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

• 作 者: Shiva Mirkalantari Nour Amirmozafari Bahram Kazemi Gholamreza Irajian
• 机 构: Tehran University of Medical Sciences, School of Medicine, Microbiology Department, Tehran, Iran“,”S
• 出 处: 亚太热带医药杂志（英文版）
• 发表日期: 2012年7期
• 关键词: Brucella Cloning virB12 pET28a pJE1.2

ABSTRACT 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 bacterialDNA was extracted by BioneerAccuPrep?GenomicDNAExtractionKit.Oligonucleotide primer pair was designed based onBrucella virB12 gene sequence withBamHI andHindIII restriction site at5′ end ofthe forward and reverse primers, respectively.DNA amplification was performed usingPrimSTAR?HSDNA polymerase and thePCR product was purified byDNAAccuPrep?GelPurificationKit.Purified DNA was cloned into pJET1.2 cloning vector.VirB12 gene fragment was excised from pJET1.2 usingBamHI/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 theBrucella virB12 gene which could be used as antigenic component for specific serological assay development.