Prokaryotic Expression Of Brucella Outer Membrane Protein Omp39 And Omp14 And Identification Of Their Immunocompetence

Brucellosis is a zoonotic disease caused by a small intracellular gram-negative bacterium which is pathogenic for humans as well as for many species of animals. This infection induces abortions, undulant fever and osteomyelitis in humans as well as livestock animals leading to severe economic losses. Every year Brucella caused about 500,000 cases of Brucellosis in mankind in the world. According to the World Health Organization statistical data, Brucellosis caused economic losses of nearly 3 billion dollars every year.Accurate diagnosis was premise for effective control and eradication of the brucellosis, but the common brucellosis serological diagnostic methods can not distinguish between the vaccine or natural infection. Development of a marker of the vaccine and find a suitable brucella diagnostic antigen has been the people's goals.Brucella outer membrane protein as the immunogenic and protective effect antigen has been well known to the world. The gene of Brucella outer membrane protein was download from GenBank. The gene was selected by BLAST analysis that showed high gene homologous of the three Brucella species(B. melitensis, B.abortus, B.suis) and the gene did not high cross-reaction with other Gram- negative bacteria, while the gene of outer membrane protein was analysised of the signal peptide and transmembrane structure by TMHMM and ConPred II Software. This test select the Omp39 and 0mpl4 of B.melitensis 16M for being the preliminary study.First, the primer of the two selected outer membrane protein genes were designed by the Premier 5.0 software. The genome of inactivated B.melitensis 16M was used as amplification templates. The amplified fragments digested with the restriction endonuclease BamH I, EcoR I were ligaed directly to the expression vector-pGEX-4T-2 which were digested with BamH I, EcoR I. The recombinant vectors were respectively transformed into Escherichia coli BL21. By colony PCR identification, recombinant vector digested identification and sequencing analysis, it showed that the recombinant expression vectors were successfully constructed. Next the two recombinant vectors were induced to express the fusion proteins at the different temperatures, different time points, different concentrations of IPTG.After expressed, the recombinant fusion proteins were checked by SDS-PAGE and Western-blotting analysis. The result showed that the immunized sera from guinea pig immunized with Brucella melitensis could specifically recognize the recombinant fusion proteins. The recombinant fusion proteins purified by electrodialysis method were coated as antigen for ELISA assay. Although the results didn't support the recombinant proteins as the possibility of Diagnosis protein, this study may provide a solid foundation for the further study on the function of proteins and brucellosis diagnostic antigens.