Cloning and Expression of Heat Shock Protein 60kDa Gene from Brucella melitensis as Subunit Vaccine

Brucellosis is caused by the bacterium Brucella and affects various domestic and wild species. GroEL (Heat Shock Protein 60kDa) as one of the major antigens that stimulate the immune system, increases Brucella survival. The aim of the current study was to clone and express GroEL in Escherichia coli in order to design subunit vaccine. Amplifying was performed using specific primers. The full-length open reading frame of this gene was cloned into the expression vector pET-32a(+) and expressed in BL21 (DE3). The expressed antigen was purified and the molecular weight of the recombinant protein was about 70 kDa. Sequencing results along with SDS-PAGE and Western analysis confirmed the expression of recombinant GroEL in the heterologous Escherichia coli. The results of colony polymerase chain reaction (PCR), enzyme digestion and sequencing showed that the GroEL antigen has been successfully cloned and sub-cloned into pET-32a(+). The results showed that Escherichia coli was able to express GroEL protein appropriately. This protein was expressed by induction with isopropyl β -D-thiogalactoside (IPTG) at concentration of 1 mM and it was confirmed by Ni-NTA column, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blotting electrophoresis. The results of this study showed that Escherichia coli can be used as an appropriate host to produce the recombinant GroEL protein. This recombinant protein may be useful to simulate immune system, to produce recombinant vaccine and diagnostic kit in future studies after it passes biological activity tests in vivo in animal model and or other suitable procedure.