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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 a conjugate of Streptococcus pneumoniae type 5 Streptococcus pneumoniae and tetanus toxoid derivative to study the effect of polysaccharide molecule size, ratio of polysaccharide protein input, reaction time and other factors on the final product of conjugation. METHODS: Shear type 5 pneumococcal capsular polysaccharide was activated with 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP). After condensation with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDAC) was coupled with tetanus toxoid (TT) linked with diacidonitrile (ADH) and purified by molecular sieve chromatography to obtain S. pneumoniae type 5 Polysaccharide and tetanus toxoid conjugate, and test its biochemical indicators, immunogenicity and so on. Results: The sheared polysaccharide had good immunogenicity, and its conjugate with TT could be well separated on molecular sieve. It also described that the amount of CDAP, the ratio of protein to polysaccharide, reaction time and so on, Molecular weight, free sugar content and polysaccharide protein ratio of the conjugate. After the mice were immunized, the geometric mean titer (GMT) of the polysaccharide-specific IgG antibody was significantly higher than that of the polysaccharide, showing the immune-enhancing effect. Conclusion: The CDAP method can be applied to the preparation of PN5-type S. pneumoniae capsular polysaccharide and tetanus toxoid conjugate vaccine.