34(3):1000–1007. doi: 10.1128/iai.34.3.1000-1007.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schurig G. G. The immune response of goats vaccinated with low and high doses of Brucella melitensis Rev 1. Vet Immunol Immunopathol. 1982 May;3(3):311–324. doi: 10.1016/0165-2427(82)90005-8. [DOI] [PubMed] [Google Scholar]
  19. Stemshorn B., Nielsen K., Samagh B. The bovine immune response to Brucella abortus. III. Preparation of antisera against a Brucella component precipitated by sera of some infected cattle. Can J Comp Med. 1981 Jan;45(1):77–81. [PMC free article] [PubMed] [Google Scholar]
  20. Stemshorn B., Nielsen K. The bovine immune response to Brucella abortus I. A water soluble antigen precipitated by sera of some naturally infected cattle. Can J Comp Med. 1977 Apr;41(2):152–159. [PMC free article] [PubMed] [Google Scholar]
  21. Stemshorn B., Nielsen K. The bovine immune response to Brucella abortus IV. Studies with a double immunodiffusion test for antibody against A2. Can J Comp Med. 1981 Apr;45(2):147–153. [PMC free article] [PubMed] [Google Scholar]
  22. Tessmer U., Dernick R. Preparative separation of poliovirus structural polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, copper staining and electroelution, and induction of monospecific antisera. Electrophoresis. 1989 Apr;10(4):277–279. doi: 10.1002/elps.1150100414. [DOI] [PubMed] [Google Scholar]
  23. Zygmunt M. S., Martin J. C., Dubray G. Analysis of immune response: comparison of immunoblots after isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cytoplasmic protein extract from Brucella. FEMS Microbiol Lett. 1990 Aug;58(3):263–268. doi: 10.1111/j.1574-6968.1990.tb13986.x. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES