| Mastitis is one of primary restriction factors for dairy cow industry development in our country. Massive research indicated that morbidity of various kinds of mastitis of dairy cows, especially the recessive mastitis, attach to 1/3 of total lactation period dairy cow, in which nearly half number of mastitises are caused by the staphylococcus aureus. This disease influence the cow's milk production and reduces the milk quality, harms the consumer health, extends the postpartam oestrus, increases dairy cow elimination speed and so on. This disease is the most primary cause that reduces the dairy cow industry economic benefit. The multitudinous researches have demonstrated that 94%-100% staphylococcus aureus isolated from mastitis milk have the capsule.The capsule in the growth process is not only the important toxicity factor, but also is the protection antigen. The capsule polysaccharide structure is verificationed, which composed of three monosaccharides, 2-acetamide -2-deoxidation-D-mann- uronicacid,2-acetamide-2-deoxidation-L-trehalose and 2-acetamide-2-deae- ration-D-trehalose.The capsule polysacch -aride has the ability that protect bacterium from phago- cytosis of immunocyte, causing S.aureus can not be killed effectively by organism immune system, moreover the capsule polysaccharide is small molecular incomplete antigen, can't be stimulate organism to creat antibody response and immunologic memory, can't be independently used in the immunoprophylaxis as the antigen.Preparing the complete antigen by binding(coupling) some kind of carrier protein through chemistry method. And it enhance the immunogenicity of small molecular, which already is the mature technical method.For example, the pneumo-coccus capsule polysaccharidecarrier protein multivalence union vaccine successfully prepared, and has been successfully applicated in the humanity clinic.Therefore, we purify the capsule polysaccharide of staphylococcus aureus and conjugatied with protein. Conjugation can enhance the capsule polysaccharide immune -ogenicity. Starting reply ability of B cell in vivo to the polysac charide antybody, then create immunologic anamnestic response by this conjug -ation.In the experiment, The CNBr as oxidizer, the ADH as bridge, the EDAS as the couplant, preparing the complete antigen through coupling the capsule polysaccharide and the protein by covalence. During complete antigen preparation process, choosing the beast dosage of CNBr,ADH and BSA, the best dialysis temperature and time in conjugating, and pH value in activating polysaccharide. The result indicated that, in the coupling process, above condition has certain influence to the coupling reaction, along with the increasing dosage of CNBr,BSA, its coupling yield is also increasing.In the dialysis time and the temperature aspect, increase the coupling yield along with the time extension, the dialysis effect that under the low temperature is quite ideal, moreover along with the temperature increasing, its coupling yield gradually depress, we think that 4℃is the best dialysis temperature, 36h is the best dialysis time; In CNBr activation polysac- charide aspect that along with the pH value increasing the coupling yield also gradually enhancing. When the pH value achieved 8.5, the coupling yield achieves maxlmum.In this experiment, we were characterizated research on the conju -gation. Which mainly used the ultraviolet spectrograph, Sepharose CL-4B chromatographic analysis and determine the coupling yield as well as identification of the animal immunology by ELISA. Through characterizated research of the conjugation, ascertainment the ultraviolet spectrograph scanning method may carry on the polysaccharide-protein coupling identification, confirmed the conjugate has antigenicity of S.aureust capsule polysaccharide, confirmed the conjugate of preparation is success. Through Sepharose CL-4B chromatographic analysis in the 206 nm and 280 nm ultraviolet scanning examinated of the conjugate. Further demonstrates that the polysaccharide and the protein successful coupling. And through the research of complete antigen immunizing mouse, confirmed that the conjugate have the immunogenicity, so this result confirmed that the conjugate successful coupling. Simultaneously this exper- iment according to the polysaccharide/protein different ratio preparation complete antigen immunizing mouse, results displayed that the primary immunity after 14 days, displayed the good immunogenicity, the induction mouse produced anti-capsule polysaccharide specificity IgG antibody, after the second and third immunizied, the antibody titer have the remarkably increasing from compared with the first immunizied. Complete antigen immune serum opsonophagocytosis titer remarkabled increase, but opsonophagocytosis titer of the polysaccharide immune serum alone using the polysaccharide immunity is 1/4~1/8 of the complete antigen immunity. The antibody titer of mouse 7 days after the first immunizied , along with the time extension, the antibody titer also increase, after three immunizied that the mouse were increased persis -tently antibody, which descripted that the mouse had the immunologic memory response. But the pure polysaccharide group has not the obvious enhancement of antibody titer after three immunities, and has not antibody response.We discover in the research on the main immune cell of mouse that the different density polysaccharide to mouse's spleen T,B lymphocyte multipli -cation has certain promoter action, but after coupling of polysa- ccharide and protein, the conjugation had remarkably promote multiplication of mouse T, B lympha cell, its best complete antigen density is 15~20μg/mL. Compares with the pure capsule polysaccharide, the complete antigen not only strengthened the capsule polysaccharide immunogenicit, but also has emerged immunologic memory respond. The stimulating lymphocyte multiplication experiment further certificated thae this characteristic of complete antigen, these have established the theory and the experimental foundation for the BsAb preparation of capsule polysaccharide coupling.
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