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目的建立一种14血清型肺炎链球菌荚膜多糖的酸沉淀法纯化工艺,以替代传统的苯酚抽提法。方法对建立的14型肺炎链球菌荚膜多糖酸沉淀法中的氯化钙终浓度(0、50、100、200、400 mmol/L)、p H值(3.5、4.0、4.5、5.0及5.7)及反应时间(0、1、2、4、6、8 h及过夜)进行优化。采用优化后的方法纯化3批14型肺炎粗制多糖,制备精制多糖,并进行理化指标、抗原性检测及核磁分析,同时与苯酚抽提纯化工艺进行比较。结果酸沉淀法的最佳工艺为:氯化钙终浓度50~100 mmol/L,p H值3.5~4.0,作用时间为过夜。采用该方法制备的3批14型肺炎精制多糖的蛋白和核酸杂质含量均较低,总磷、总氮、氨基己糖含量、多糖分子质量均符合企业注册标准,抗原活性和核磁共振图谱的分析结果表明其多糖结构无异常。结论酸沉淀法具有操作简便、效果显著及不需使用有毒有害试剂等优点,可用于纯化14型肺炎链球菌荚膜多糖。
Objective To establish an acid precipitation purification process for 14 serotypes of Streptococcus pneumoniae capsular polysaccharide to replace the traditional phenol extraction method. Methods The final concentration of calcium chloride (0,50,100,200,400 mmol / L), p H value (3.5, 4.0, 4.5, 5.0 and 5.7) in the established pneumococcal capsular polysaccharide 14 ) And reaction time (0, 1, 2, 4, 6, 8 h and overnight). The crude polysaccharides of 3 types of pneumonia pneumoniae were purified by the optimized method to prepare refined polysaccharides. The physical and chemical indexes, antigenicity and nuclear magnetic resonance (NMR) analysis were compared with that of phenol extraction and purification. Results The optimum conditions of acid precipitation were as follows: the final concentration of calcium chloride was 50-100 mmol / L, p H value was 3.5-4.0, and the action time was overnight. The content of protein and nucleic acid impurities of the three batches of 14 type pneumonia refined polysaccharides prepared by the method were low, and the content of total phosphorus, total nitrogen, hexose hexose and molecular weight of polysaccharide all met the standards of registered enterprises, antigen activity and nuclear magnetic resonance spectroscopy The results show that the polysaccharide structure is normal. Conclusion The acid precipitation method has the advantages of simple operation, significant effect and no need to use toxic and harmful reagents, which can be used to purify Streptococcus pneumoniae type 14 capsular polysaccharide.