Study On Protective Immunity Of The In Vivo Infectious Factor Of Brucella Abortus

Brucellosis is a chronic infectious disease caused by Brucella. It is the world’s most popular wide disease. As one of the most damaging zoonotic diseases of statutory B infectious diseases, it has seriously threated to human and animals health. The live attenuated vaccine is the most widely used for Brucella currently, which can provide better protective immunity, but the virulence is easy to back and to be strong; Inactivated vaccine can only provide short-term protection; Subunit vaccine’s component is clear and has side effects, it will have broad prospects for its easily controlled production.The virulence factors and intracellular parasitic mechanism of Brucella have become the focus of research scholars. Studies have shown that many of the genes in the genome of Brucella are closely related to growth, invasion, parasitic and immune function of Brucella, but22%of the coding genes’function are still unknown. In vivo-induced antigen technology was used and screened33individual infectious agents in preliminary study of the laboratory. In order to provide a reference for screening better protective antigen to prevent and control the brucellosis,further research was done on the part of the infectious agent in the body’s immune protection.Brucella infectious agents YaeC, Asly, EcoA, MDH, SDR, Fumhy, nitrite, EnvZ, GntR, Arg and Sugar were screened by IVIAT, expressed in E.coli and purified. The infectious agents were amplified and connected to the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid, which was induced by IPTG, purified by affinity chromatography after transformed into E. coliBL21(DE3) competent cells.Binding activity of10kinds of recombinant proteins with fibronectin protein and plasminogen fibronectin were detected, using MDH protein as positive control, while BSA as a negative control.Fibronectin and fiber connected plasminogen binding activity experiments showed that the protein of Asly, EcoA, MDH, SDR, Fumhy, nitrite, EnvZ, Arg and Sugar can be combined with fibronectin and fibronectin plasminogen. The results suggested that these proteins might play a role in the process of Brucella adhesion and invasion of host cells, they were maybe involved in the pathogenesis, intracellular survival and proliferation of Brucella.In order to analyze the protective immunity of the recombinant protein, the indicators of humoral immunity and cellular immunity in mice, Balb/c mice were immunized with recombinant emulsified protein, the antibody titers and the cytokines in peripheral blood in mice were detected after immunization at different times, and peripheral blood lymphocyte subtypes were identified. The result showed that higher levels of specific antibodies could be produced after the second immunization. The cytokine detection results showed that the contents of IFN-γ in the serum of Asly, Nitrite and Sugar immunized groups were significantly higher than that of the control group (p<0.01) after challenge with Brucella. In order to protect the host against Brucella, IFN-γ was mainly produced by T cells and NK cells within the host cell. The content of IL17A was also significantly higher than control group(p<0.05). The rise content of IL17A showed IL17A was activated, the process of which mediated tissue inflammation to protect body from infection by speeding up the body to clear and kill Brucella.At10days after immunized the mice were infected by B. abortus2308, to count the number of bacteria in the spleens of mice and evaluate the effects of recombinant protein on bacteria in spleen of immunized mice. The results of immune protection experiment showed that Asly, Nitrite and Sugar could protect mice against Brucella infection. Bacteria in spleen of mice immunized with Asly decreased by2times than control group,7times for Nitrite, and even14times for Sugar. Flow Cytometry results showed that CD4+/CD8+ratio of Asly and Sugar immunohistochemistry was significantly lower than the control group, while the Nitrite is opposite. The result suggested that recombinant proteins of Asly and Sugar could induce specific CD8+lymphocyte responses, and Nitrite induced CD4+T lymphocyte responses.In this study, three protective antigens against B. abortus2308were selected out. Humoral and cellular immune parameters were studied after protective antigens were immuned. The results obtained in this experiment provided the valuable reference for the study of the prevention of brucellosis vaccine.