Prokaryotic Expression Of Brucella Outer Membrane Protein Omp15 And Identification Of It's Immunocompetence

Brucellosis is a zoonotic disease that was caused by brucella spp..It is severe affectting humans health and the development of animal husbandry.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,The Brucella outer membrane was investigated to seek immunogenic and protective antigens for potential diagnostic and vaccine applications. The gene of Brucella outer membrane protein was downloaded 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 ConPredâ…¡Software.This test select the Omp15(predicted secreted protein)of B.melitensis 16M for being the preliminary study.In this study,A fragment of about 423bp was obtained by touchdown PCR(TD-PCR) from the B.melitensis 16M genomic DNA,The amplified fragments were digested with the restriction endonuclease BamH I,EcoR I.then linked to expression vector pGEX-4T-2 and formed a pGEX-4T-2/Omp15.Identification by the sequencing showed the correct inserting position of the interesting gene in the vector.The recombinant fusion protein(pGEX-4T-2/Omp15)was expressed in the form of inclusion bodies at high level in E.coli BL21 induced by 0.5 mmol/L IPTG for 5h.The molecular weight of recombinant fusion protein is about 41 kDa.The inclusion bodies were washed with different concentrations of urea for 3-4 times,and then dissolved in 8M urea for denaturant.The purification of pGEX-4T-2/Omp15 was done by electrodialysis method. Then purified protein was coated as antigen and tested by ELISA.The positive immunized sera from guinea pig immunized with B.melitensis and rabbit anti-guinea pig IgG.were used to test if the protein could be used as diagnosis protein.After induced by IPTG in E.coli BL21,the recombinant fusion protein was expressed in the recombinant bacteria,and the immunized sera from guinea pig immunized with B. melitensis could specifically recognize the recombinant fusion proteins pGEX- 4T-2/Omp15 in western-blotting analysis.Although we were unable to determine finally the possibility of the Omp15 as a diagnostic protein,this result may provide a solid foundation for the function of protein and search for brucella diagnostic antigens in the future.