Construction And Immune Effect Evaluation Of Brucella S19 Recombinant Vaccine

Brucellosis,caused by Brucella,is an animal-borne zoonotic disease.The disease is not only a great hazard to the livestock industry,but also poses a threat to public health security.Livestock vaccination is the main means of prevention and control of disease,especially in developing countries.However,existing vaccines include S19,which is commonly used internationally,and S2 and M5 are developed in China.The immune protective effectiveness provided of existing vaccines is not satisfactory.Therefore,improving and enhancing the immunogenicity and protective efficacy of vaccines has become the main direction for the development of new Brucellosis vaccines.American scholars overexpress Brucella's protective antigen SOD in strain RB51 to enhance its vaccine efficacy.In this study,brucella vaccine strain S19 as the research object,taking the broad-host-range plasmid to transform the Omp31,BCSP31 and UGPase genes into the S19 strain,and achieve over-expression of the self-protein.American scholars over-expression of a protective antigen SOD of Brucella.abortus in strain RB51 to enhance its vaccine efficacy.Mice vaccinated with RB51-SOD,but not RB51,developed antibodies and cell-mediated immune responses to SOD.Strain RB51-SOD vaccinated mice developed significantly more resistance to infection by the virulent strain 2308 than those vaccinated with strain RB51 alone.The immunogenicity and protective effects were evaluated by animal experimentsand the application potential of recombinant overexpressing strains as vaccine candidate strains was analyzed,which provides a new strategy for the prevention and control of brucellosis.The main research are as follows:First,a recombinant broad-host-range plasmid pBBR1MCS-PT containing the double-promoter pR/pL-terminator sequence rrnbT1T2 was constructed,and inserted into the Omp31,BCSP31 and UGPase genes to construct the recombinant expression plasmids pBBR1MCS-PT-Omp31,pBBR1MCS-PT-BCSP31 and pBBR1MCS-PT-UGPase,The recombinant plasmid was electrically transformed into Brucella S19,and the recombinant strains S19-Omp31,S19-BCSP31 and S19-UGPase were obtained by resistance screening and PCR.The SDS-PAGE and Western Blot were used to identify the recombinant Brucella expressing proteins of Omp31,BCSP31 and UGPase.Recombinant strains were serially passaged until the 15th generation without gene loss,had good genetic stability.The bacterial curve showed that the growth of the recombinant strain and the parental strain were basically the same,indicating that gene overexpression did not change the characteristics of Brucella.Next,this study conducted research on the safety and immunogenicity of recombinant strains.Animal safety test results showed that there was no significant difference in body weight,spleen weight and spleen number between S19-Omp31 group,S19-BCSP31 group and parental S19 group when Balb/c mice were immunized with the same dose(P>0.05);However,the S19-UGPase group had a significantly higher number of spleen bacteria than the S19 group(1�10~8 CFU/mL and 1�10~9 CFU/mL),indicating that the recombinant S19-Omp31 and S19-BCSP31 strains have good safety.The results of humoral immunity showed that the three groups of recombinant strains could effectively produce specific antibodies against S19 strain,in which the antibody levels reached the peak in the S19-UGPase group and the S19-BCSP31 group at the4th week.The S19-Omp31 group in the 5th week,its antibody titer was significantly higher than that of the S19 group(P<0.05).Meanwhile,the protein specific antibody results showed that the specific antibody levels in the S19-Omp31 group and the S19-BCSP31 group were significantly higher than those in the S19 group(P<0.01).Cellular immunity showed that both S19-Omp31 and S19-BCSP31 groups up-regulated the secretion of IFN-?,INF-?,IL-2,IL-4 and IL-12 cytokines,and induced the body to produce Th1 type(CD3~+CD4~+IFN-?~+)cellular immune response.The secretion of IL-2 and IL-12 in S19-UGPase group was significantly higher than that in S19 group(P<0.05),and the level of IFN-?was slightly lower than that in S19group(P<0.05).INF-?,IL-4 and IL-10 was no Significantly different in secretion levels between the S19 group.Finally,the immune protection efficacy of the recombinant strain was evaluated by virulent Brucella 16M strain challenge.The results showed that the immune protection units of S19-Omp31 group and S19-BCSP31 group were 2.25 and 2.08,which were significantly higher than that of S19 group(1.68).The immune protection unit of the S19-UGPase group was 1.74,which was not significantly different from that of S19 group.This indicates that the recombinant S19-Omp31 and S19-BCSP31strains provide good immune protection efficacy.In summary,the three recombinant Brucella strains(S19-Omp31,S19-BCSP31and S19-UGPase)constructed in this study have good genetic stability,among which S19-Omp31 and S19-BCSP31 strains has good immunogenicity,can provide the desired immune protection by virulent attack.The results of this study will provide a strategy for the development of Brucella vaccine.