The zinc protease gene was isolated and amplified by PCR using extracted B.melitensis genomic DNA as a template. A primer pair was designed from this gene containing restriction sites of NdeI and BamHI at 5'and 3' end, respectively. Those Primers were used to amplify an insert of 927 pb. This amplicon was digested and inserted into the NdeI-BamHI cut expression vector pET-15b. This recombinant plasmid was transformed into Escherichia coli BL21 (DE3) for protein expression. Following induction by several amounts of IPTG, the expression of this protease induced bacterial cell death, two hours after expression induction.

The thio disulfide oxidoreductase gene was amplified from Brucella melitensis genomic DNA by polymerase chain reaction (PCR) technique using specific primers and inserted into expression vector pET-15b, then the pET-15b / dsbA Sonstruction was transformed into E. coli BL21(DE3). This gene was highly overexpressed by IPTG induction. SDS-PAGE analysis showed that the expressed protein is about 28 kDa. Finally, recombinant protein was purified with a metal-chelating affinity column.