Construct Of A DNA Vaccine Encoding Brucella SOD, L7/L12, OMP25, OMP31Gene And Analysis Of Immunogenicity In Mice

Brucellosis is a zoonotic disease caused by Brucella. Brucella has a wide host range,including a variety of livestock, poultry, wildlife and people, causing symptoms such as fever,abortion, infertility, chronic arthritis and nerve damage. Brucellosis exists in many countries andregions in the world, resulting in huge economic losses. Therefore, prevention of brucellosis isvery important. Prevention is mainly achieved through vaccination.In this study, a virulent strain of B. melitensis M28as a template, PCR amplification of SOD,L7/L12, OMP25and OMP31gene. The recovered and purified of PCR products were digested byEcoRⅠand BglⅡ, then subcloned into pCAGG plasmidand constructed eukaryotic expressionplasmid pCAGG-SOD, pCAGG-L7/L12, pCAGG-OMP25, pCAGG-OMP31. The encoding SODgene and OMP25gene of B. melitensis virulent strain M28were amplified by PCR. The productswere subcloned into EcoR Ⅰ andXhoⅠ digestion sites of plasmid pET-32a, recombiningexpression plasmid pET-SOD and pET-OMP25. pET-SOD and pET-OMP25plasmids were inducedby IPTG and expressed in BL21(DE3) competent cells. The recombinant protein of rSOD andrOMP25were separated by the nickel sepharose affinity chromatography.In mice experiments, the BALB/c mice were immuned by pCAGG-SOD、pCAGG-L7/L12、pCAGG-OMP25and pCAGG-OMP31as a DNA group, while setting the attenuated vaccineM5-90group, PBS group, pCAGG group as controls. Specific antibody levels in serum in micewere detected by i-ELISA. The level of cellular immunity in mice were detected by FCM andELISPOT.The results of this study showed that the molecular weights of rSOD was34ku and rOMP25was43ku. I-ELISA showed that specific IgG antibody levels of DNA group were significantlyhigher than M5-90group (p<0.05). ELISPOT showed that the number of cells secreting IFN-γ ofDNA group were significantly higher than M5-90group (p<0.05). FCM showed that miceimmunized with DNA vaccine or M5-90vaccine could increase the levels of CD4+T cell and CD8+T cell. CD4+T cell and CD8+T cell levels of DNA group increased more significantly.This study indicated pCAGG-SOD, pCAGG-L7/L12, pCAGG-OMP25, pCAGG-OMP31induce both specific neutralizing antibodies and T cell responses in mice, thus appear to be potential Brucellosis vaccine candidates in future.