Study On The Technique Of Enhancing The Immunogenicity In Capsular Polysaccharide CP5 And CP8 Of Staphylococcus Aureus

Mastitis severely affect the development of dairy industry. Staphylococcus aureus is one of the major mastitis pathogens, 94% -100% of Staphylococcus aureus isolated from the milk of cows suffering from mastitis have the capsule, 70% -80% of staphylococcus aureus capsular polysaccharide is type 5 or 8, so CP5 and CP8 had been considered to be a major factor of mastitis. Capsular polysaccharide on the surface of Staphylococcus aureus is relate to bacterial virulence and antiphagocytic, so staphylococcus aureus can not be effectively killed by immune system.To develop specific capsular polysaccharide vaccine which can stimulate immune response and immune memory response is an effective way to prevent mastitis. However, capsular polysaccharide is small molecule and is incomplete antigen, it can not stimulate immune response and immune memory response,so capsular polysaccharide can not be served as an independent antigen for immunization. Carrier protein molecules can be conjugated with small molecules capsular polysaccharide by chemical method.By this way, macromolecular substances can be synthesized to stimulate specific capsular polysaccharide antigen.There have been successful reports at home and abroad.In the course of experiment, we chose ADH as inter-bridge and EDAC as coupling agent to bide capsular polysaccharide and different carrier protein together.Bacterial flagellin is perfect adjuvant, it can enhance the immune response. So, in the field of choosing carrier, flagellin was chosen as the main object to study. In order to increase lysine content in flagellin, we add a polylysine to flagellin N-terminal.In this way, polysaccharide derivatives'attack power to flagellin protein can be enhanced, forming a continuous polysaccharide fragments. Through continuous polysaccharide fragments and adjuvant effect of flagellin, more effective conjugates can be got.In the assessment process of macromolecular conjugates, we chose thin-layer chromatography, phenol sulfuric acid, sepharose CL-6B chromatographic analysis to assess the conjugates.The success of conjugate was proved through the identification and the binding rate was determined.Mice were immunized by Staphylococcus aureus capsular polysaccharide conjugate, then blooded. ELISA method, MTT method and mouse toxicity test were chosen to detect immunogenicity and protective effect of capsular polysaccharide conjugates.The results showed that two weeks after the second immunization, Polysaccharide conjugates have shown immunogenicity, and induced specific IgG, IgM capsular polysaccharide antibodies. Two weeks after the third immunization, antibody levels had significantly increased compared to the second immunization, it indicated that an immune memory response was generated in mice. While, after the third immunization, the separate polysaccharide group could not increase antibody levels and could not produce immune memory response. Among the mice immunized by polysaccharide conjugates, the antibody level of Lys-flic-cp group was significantly higher than the flic-cp, flic-m-cp and BSA-cp groups, the antibody level of flic-cp group had significantly higher lever than flic-m-cp and BSA-cp groups. It indicated that flic played the role of immune adjuvant and could enhance humoral immunity, and adjuvant effect of lys-flic was stronger than flic.During the study of cell-mediated immunity in mice, we proved that CP5 and CP8 could promot proliferation of mouse spleen lymphocytes. However, capsular polysaccharide- carrier protein conjugates was significantly promote the proliferation of mouse spleen lymphocytes. Compared to flic-cp, bsa-cp and flic-m-cp, lys-flic-cp played a more prominent role in the promotion of T lymphocyte proliferation.Stimulation index of flic-cp group was significantly higher than bsa-cp and flic-m-cp groups. It indicated that the lys-flic had more immune enhancement ability than flic. In this experiment, the mice 14 days after the third immunity was attacked with Staphylococcus aureus type 5 or 8. The results showed that the survival rate of mice immunized with the capsular polysaccharide conjugates is higher than the control group.The mice immunited with lys-flic-cp conjugates had the highest survival rate, it suggested the immune enhancement of lys-flic.