Cloning, nucleotide sequence, and expression of the Brucella melitensis sucB gene coding for an immunogenic dihydrolipoamide succinyltransferase homologous protein

Abstract

The Brucella melitensis sucB gene encoding the dihydrolipoamide succinyltransferase (E2o) enzyme (previously identified as an immunogenic protein in infected sheep) was cloned and sequenced. The amino acid sequence predicted from the cloned gene revealed 88.8 and 51.2% identity to the dihydrolipoamide succinyltransferase SucB protein from Brucella abortus and Escherichia coli, respectively. Sera from naturally infected sheep showed antibody reactivity against the recombinant SucB protein.

FIG. 1

Immunoblotting after SDS-PAGE of E. coli (pMZ4503) cells expressing the sucB gene of B. melitensis 16M with anti-SucB MAb (lane 1), with sera from brucellosis-negative sheep (lanes 3 and 4), and with sera from naturally infected sheep (lanes 4 to 10), all adsorbed with E. coli JM109 cells carrying the control vector pGEM7Zf+. Lane 2, immunoblotting after SDS-PAGE of E. coli JM109 carrying the control vector pGEM7Zf+ with anti-SucB MAb.

FIG. 2

Alignment of the B. melitensis (B. mel) 16M sucB and B. abortus (B. ab) S19 sucB nucleotide sequences. Identical nucleotides are indicated by stars, and gaps are indicated by hyphens.

FIG. 3

Organization of the B. melitensis 16M suc genes. The 6,246-bp NotI-BamHI fragment comprised, besides the sucB gene, part of the sucA gene, ORF1 coding for an amino acid efflux-like protein, and part of the lpdA gene. Arrows indicate the direction of transcription.