Study On Macrophage And Dendritic Cells Affected By Mannose-hexose-modified Brucella P39 Protein

Objective: Brucellosis is a worldwide zoonotic infectious disease.The number of new infections in the world is approximately 500,000 each year.More than 170 countries have reported human and livestock epidemics worldwide.However,at present,the brucellosis vaccine is mostly attenuated vaccine with certain virulence.With the deepening of research,the predominant antigen proteins of Brucella proteins known so far include outer membrane proteins(OMPs),nuclear proteins L7/L12,surface proteins(BCSP),and cytoplasmic binding proteins(P39).P39 protein is one of the preferred T cell antigens and a recognized Brucella dominant antigen,but its immune protection is inadequate.It is found that glycosylation can improve the immunogenicity of proteins.In this experiment,the effect of sugar modification on the immunogenicity of proteins was studied by mannohexose-modified P39 protein,which provided a theoretical basis for the study of Brucella glycogen modified protein antigen vaccines.Methord: In this experiment,Brucella P39 protein was taken as the research object.The P39 gene fragment was obtained by the design of primers and application of PCR amplification.The target gene was ligated into the plasmid expression vector.After the recombinant plasmid was constructed,IPTG induced the expression of the recombinant protein.The target protein P39 was purified by using GST column.The protein was performed mannose hexasaccharide modification by using EDC/NHS chemical conjugation method,and the ratio of its sugar to protein modification was detected.After the mice were immunized with mannose-hexose-modified protein by using MTT assay and indirect ELISA,experiments such as cell proliferation,cytokine detection,and antibody titer detection were performed.In addition,the effect of the sugar-modified protein on dendritic cells and macrophages was studied by using the above-described method.The FITC-labeled mannohexaose modified protein explored macrophage phagocytic protein status.Western Blot detected cell surface receptor TLR2,TLR4,MyD88 and TRAF6 expression.Result: In this experiment,the 1206-bp fragment of the target gene was amplified from the Brucella strain 16 M by PCR and the target gene fragment was successfully ligated into the pGEX-6P-1 expression vector.Positive strains were induced by IPTG,and the P39 gene was expressed in the prokaryotic expression system.The soluble form protein was successfully expressed.Through the selection of the inducer concentration and the induction temperature,the P39 protein expression was the highest when the final concentration of IPTG was 1 mmol/L at 28°C.The target protein was purified by GST affinity column method.The size of the target protein was 45 kDa,which was in accordance with the theoretical predictions.A single component of the protein was successfully purified.The glycosylation reaction of the target protein P39 was performed and the actual ratio of sugar to protein was calculated to be 2.3:1 based on the measurement results.Mannohexose-modified protein molecules were phagocytosed faster by macrophage RAW264.7.At the 6h of protein,the number of phagocytos was higher than that of the unmodified protein group.Mannohexaose modified protein could significantly promote the proliferation of spleen lymphocytes in mice.In addition,when the protein stimulated macrophages and dendritic cells,the cell proliferation was also significantly enhanced.The mannohexasaccharide-modified target protein detected higher antibody concentration in the fifth week and was higher than the antibody concentration produced by the unmodified protein-stimulated mouse.After mice were stimulated with mannose-hexose-modified P39 protein,the contents of cytokines IFN-?,IL-2,IL-4 and IL-10 in the culture supernatant of mouse splenocytes were significantly increased comparing with the unmodified protein.High levels of IL-10 were detected after stimulation of dendritic cells by the mannohexose-modified proteins.The differences were significant.Mannose-hexose-modified P39 protein could activate the expression of TLR2 receptors.Conclusion: The mannohexose-modified P39 protein not only promotes the proliferation of lymphocytes,macrophages and dendritic cells,but also can significantly increase the level of cellular and humoral immunity by TLR2 receptor pathway.