Study The Extraction, Purification Of CP8and The Immunogenicity Of Conjugate In CP8from Staphylococcus Aureus

Staphylococcus aureus is one of the most significant pathogens which causes the bovine mastitis. And we considerd the major virulence factors is capsular polysaccharide and adhesin of S. aureus. When S. aureus invasion of the body, it can’t be cleared easily by phagocytic cell, because of resisting the phagorytosis of capsular polysaccharide. Further more, the bacterial adhesin recognised and adhered to the organization and definite molecule of cell. The infection is hard to control when bacterium produce enzymes and toxins after the success of colonization. In the experiment, Coupling the capsule polysaccharide and adhesin to prepare the conjugates through chemical method. So it laid the theoretical and experimental basis of the divalent virulence factors vaccine of CP and adhesin in S. aureus.We added different nutrients in Columbia solid medium in order to optimize the culture conditions of S. aureus in the text. Using the method of plus enzyme to extracted CP8from S. aureus. We purified CP using through column chromatography methods. Its contents were detected by the method of Phenol-vitriolic colorimetry. On the basis of get the purified CP8, we chose ADH as bridge, EDC as the couplant, the purified ClfA-FnBPB as carrier protein to bide CP and ClfA-FnBPB together in order to prepare conjugates. In the couping process to determine the best mass ratio of conjugate. Secondly, the use of UV absorbance and Phenol-vitriolic colorimetry method to qualitative and quantitative analysis and calculate the coupling ratio. Finally, the BalB/c mice were immunized with the conjugates, ELISA method and mouse toxicity test were chosen to detect antibody levels and protective effect of CP conjugates.The result showed that CP8S. aureus had the most vigorous growth in Columbia solid medium which added1%lactose,2%NaCl and10%fetal bovine serum. We used these conditions to cultured strains which extracted capsular polysaccharide. The purity of the crued CP was34.43%, the purity of the refined CP was79.68%. The nucleinic acid and protein total concentration was0.1365mg/ml. The success of conjugates were proved that CP/ClfA-FnBPB mass ratio was one to two through chemical method and the coupling ratio was1.89. Using ELISA method, the second immunity after7days, conjugate group showed immunogenicity, and antibody titers can up to1:6400. so an immune response was generated in mice. The antibody levels did not appear also in negative control group and CP group. It can stimulate immune response in fusion protein group after the second immunization, but the effect was not obvious and antibody titers is only1:100..The results of mouse toxicity test showed that the conjugate group had the highest survival rate, it can reach80%. The survival rate of negative control group, CP group and fusion protein group were20%,30%,60%. So the conjugates have good immunogenicity. which reach the target of this experiment.