摘要
目的评价8型肺炎球菌荚膜多糖(capsular polysaccharide of Streptococcus pneumoniae type 8,CPS8)分子大小对多糖蛋白结合物物理化学(理化)性质的影响,并对结合效果最优的多糖蛋白结合物进行抗原一致性评价。方法采用高压均质法制备不同分子大小的CPS8;采用1-氰基-4-二甲氨基吡啶四氟硼酸酯活化法,制备以白喉类毒素无毒突变体CRM197为载体的CPS8蛋白结合物CPS8-CRM197,并检定其理化性质;采用核磁共振氢谱及核磁共振碳谱分析降解前后多糖糖链结构的变化情况,同时采用抑制ELISA评价CPS8-CRM197结合物的抗原一致性。结果高压均质法制备的CPS8相对分子质量在69000~268000;CPS8相对分子质量>200000时会过度交联,<100000时结合效果较差;CPS8在相对分子质量100000~200000之间的CPS8-CRM197结合物游离多糖含量均<10%,游离蛋白含量均<5%。核磁共振氢谱及核磁共振碳谱结果表明,降解前后多糖糖链结构未发生改变,特异基团得到完整的保留;抑制ELISA表明不同CPS8-CRM197结合物对8型肺炎球菌抗血清抑制的半数效应浓度在同一数量级内,分别为0.005、0.003、0.005μg/ml,抗原性具有一致性。结论多糖结合疫苗制备工艺中,应将CPS8分子大小控制在相对分子质量100000~200000之间,该范围内多糖所制备的多糖蛋白结合物理化性质较优、抗原一致性较好。
ObjectiveTo evaluate the effects of molecular size of capsular polysaccharide of Streptococcus pneumoniae type 8(CPS8)on the physicochemical properties of polysaccharide-protein conjugate,and evaluate the antigen consistency of the polysaccharide-protein conjugate compound with the best binding effect.MethodsCPS8s with different relative molecular masses were prepared by high pressure homogenization.CPS8 was activated by 1-cyano-4-dimethylaminopyridine tetrafluoroborate and then conjugated with nontoxic mutant of diphtheria toxoid CRM197 to form conjugate CPS8-CRM197.The physicochemical characteristics of different CPS8-CRM197s were analyzed.1H nuclear magnetic resonance spectroscopy(1H-NMR)and 13C nuclear magnetic resonance spectroscopy(13C-NMR)were used to analyze the changes of polysaccharide chain structures before and after degradation.The antigenicity of CPS8-CRM197 was evaluated by inhibition ELISA.ResultsThe relative molecular masses of CPS8s prepared by high pressure homogenization were 69000-268000.CPS8 was over crosslinked when relative molecular mass was higher than 200000,and the binding degree was low when the relative molecular mass was lower than 100000.The contents of free polysaccharide and free protein for different CPS8-CRM197s were all less than 10%and 5%,respectively,when the relative molecular mass of CPS8 was 100000-200000.The 1H-NMR and 13C-NMR results showed that the structure of the sugar chain remained unchanged and the specific groups were fully preserved before and after degradation of CPS8.Inhibition ELISA results showed that median effective concentrations of inhibition by different CPS8-CRM197 conjugates to Streptococcus pneumoniae type 8 antiserum were within the same order of magnitude,at 0.005,0.003,and 0.005μg/ml,respectively.The antigenicity was consistent.ConclusionsIn the preparation of polysaccharide conjugated vaccine,the relative molecular mass of CPS8 should be controlled within a certain range of 100000-200000.The polysaccharide-protein conjugates prepared from polysaccharides within this range has good physical and chemical properties,and good antigen consistency.
作者
苏璇
李硕
题靖
梁阳
赵硕
韩伟
陈鸣
徐磊
任涛
杨朝晖
Su Xuan;Li Shuo;Ti Jing;Liang Yang;Zhao Shuo;Han Wei;Chen Ming;Xu Lei;Ren Tao;Yang Zhaohui(R&D Center,Beijing Minhai Biotechnology Co.,Ltd.,Beijing Key Laboratory of Study on the New Technique for Conjugate Vaccine,Jiangnan University-Beijing Minhai Biological Sugar Vaccine Joint Research and Development Center,Beijing 102600,China)
出处
《国际生物制品学杂志》
CAS
2023年第4期186-191,共6页
International Journal of Biologicals
关键词
8型肺炎球菌荚膜多糖
分子大小
CRM197结合物
抗原性
Capsular polysaccharide of Streptococcus pneumoniae type 8
Molecular size
CRM197 conjugate
Antigenicity
