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目的:研究1型肺炎球菌多糖的氨基、羧基和羟基的结合反应活性,制备1型肺炎球菌多糖-蛋白质结合物,并研究其抗原性和免疫原性。方法:将天然多糖降解,用EDAC直接缩合法验证多糖分子上氨基的反应活性。采用EDAC缩合法,利用降解多糖分子上的羧基与破伤风类毒素己二酸酰肼衍生物(TTAH)的氨基和肼基缩合,以CDAP活化降解多糖的羟基,并与破伤风类毒素(TT)或TTAH直接结合,纯化结合物并对结合物抗原性和免疫原性进行分析。结果:天然多糖重均相对分子质量由4.175×105g·mol-1降为8.917×104g·mol-1,其结构、特异基团含量和抗原性未改变;降解多糖与EDAC混合后,相对分子质量未增加;3种方法制备的结合物,其多糖抗原性未发生变化,免疫原性显著高于天然多糖(P<0.05),3针免疫后Ig G抗体水平均有剂次加强效应,呈T细胞依赖型免疫应答。不同方法制备的结合物,免疫原性无显著差异。结论:采用本研究的方法,可降解1型肺炎球菌多糖并保留天然多糖特性;多糖分子上的氨基无结合反应活性,羧基和羟基有结合反应活性;EDAC缩合法与CDAP活化法所制备结合物,赋予了多糖典型的T细胞依赖型抗原的特征,不同方法及制备的结合物免疫原性无显著差异。间隔剂在本研究中对结合物免疫原性无影响。
OBJECTIVE: To study the binding activity of amino group, carboxyl group and hydroxyl group of pneumococcal type 1 pneumococcal polysaccharide to prepare pneumococcal type 1 pneumococcal polysaccharide - protein conjugate and study its antigenicity and immunogenicity. Methods: Natural polysaccharides were degraded and the reaction of amino groups on polysaccharides was verified by EDAC direct condensation method. EDAC condensation method was used to degrade the hydroxyl groups of polysaccharides by CDAP activation by condensing the carboxyl groups on the degraded polysaccharides with the amino group and hydrazino group of the tetanus toxoid adipic acid hydrazide derivative (TTAH) ) Or TTAH, the conjugate is purified and the antigenicity and immunogenicity of the conjugate are analyzed. Results: The weight-average molecular weights of native polysaccharides decreased from 4.175 × 105g · mol-1 to 8.917 × 104g · mol-1, and the structure, specific groups and antigenicity of polysaccharides did not change. After the polysaccharides were mixed with EDAC, the relative molecular mass (P <0.05). The level of Ig G antibody after 3-needle immunization showed dose-boosting effect with T Cell-dependent immune response. Conjugates prepared by different methods showed no significant difference in immunogenicity. Conclusion: The method of this study can degrade the type 1 pneumococcal polysaccharide and retain the characteristics of natural polysaccharides; polysaccharide molecules on the amino non-binding reactivity, carboxyl and hydroxyl binding activity; EDAC condensation method and CDAP activation prepared conjugates , Which conferred the characteristic of typical T cell-dependent antigen of polysaccharide. There was no significant difference in the immunogenicity between different methods and the prepared conjugates. Spacers have no effect on the immunogenicity of the conjugates in this study.