Construction Of Novel Vaccines For Brucella And Preliminary Evaluation Of Their Immune Protective Effects

Several strategies have been pursued in the development of brucellosis vaccines. These include development of subunit vaccines, DNA vaccines, and vaccination with live bacterial vectors expressing Brucella antigens. Critical to any vaccine development strategy is the finding of outer membrane protein which have immunogenicity.Brucella strain 16M genome sequence was analyzed by PSORT and CELLO to predict the genetic sequence of outer membrane proteins. 17 outer-membrane genes and ribosome protein L7/L12 were selected to amplify in polymerase chain reactions. The PCR product was cloned into the prokaryotic expression vector pET32a(+), which were then transfected into the competent cells E.coli BL21(DE3), The protein expression was induced by IPTG and examined with SDS-PAGE and Western blot, and purified by HisTrapTMHP. Recombinant protein vaccines were preparated. Also, the 18 gene fragments and hGM-CSF gene, were cloned into the eukaryotic expression vector PVAX1, which were then transfected into MDCK cells. The protein expression was examined with IFA. DNA vaccines were preparated. Then the Balb/c mice was immunized with these candidate vaccines, and indirect ELISA, ELISPOT, FCM techniques were used to evaluate immune response.The results showed that: 31 outer-membrane proteins were predicted by PSORT and CELLO. Using the prokaryotic expression system, 18 recombinant plasmids were successfully constructed, of which 13 were strongly expressed. BMEI0402,BMEI0454, BMEII0983,BMEI0653,BMEI1777, BMEI1980,BMEII0381 and BMEII0472 were exsited in soluble form, BMEI1830, BMEI1305, BMEI1306, BMEII1120 were in inclusion body. BMEII0983,BMEI0653,BMEI1777,BMEI1980,BMEII0381,BMEII0472,BMEI1829 were selected to purify, and the purified proteins were reached a purity of more than 90%. 18 eukaryotic expression recombinant plasmids were successfully constructed, of which 12 were expressed in MDCK cells. The Balb/c mice was immunized with the DNA vaccines and recombinant vaccines of BMEII0381, BMEII0472, BMEI0653, BMEI0748, BMEI1777 and BMEI1980 produced a strong immune response. DNA vaccines were mainly elicited cellular immunity, and the number of specific IFN-γsecreting cells was more than IL-4 secreting cells by ELISPOT assay. Antibody subtype classification assays of the immunized mice showed that the ratio of IgG2a to IgG1 was higher than 1. The value of CD4+/CD8+ was decreased compared with negative control group by FCM. But the recombinant protein and DNA protein mixed vaccines produced humoral immunity, and the number of specific IFN-γsecreting cells was less than IL-4 secreting cells. The IgG1 level much higher than the IgG2a level of the immunized mice, and the value of CD4+/CD8+ was increased compared with negative control group. The candidate vaccines produced both cellular and humoral immune response, of which BMEI1980 and BMEI1777 were more excellent than others proteins in immunogenicity.The results in this study might pave the way for the further study on developing effective prevention of brucellosis based on vaccine strategies.