层析法提纯A、C、Y、W135群脑膜炎球菌荚膜多糖工艺的建立

摘要

Objective To develop the chromatographic methods for purifying capsular polysaccharides from Neisseria meningitidis serogroups A,C,Y,W135.Methods Group A capsular polysaccharide was purified by ceramic hydroxyapatite and DEAE sepharose FF chromatographic column in PBS system.Group C capsular polysaccharide was pre-treated with sodium deoxycholate (SDC),then purified by ceramic hydroxyapatite column in PBS system.Groups Y and W135 capsular polysaccharides were dissolved in equilibration buffer system containing SDC,then purified by Capto Adhere and Capto DEAE columns in series.The purified polysaccharides were desalted with Sephadex G-25 Medium column,freeze-dried,and subjected to control tests according to Chinese pharmacopoeia (2015 edition,volume Ⅲ).Results After purification,the protein contents in groups A,C,Y,W135 polysaccharides decreased to 3.7,4.2,5.4 and 5.3 mg/g,respectively,and the nucleic acid contents decreased to 1.2,3.0,1.1 and 0.8 mg/g,respectively.All the above contents and phosphorus,sialic acid,and O-acetyl group contents met the pharmacopoeia requirements.Conclusion The chromatographic methods for purifying capsular polysaccharides from Neisseria meningitidis groups A,C,Y and W135 are well developed.%目的 建立A、C、Y、W135群脑膜炎球菌荚膜多糖层析纯化工艺.方法 采用陶瓷羟基磷灰石和DEAE Sepharose FF层析柱,在PBS体系中纯化A群多糖.以0.5％脱氧胆酸钠预处理C群多糖,用陶瓷羟基磷灰石层析柱在PBS体系中纯化C群多糖.以含有脱氧胆酸钠的平衡缓冲液溶解Y和W135群多糖,再用Capto Adhere和Capto DEAE层析柱串联纯化.纯化的多糖经Sephadex G-25Medium层析柱脱盐后冻干,按中国药典2015年版三部的要求进行检定.结果 经过层析纯化,A、C、Y、W135群多糖的蛋白含量分别降至3.7、4.2、5.4和5.3 mg/g,核酸含量分别降至1.2、3.0、1.1和0.8 mg/g,均符合药典要求.此外,磷、唾液酸和O-乙酰基含量等指标亦均符合药典标准.结论 建立了A、C、Y、W135群脑膜炎球菌荚膜多糖的层析纯化工艺.