Molecular cloning of virB12 gene of Brucella melitensis 16M strain in pET28a vector.

OBJECTIVE

To clone the virB12 gene in pET28a expression vector for production of recombinant protein to be used as antigenic component for future serological test development.

METHODS

Brucella melitensis (B. melitensis) 16M strain was cultured and bacterial DNA was extracted by Bioneer AccuPrep® Genomic DNA Extraction Kit. Oligonucleotide primer pair was designed based on Brucella virB12 gene sequence with BamHI and HindIII restriction site at 5' end of the forward and reverse primers, respectively. DNA amplification was performed using PrimSTAR® HS DNA polymerase and the PCR product was purified by DNA AccuPrep®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 (+).

RESULTS

Brucella virB12 gene was successfully cloned in pJET1.2 and then in pET28a (+) plasmids. PCR and restriction enzyme digestion confirms the procedure.

CONCLUSION

We cloned and expressed the Brucella virB12 gene which could be used as antigenic component for specific serological assay development.