金黄色葡萄球菌CP8-ClfA-FnBPB偶联物的制备及其免疫学特性

Preparation and immunology of conjugates of CP8 from Staphylococcus aureus

以己二酰肼为间桥,碳二亚胺为偶联剂,将纯化的血清8型荚膜多糖和纯化的ClfA-FnBPB融合蛋白偶联制备偶联物,并在偶联的过程中确定其最佳质量比和偶联比。用制备的偶联物免疫小鼠,同时用间接ELISA法检测抗体水平,并对三免后2周的小鼠进行攻毒保护试验。结果显示,用化学方法制备的多糖-蛋白偶联物在其质量比为1∶2时可成功偶联,计算得到偶联比为1∶1.89。采用间接ELISA法检测抗体水平,二免后7d,偶联物免疫组可产生抗体,且抗体效价最高可达1∶6 400。阴性对照组和单独多糖组均没有抗体产生,而单独融合蛋白组在二免后产生了抗体反应,但抗体效价较低,最高时仅为1∶100。对小鼠进行攻毒保护的结果表明,偶联物组的攻毒保护率最高,可达80%,而阴性对照组、单独多糖组以及单独融合蛋白组的保护率分别为20%、30%和60%。

On the basis of this chemical method,we chose ADH as bridge,EDC as the couplant to bide purified CP and purified ClfA-FnBPB together in order to prepare conjugates. In the couping process to determine the best mass ratio and coupling ratio of conjugate were determind. Then 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 the conjugates were made in the CP/ClfA-FnBPB mass ratio of one to two through chemical method and the coupling ratio was 1 to 1.89. Using ELISA method, 7 days after the second immunity, conjugate group showed immunogenicity, and antibody titers can up to 1 ： 6 400. The antibody levels did not appear 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, andantibody titers is only 1 ： 100. The results of mouse toxicity test showed that the conjugate group had the highest survival rate,it can reach 80 %. The survival rate of negative control group,CP group and fusion protein group were 20% ,30% and 60% ,respectively.