| Brucellosis caused by Brucella is a worldwide severe zoonosis, belongs to the second class infectious disease, which is one of the world’s five major human and animal infectious diseases(rabies, anthrax, brucellosis, tuberculosis and swine epidemic encephalitis B).Vaccination is one of the important measures for Brucellosis’ prevention and control, but the vaccine still exists defects in all aspects and can not be widely used. Therefore, the development of new vaccines is the main goal of brucellosis research. There are 7 certein outer membrane proteins(OMPs) at present, and researches shown that OMP16 and OMP19 are important protective antigens. In this study, we purified Brucella OMP16 make sure of its optimal acquisition conditions. Then, we added OMP16 into RAW264.7 and detected the main factor of apoptosis, Caspase-3, the main factors of ERS, GRP78 and CHOP, and immune factors, IL-1β, IL-6, TNF-α, in order to explore the apoptosis and immunocompetence influence of OMP16 on RAW264.7. Meanwhile, we used OMP16 to immune Balb/c mice to detect the immunizing potency in serum, and infected mice with Brucella to observe their clinical features, change of spleen, immune factors, in order to explore the immunogenicity of OMP16. The main results are as follows:1. Using prokaryotic expression and NTA resin affinity column to purifying Brucella OMP16. The concentration of purified OMP16 was detected by SDS-PAGE. Using conditions like 22℃, 26℃, 30℃ to induce OMP16 at 7 h, 8 h, 9 h, 10 h, and also different imidazole elution concentration like 20 mmol/L, 50 mmol/L, 100 mmol/L, 250 mmol/L, 500 mmol/L,respectively. Finally, make sure of the optimal conditions are: induce OMP16 at 22℃ for 8 h and 100 mmol/L and 250 mmol/L twice for elution.2. Using different concentrations of Brucella OMP16 to stimulate RAW264.7 at different time. Though MTT and Hoechst assaies, we screening out the optimal concentration(10μg/mL) of OMP16 and optimal action time(36 h). We set OMP16 stimulate RAW264.7 group and control group, using immunofluorescence and Western blot assaies to detect the main factor of apoptosis, Caspase-3 in OMP16 stimulated RAW264.7 and the results shown thatCaspase-3 has a highly significant up-regulation(P < 0.001). Using RT-qPCR and Western blot to detect the main factors of ERS, GRP78 and CHOP both increased,and the results of FCM shown that OMP16 can induce significant apoptosis in OMP16 stimulated RAW264.The results above shown that Brucella OMP16 can induce apoptosis in RAW264.7 and the ERS was participated in the regulation of this apoptosis. Meanwhile, we use RT-qPCR and found that the immune factors, IL-1β, IL-6 and TNF-α in OMP16 stimulated RAW264.7 all increased in different level, which shown that Brucella OMP16 can motivate the immunocompetence of RAW264.7.3. Using Brucella OMP16 to immume Balb/c mice, and infected them with B.suis.S2 while their immunizing potency has reached 1∶106. At the third day of infection, the infected group started to show some symptoms like spiritual malaise, weight loss, reduction of food intake, afraid of cold and shivery, and grouped together, while the immuned group doesn’t shown these symptoms mentioned above. Using RT-qPCR to detect the immune factors,IL-1β, IL-6, TNF-α in mice spleen at day 7 and also the immune factors in serum by ELISA kit. The results shown that the infected group have a remarkble increasement of immune factors, but the immuned group and control group were almost the same. These results shown that Brucella OMP16 has a good immunogenicity and it has certein immune protection against Brucella infection. |
