Identification Of B.melitensis 16M Protein Immunodominant Antigens

Brucellosis, is a highly contagious disease caused by the gram-negative intracellular bacteria of the genus Brucella. As an important zoonosi, it poses a great threat to human health and animal husbandry. For a long time, naturally infected animals and vaccinated animals are difficult to differentiate because existing vaccines lack diagnostic markers, and researchers have not yet developed effective differential diagnostic antigens. Because the existence of infected animals in the wild,the eradication of brucellosis has been prohibited. The prevention and control of brucellosis is in desperate need of more safe, effective and distinguishable new vaccines, and the develpoment of the diagnosis tests.In this study,we targeted nine proteins among the membrane proteins, there are OMP31,BP26,L7/L12,BCSP31,Dnak,cuznSOD,GroEL,GroES and P39 .Then amplified their genes by means of polymerase chain reaction (PCR). The amplified products were purified with agarose gel extraction kit. Following digestion with the restriction endonuclease Nde I and Xho I, these target genes were inserted into the expression vector pET-21b or pET-32a respectively. The recombinant plasmids were transformed into Escherichia DH5a and validated by PCR identification, double enzyme digestion and sequencing analysis, which showed that the target genes were successfully inserted into the vector. Then, the nine recombinant vectors were transformed into Escherichia Rosetta and induced to express the fusion proteins at different time points with different concentrations of IPTG., SDS-PAGE revealed the target genes were over-expressed in E.coli system. Western blot indicated that these fusion proteins could react specifically with the sera obtained from bovine immunized with Brucella. After purifying with affinity chromatography, these fusion proteins were coated as antigen for enzyme-linked immunosorbent assay (ELISA), respectively. Then, the BP26-iELISA was successfully established for the detection of antibodies against Brucella. The detectable rate of this assay could be up to 96% , indicating bp26 fusion proteins possess well immunogenicity and could be used as diagnostic antigens in clinics.After immunizing BALB/c mice with these fusion proteins respectively, cfu/spl were detected. All these proteins could confer certain immune protection, with OMP31,BP26,GroEL,GroEs,P39 induced immune protection much stronger. This study provides a preliminary basis for the development of Brucella subunit vaccine and DNA vaccine.