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目的对C群脑膜炎球菌多糖(group C meningococcal polysaccharide,GCMP)蛋白结合物的5种制备方法进行比较。方法以GCMP作为多糖抗原,破伤风类毒素(tetanus toxoid,TT)和重组绿脓杆菌外毒素A(recombinant exoprotein A from Pseudomonas aeruginosa,r EPA)作为载体蛋白,采用5种结合路线制备7种结合物,并对其生化指标、免疫原性及磷酸铝佐剂的免疫增强效果进行检测。结果不同的结合方法均可将不同载体蛋白与多糖有效结合,在小鼠体内均具有良好的免疫原性。7种结合物的分子大小分布、游离多糖含量、多糖/蛋白值、回收率及免疫原性等指标有较大差异;采用相同结合方法,以TT和r EPA作为载体蛋白制备的结合物的生化指标及免疫原性无差异;磷酸铝佐剂可显著提高结合物的免疫原性。结论在选择GCMP蛋白结合物制备方法时应综合考虑原料多糖的化学结构、载体蛋白类型、结合路线、结合物纯化方法、质量控制方法和剂型等因素。本实验为GCMP蛋白结合物制备路线的选择提供了实验依据。
OBJECTIVE To compare five methods of preparation of the group C meningococcal polysaccharide (GCMP) protein conjugates. Methods GCMP as polysaccharide antigen, tetanus toxoid (TT) and recombinant exoprotein A from Pseudomonas aeruginosa (r EPA) were used as carrier proteins to prepare seven kinds of conjugates , And its biochemical indicators, immunogenicity and aluminum phosphate adjuvant immune enhancement test. Results The different binding methods could effectively bind different carrier proteins and polysaccharides and had good immunogenicity in mice. The molecular size distribution, free polysaccharide content, polysaccharide / protein value, recovery rate and immunogenicity of 7 kinds of conjugates were quite different. The bioconjugation of the conjugates prepared by the same binding method using TT and r EPA as carrier protein Indices and immunogenicity were not different; aluminum phosphate adjuvant can significantly improve the immunogenicity of conjugates. Conclusion The chemical structure, carrier protein type, route of combination, purification method of conjugate, quality control method and dosage form of polysaccharide should be considered in the selection of preparation method of GCMP conjugate. This experiment provides the experimental evidence for the selection of GCMP protein conjugate preparation route.