The Effect Of Glycosylation On The Immunogenicity Of The Brucella Protein Ahcy

Brucellosis is caused by brucella all over the world,especially in developing countries.At present,there is no effective commercial vaccine for human,and the animal vaccines are always attenuated vaccines with toxicity and pathogenesis and hardly different from nature infection.The effective vaccine is main control method for animal brucellosis.The protein Ahcy is immune protection candidate antigens of sheep Brucella screened by immune proteome protocol.In this paper,the Ahcy protein was purified and then conjugated with different oligosaccharide,the characteristics of modified proteins and the cellular immunity and humoral immunity was evaluated for development of new brucella vaccines.Methord: The protein was purified by affinity chromatograp Hy method,and glycosylation modification by EDC/NHS method.And then the p Hagocytosis of the modified proteins by murine macrop Hage was estimated.The effect of protein oligosaccharide modification on immunology was evaluated by mice.The spleen lymp Hocyte proliferation,antibody dynamics and cytokines were detected by MTT and ELISA.And the mice immuned with modified proteins were attacked by wide strain Brucella melitensis M16.And the spleen colonies were calculated by BSA medium culture.Finally,the p I changes of modified protein were detected by HPLC and two-dimensional electrop Horesis,and the structure information affected by modification was measured by circular dichroism.Result:1、The candidate antigens were modified with mannose and chitosan oligosaccharides by EDC/NHS method,and the maximum conjugation ratio of oligosaccharide to protein was 1000:1.When macrop Hage was cultured with 0.3 μ mol/ml conjugated proteins modified by FITC,the fluorescence intensity of six chitin modified protein Ahcy were stronger than mannose at 4h,but there were no difference on RS-α.Manno-tetraose,manno-pentaose,manno-hexaose modification caused stronger absorption on Ahcy.2、The results of glycoprotein on spleen lymp Hocytes in mice showed that the Ahcy protein modified by mannohexaose can significantly improve the lymp Hocyte proliferation,but chitin modification significantly inhibited lymp Hocyte proliferation than mannose.3 、 The dynamics of antibody titer in mice showed that when the ratio of oligosaccharides and protein is 1000:1,on the fourth week,the antibody reached a maximum of 123000:1.When in the sixth weeks,mannohexaose and chitohexaose modified two protein to immune mice could induced higher level of antibody IGg,the antibody level of mannohexaose modified protein Ahcy could reach maximum.But the antibody level of chitohexaose modified protein RS-α could only detected in the fifth weeks.The cytokines level of IFN-γand IL-2 for mannohexaose modified protein Ahcy weres higher than protein Ahcy,which indicated Th1 cellular immune level was enhanced.According to wide strain attack experiment found the group of protein Ahcy modified by mannohexaose spleen bacterium numbers was 4.185±0.033(log CFU/g),compared with the group of PBS was 4.263±0.100,indicated that protein Ahcy modified by mannohexaose could effectively protect the mice infection.4、The properties of glycoprotein recovered that the isoelectric point improved from 5.3 to 6.1.The reverse HPLC detection showed that the protein modified by mannohexaose the peak time was earlier than protein Achy,which indicated glycosylation modification could increase the polarity of protein;and according to the result of circular dichroism found the disorder domains were increasing after modified by mannohexaose.Conclusion: The results of glycoprotein on spleen lymp Hocytes in mice showed that the Ahcy protein modified by mannohexaose can significantly improve the lymp Hocyte proliferation,The dynamics of antibody titer and the cytokines level in mice showed that mannohexaose modified protein Ahcy weres can effectively promote the level of cellular immune and humoral immune.through the immune protection indicated that protein Ahcy modified by mannohexaose could effectively protect the mice infection.All of these laid the foundation experiment of the protein Ahcy modified by mannohexaose as brucella vaccine candidate antigens.