Prokaryocyotic Expression of Outer Membrane Protein 25 of Brucella

To clone and construct the recombinant expression plasmid for 25 ku Outer membrane protein(OMP25) of Brucella in E.coli,The gene segments of Brucella OMP25 were amplified and obtained by polymerase chain reaction(PCR),then sequenced after TA cloning.Subsequently the target gene was cloned into prokaryotic expression vector PET-32a and identified by restriction enzyme digestion and DNA sequencing.The recombinant plasmid was transformed into E.coli Rosetta.After induced by ITPG,the recombinant protein expressed by OMP25 gene in E.coli DH5α and Rosetta was identified by SDS-PAGE analysis.The results of DNA sequencing showed that the sequence and cloning site of the insert for OMP25 gene were exact.The recombinant expression plasmid PET-32a-OMP25 was constructed,and the protein including bodies of OMP25 43 ku fusion was successfully expressed induced by IPTG.The OMP25 gene of Brucella was cloned into PET-32a,and then recombinant plasmid pET32a-OMP25 was constructed and the recombinant OMP25 protein was successfully expressed in E.coli Rosetta.The western-blotting analysis showed that the recombinant OMP25 reacted specifically with brucella positive serum.It indicated the recombinant protein processed favorable immunogenicity.