Study The Effect Of Glycosylation On Immunogenicity Of Protein Rsα And AhcY

Brucellosis is still popularity in many parts of the world-wide, it can not be completely removed particularly in developing countries. Vaccination has been an important means of controlling animal brucellosis. Rsα and AhcY are sheep bracella immune protective antigen protein screened by immunoproteomics, which can be used as brucellosis vaccine candidates. Rsα and AhcY are modified by mannose. Affect of saccharide modification on protein immunogenicity are studied to explore new Brucella saccharide-modified protein vaccine.Methord:The purified protein Rsα and AhcY were obtained. Rsα and AhcY were modified by six kinds of mannose through EDC / NHS coupling chemistry method. BCA method and the phenol-sulfuric acid method were used to measure molar concentration of protein and saccharide, and then the reaction molar ratio of protein and saccharide chain were determined.Raw264.7 cells are stimulated by FITC-labeled mannose modified protein. The speed saccharide-modified protein being phagocytosised detected by fluorescence microscopy, the amount of phagocytosis protein measured by flow cytometry. The saccharide-modified protein mixed with Freund’s adjuvant was immunized Balb/c mice: after the fourth week of the immunization, MTT assay was conducted to test spleen lymphocyte proliferation; the secretion cytokines of glycoproteins stimulated splenocytes from immunized mice were measured; mice serum antibody titer was detected from 2th to 8th weeks by indirect EILSA. Spleen cell surface receptor was detected.Result:When mannose and protein were feed in 1000:1 molar ratio, the reaction molar ratio of protein and saccharide chain is stable. Reaction ratios of mannose, mannose disaccharide, mannose trisaccharide, mannose tetrasaccharide, mannose pentasaccharide, mannose hexasaccharide with Rsα were 7.5:1、3.6:1、1.7:1、2.0:1、3.2:1、4.8:1.The reaction molar ratio of Mannose, mannose disaccharide,mannose trisaccharide, mannose tetrasaccharide,mannose pentasaccharide, mannose hexasaccharide and protein AhcY are 7.6:1、8.7:1、9.4:1、5.5:1、5.0:1、4.9:1. pentasaccharide modified Rsα and tetra, hexa-saccharide modified AhcY engulfed by cells faster. Measured more pentasaccharide modified Rsα and tetra, hexasaccharide-modified protein AhcY being phagocytoes than the original protein at 6 houRs. Proliferation of disaccharide, hexa-saccharide modified AhcY immunized mice spleen lymphocyte is significantly higher than the original protein group. And hexa,disaccharide modified AhcY immunized mice highest antibody Ig G titer is the twice times of original protein group, humoral antibody titer level was increased. Cytokines IFN-r, IL2,IL10 produced by disaccharide modified AhcY group are higher than the original group, Th1,Th2 cell immune level are both improved.Tetra, petna, hexasaccharide modified AhcY protein stimulates the higher level production of IFN-r, IL2 than the original protein, which improve the level of Th1 cell immune. Disaccharide modified AhcY protein can activate high TLR4 receptor expression, hexasaccharide modified Ahc Y activates high TLR2 receptor expression.Conclusion:In summary, the present study identify a hexasaccharide modified protein, it’s regognised fast and it’s immunogenicity being increased. Immunogenicity of disaccharide modified AhcY is increased. Pentasaccharide modified Rsα and tetraccharide modified Ahc Y are identified by receptor quickly.