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Molecular and immunological characterisation of recombinant Brucella abortus glyceraldehyde-3-phosphate-dehydrogenase, a T- and B-cell reactive protein that induces partial protection when co-administered with an interleukin-12-expressing plasmid in a DNA vaccine formulation

GRACIA M. S. ROSINHA, ANDERSON MYIOSHI^*, VASCO AZEVEDO^*, GARY A. SPLITTER and SERGIO C. OLIVEIRA

Department of Biochemistry and Immunology and Institute for Investigation in Immunology – Millenium Institute, and *Department of General Biology, Federal University of Minas Gerais, Belo Horizonte, Brazil and Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, WI 53706, USA

Corresponding author: Dr S. C. Oliveira (e-mail: scozeus{at}icb.ufmg.br).

Received 25 Feb. 2002; accepted 14 March 2002.

Abstract

To identify antigens of Brucella spp. that are potentially involved in stimulating a protective T-cell-mediated immune response, previous studies identified 10 clones from a Brucella abortus 2308 genomic library with primed lymphocytes as probes. One selected positive clone (182) contained an insert of 1.2 kb which was identified, sequenced and characterised. The deduced amino acid sequence of the open reading frame (ORF) revealed 82% and 81% identity to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes from Agrobacterium tumefaciens and Xanthobacter flavus, respectively. Southern blot analysis demonstrated that the gap gene is present in only one copy in the Brucella genome. B. abortus GAPDH was then expressed in Escherichia coli as a fusion protein with the maltose-binding protein (MBP). To demonstrate the functional activity of Brucella GAPDH, E. coli gap mutants were transformed with a Brucella pMAL-gap construct. Genetic complementation was achieved and as a result E. coli mutants were able to grow on glucose or other carbon source medium. The humoral and cellular immune responses to the recombinant (r) GAPDH were characterised. In Western blots, sera from naturally infected cattle and sheep showed antibody reactivity against rGAPDH. In response to in-vitro stimulation by rGAPDH, splenocytes from mice vaccinated with rGAPDH or B. abortus S19 were able to produce -interferon and tumour necrosis factor- but not interleukin (IL)-4. Furthermore, gap associated with murine IL-12 gene in a DNA vaccine formulation partially protected mice against experimental infection.

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