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

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• 作者：Shiva Mirkalantari^a ; Nour Amirmozafari^a ; ^{Amirmozafari@yahoo.com} ; Bahram Kazemi^b ; Gholamreza Irajian^a
• 关键词：Brucella ; Cloning ; virB12 ; pET28a ; pJE1.2
• 刊名：Asian Pacific Journal of Tropical Medicine
• 出版年：2012
• 期刊代码：pca68_19957645
• 类别：med
• 出版时间：July, 2012
• 卷：5
• 期：7
• 页码：511-513
• 文件大小：334 K

摘要

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

m>Brucella melitensis (B. melitensis)m> 16M strain was cultured and bacterial DNA was extracted by Bioneer AccuPrep庐 Genomic DNA Extraction Kit. Oligonucleotide primer pair was designed based on m>Brucellam> 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 (+).

Results

m>Brucellam> 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 m>Brucellam> virB12 gene which could be used as antigenic component for specific serological assay development.