Amplification, cloning, and expression of Brucella melitensis bp26 gene isolated from Markazi province in order to produce BP26 recombinant protein

Azizpour Mm, Hosseini Sd, Akbary, Basiri, Nezamabadi, Eskandari, Sarikhani
2013 Arak Medical University Journal (AMUJ)   unpublished
Brucellosis is a debilitative disease that imposes heavy costs on the economy and society. Therefore, using the most accurate and efficient method to diagnose this disease is essential. In Iran, Brucella melitensis is the common causative agent for brucellosis and BP26 protein of this bacterium has a good level of antigenicity. Thus, the aim of this study is to produce Brucella melitensis recombinant BP26 protein with a PET28a expression vector. Materials and Methods: In this
more » ... applied-fundamental study, genomic DNA was isolated from bacterial culture through proteinase K (pK) and phenol/chlorophorm protocol. Then, two pairs of primers were designed based on the known sequence in the gene bank for amplification of Brucella melitensis bp26 gene and PCR reaction was set up and optimized. The PCR product was cloned first into PTZ57R/T vector and accessed on the PET28a vector and sequenced. The recombinant vector was transformed and expressed into E. coli BL21 (DE3). Then, the recombinant protein was purified with Ni-NTA column of chromatography against His tag. Results: The size of PCR product was in accordance with the part of bp26 gene size in the gene bank. The bp26 gene without adding IPTG had little expression and 3 hours after adding IPTG with a 1 Mm concentration to culture media, extreme expression was observed. Conclusion: The production of recombinant BP26 protein from isolated Brucella melitensis native to Markazi province was done. Noticing the importance of BP26 protein and its significance for future studies on providing brucellosis diagnosis kits, its production was made possible.