Construction And Immunogenicity Analysis Of Different Subunit Vaccines Of Brucella L7/L12-Omp16 Fusion Gene

Brucellosis is a common zoonosis caused by Brucella.Which widely spreads and causes great harm to animal and human.This disease leads to undulant fever and chronic infection,abortion and infertility,which seriously endangers human health and animal husbandry development.The present studies about Brucella vaccines mainly include inactivated vaccines,live attenuated vaccines and subunit vaccines.However,the inactivated vaccine induced poor immunity.The live attenuated vaccines also have strong adverse reaction,which is difficult to distinguish the infected animals from vaccinated animals,and the recovery of toxicity.Due to these shortcomings,the study on an effective subunit vaccine has become.In this study,the gene fragments of L7/L12 and Omp16 were obtained by PCR amplification and the prokaryotic expression plasmids of pET-28a-L7/L12 and pET-28a-Omp16 were constructed.And the rL7/L12 protein and rOmp16 protein were obtained after induced by IPTG.The L7/L12-Omp16 fusion gene fragment was amplified by overlap PCR.Then the eukaryotic expression plasmid of pVAX1-L7/L12-Omp16 was constructed and transfected into 293T cells.The indirect immunofluorescence test was performed.The results showed that the DNA vaccine could express highly in eukaryotic cells.At the same time,the recombinant plasmid of pMV306-L7/L12-Omp16 was constructed and transformed into competent cells of BCG to prepare recombinant BCG vector vaccine.Western Blot validation showed that L7/L12-Omp16 fusion protein could be correctly expressed in BCG.Based on L7/L12 and Omp16 proteins,the recombinant protein vaccine,DNA vaccine and vector vaccine were used to immunize BALB/c mice with different strategies for immunogenicity analysis.The levels of IgG,IgG1 and IgG2a antibodies in serum were detected by ELISA.The results showed that the antibody titer of the DNA-Protein group was highest and the antibody titer of the rBCG group and the rBCG-DNA group was low.Thus,the DNA-Protein immunization strategy can enhance the humoral immune response levels,and vector vaccines induce weak humoral immunity.The spleen lymphocytes of mice were stained for CCK-8 and Proliferation Dye eFlour^TM 450 to detect the proliferation activity of spleen lymphocytes in each group.The results showed that the immunization strategies of DNA-Protein group and rBCG-DNA group can enhance the proliferation activity of lymphocytes.The secretion of IFN-?,TNF-?,IL-2 and IL-17 in CD4+T and CD8+T cells was detected by FCM.The results showed that the levels of IFN-?,TNF-?,IL-2and IL-17 were detected in both DNA vaccine and vector vaccine immunized groups were significantly higher than those of the PBS group.DNA vaccines and vector vaccines could induce a strong cellular immune response and the immunization strategies of DNA-Protein and rBCG-DNA could enhance cellular immune response level.ELISPOT detection of IFN-?secreting cells in each immunized group was consistent with FCM detection of IFN-?.The immunization strategies of DNA-Protein and rBCG-DNA could enhance the cellular immune response.In conclusion,the recombinant protein vaccines,DNA vaccine and vector vaccine based on Brucella proteins of L7/L12 and Omp16 were constructed in this study.The BALB/c mice were immunized with different immunization strategies.DNA vaccine and vector vaccine could induce a strong cellular immunity,but the humoral immunity was poor.Compared to the use of a single vaccine,the immunization strategies of DNA-Protein and rBCG-DNA could enhance the humoral immune and cellular immune response level.