Brucella melitensis (B. melitensis) 16M strain was cultured and bacterial DNA was extracted by Bioneer AccuPrep庐 Genomic DNA Extraction Kit. Oligonucleotide primer pair was designed based on Brucella virB12 gene sequence with BamHI and HindIII restriction site at 5鈥?end of the forward and reverse primers, respectively. DNA amplification was performed using PrimSTAR庐 HS DNA polymerase and the PCR product was purified by DNA AccuPrep庐Gel Purification Kit. Purified DNA was cloned into pJET1.2 cloning vector. VirB12 gene fragment was excised from pJET1.2 using BamHI/HindIII and subsequently subcloned into pET28a (+).
Brucella virB12 gene was successfully cloned in pJET1.2 and then in pET28a (+) plasmids. PCR and restriction enzyme digestion confirms the procedure.
We cloned and expressed the Brucella virB12 gene which could be used as antigenic component for specific serological assay development.