BACKGROUND AND OBJECTIVESBrucellosis (malt fever disease) is one of the most important diseases that is transmitted between humans and animals, this disease is of special interest in Iran in terms of both economic and public health aspects. The use of recombinant vaccines is one of the newest ways to deal with the side effects of common vaccines and to improve immunity. Therefore, in this study, the design and preparation of HBHA-Omp۲۵ immunological conjugated construct consisting of Omp۲۵ extramembrane antigen and HBHA molecular adjuvant and the feasibility of expressing this construct in
prokaryotic expression system are investigatedMATERIALS AND METHODSIn the present study, after HBHA and Omp۲۵ gene fragments were extracted, they were amplified through PCR reaction. Also, SOE-PCR reaction was used in order to perform chimera and connect HBHA and Omp۲۵ gene fragments. Enzymatic digestion of HBHA-Omp۲۵ recombinant chimera fragment and pET۲۲b (+) vector was performed through NCOI and ECORI cutting enzymes. It should be noted that HBHA-Omp۲۵ fragment was placed inside the vector during the ligation process. In order to multiply the recombinant vector containing the HBHA-Omp۲۵ chimeric fragment, the said fragment was transferred into the susceptible E.coli (DH۵α) cell using the heat shock process, and then after extracting the plasmid, the recombinant plasmid was again transferred into the E.coli BL۲۱ (DE۳) susceptible cell. In addition, induction of gene expression was also done using ۱ mM IPTG. Finally, the produced protein was purified using a nickel column and visualized. Also, gene expression was evaluated using SDS-PAGE gel.RESULTS AND DISCUSSIONAccording to the results obtained from the PCR, SOE-PCR and Colony-PCR reactions, the amplification of gene fragments, chimeras and the process of transferring the recombinant vector in susceptible DH۵α and BL۲۱ (DE۳) cells through heat shock were successful. Finally, the obtained results showed that the expression and purification of the recombinant protein has been done successfully.CONCLUSIONThe results of this research were that the production of HBHA-Omp۲۵ recombinant protein was successfully carried out. And the resulting structure has the ability to be used as a candidate for making recombinant vaccines against Brucella melitensis bacteria.