Cloning,expression and antigenicity of recombinant protein OMP31 of Brucella melitensis

Objective To construct a prokaryotic vector for the expression of OMP31 protein of Brucella melitensis.Methods A DNA fragment coding OMP31 of Brucella melitensis was amplified by PCR and inserted into the vector of pET-30a(+).The resultant recombinant plasmid,designated as pET-30a(+)/OMP31,was verified by double digestion using restriction endonucleases NdeⅠand XhoⅠand direct DNA sequencing.Then the plasmid was transfeced into BL21(DE3) competent cells for the expression of the OMP31 protein.After induction with different concentrations of isopropyl β-D-thiogalactopyranoside(IPTG),the collected cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Results We successfully constructed an expression vector of pET30a(+)/OMP31.An IPTG-induced expression of the OMP31 protein(31 KDa in molecular weight)was identified with SDS-PAGE.Only appearing in the inclusion bodies,the highly pure OMP31 protein could be obtained by refolding.Western blot assay showed that the refolded protein could be recognized by the anti-serum against Brucella melitensis.Conclusion The recombinant protein of OMP31 with a C-terminal hexa-His-tag was successfully expressed in E.coli BL21 as inclusion bodies.The refolded protein is of good immunogenicity.