摘要
目的:研制5型肺炎链球菌荚膜多糖(PN5PS)与白喉无毒突变体(CRM197)结合疫苗。方法:采用还原胺化法制备结合物,即以肺炎5型多糖的邻位羟基作为活化位点,用高碘酸钠进行氧化,形成的醛基与CRM197蛋白上的氨基生成席夫碱反应,在氰基硼氢化钠的作用下形成共价结合物,并将制备的结合物与两种不同铝佐剂吸附,用间接ELISA法检测大鼠血清中针对肺炎5型多糖的抗体效价,比较结合疫苗与不同佐剂吸附前后的免疫原性。结果:当多糖活化度为7. 3时,总糖及蛋白的回收率较高,游离糖含量较低,糖蛋白结合比在1. 2~2.7时结合疫苗具有较强的免疫原性,结合比为1. 9时结合疫苗的免疫原性最强;结合物吸附磷酸铝佐剂后所产生的抗体水平略高于氢氧化铝。结论:通过还原胺化法制备的PN5PSCRM197结合疫苗具有较好的免疫原性。
Objective: To develop the method of combination of pneumococcal type 5 capsular polysaccharide(PN5 PS) with diphtheria non-toxic mutant(CRM197) conjugate. Methods: The PN5 PS was conjugated to protein with reductive amination method. The vicinal hydroxyl group of pneumonia type 5 polysaccharide was used as the activation site. It was oxidized by sodium periodate,and the aldehyde produced Schiff base reaction with amino of CRM197 protein to form a covalent conjugate under the action of sodium cyanoborohydride. Conjugates were adsorbed to different aluminum adjuvants and immunogenicity was evaluated in rat model.Antibody titers against the pneumococcal type 5 capsular polysaccharide in rat sera were determined by indirect ELISA. Results: Under the condition that degree of oxidation(DO) was 7. 3,the recovery of conjugate was optimal,and the content of free Ps was low. The immunogenicity of the conjugate vaccine was good when the proportion of polysaccharide and protein in the product is 1. 2-2. 7 and the best when the proportion of polysaccharide and protein in the product was 1. 9. Aluminum phosphate induced higher antibody titer compared with the aluminum hydroxide. Conclusion: The immunogenicity of the PN5 PS protein conjugate vaccine prepared with reductive amination method is good.
引文
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