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

作者：Mirkalantari Shiva; Amirmozafari Nour^*; Kazemi Bahram; Irajian Gholamreza

来源：Asian Pacific Journal of Tropical Medicine, 2012, 5(7): 511-513.

DOI：10.1016/S1995-7645(12)60089-3

• Brucella
• Cloning
• virB12
• pET28a
• pJE1.2

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 (R) Genontic DNA Extraction Kit. Oligonucleotide, primer lair was designed based on Brucella virB 12 gene sequence with BamHI and HindIII restriction site at 5 end of the forward and reverse primers, respectively. DNA amplification was performed using PrimSTAR (R) US DNA polymerase and the PCB product was purified by DNA AcettPrep (R) Cel Purification Kit. Purified DNA was cloned into pJETI.2 cloning vector. VirB12 gene fragment was excised front pJETI.2 using BamHI/HindIII and subsequently subeloned into pET28a (+). Results: Brucella virB12 gene was successfully cloned in pJETI.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.

• 出版日期2012-7