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目的:制备5型肺炎链球菌多糖与破伤风类毒素衍化物的结合物,探讨结合过程中多糖分子大小、多糖蛋白投入比、反应时间等因素对结合终产物的影响.方法:应用1-氰基-4-二甲氨基-吡啶四氟硼酸酯(CDAP)活化剪切后的5型肺炎球菌荚膜多糖,在缩合剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDAC)作用下与连接了二酰乙二腈(ADH)的破伤风类毒素(TT)进行偶联反应,经分子筛层析纯化,得到5型肺炎链球菌多糖与破伤风类毒素的结合物,并检测其生化指标,免疫原性等.结果:剪切后的多糖免疫原性良好,其与TT的结合物在分子筛上可以很好分离；描述了多糖活化时CDAP的投入量,蛋白与多糖投入比,反应时间等对结合效率、结合物分子量、游离糖含量和结合物的多糖蛋白比影响.合成的结合物在免疫小鼠后,多糖特异性IgG抗体几何平均滴度(GMT)较多糖显著提高,显示免疫增强效应.结论:CDAP方法可以应用于PN5型肺炎链球菌荚膜多糖与破伤风类毒素结合疫苗的制备.“,”Objective:To prepare pneumococcal PN5 polysaccharide vaccine using CDAP method with TT as the carrier protein,and to investigate the effect of polysaccharide size,polysaccharide/protein input ratio,reaction time and other parameters on conjugation.Methods:Size reduced type 5 capsular polysaccharide of Streptococcus pneumoniae (PN5PS) was activated by CDAP,and then chemically combined with ADH-coupled TT directly at different conditions.After passing through gel filtration column,the conjugates were characterized and its immunogenicity was evaluated in NIH mice.Results:CDAP/PS ratio,PS/TT ratio and the reaction time were important parameters for the conjugation process,leading to different PS/PR ratio,free polysaccharide contents,average molecular size,yields of protein and polysaccharide,respectively.The conjugates induced significantly higher antiPN5PS IgG titers than PN5PS alone with booster effect.Conclusion:The proposed method is suitable for PNSPS protein conjugate vaccine development with high yield,low free polysaccharide and well controlled process with good consistency.