5型肺炎链球菌荚膜多糖与破伤风类毒素的结合及其影响因素探讨  

Preparation of Streptococcus pneumonia type 5 capsular polysaccharide-TT conjugate and the key synthetic parameters

作　　者：尹丹丹[1] 周觉非 刘威[1] 邹莎莎[1] 杨溢尧 马诚[1] 崔长法[1] 江山[1] 张雪梅[1] 葛永红[1] 

机构地区：[1]成都生物制品研究所有限责任公司,成都610023

出　　处：《中国新药杂志》2013年第16期1895-1900,共6页Chinese Journal of New Drugs

基　　金：国家国际科技合作项目(2012DFB33450)

摘　　要：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 polysaceharide size, polysaccharide/protein input ratio, reac- tion time and other parameters on conjugation. Methods ： Size reduced type 5 capsular polysaecharide of Streptococ- cus 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 immuno- genicity 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 mo- lecular size, yields of protein and polysaecharide, respectively. The conjugates induced significantly higher anti- PNSPS IgG titers than PNSPS 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.

关 键 词：肺炎链球菌 荚膜多糖 结合疫苗 破伤风类毒素 

分 类 号：R18[医药卫生—流行病学]