伤寒Vi多糖柱层析纯化工艺的优化

目的优化伤寒Vi多糖柱层析纯化工艺,以替代传统苯酚抽提、乙醇分级沉淀法。方法采用DEAE离子交换层析柱,分别在缓冲液(20 mmol/L Tris-Cl)pH值为6.0、7.0和8.0的条件下纯化1批伤寒Vi多糖粗糖,并与传统苯酚抽提、乙醇分级沉淀法进行比较。按《中国药典》三部(2010版)要求检测纯化样品的蛋白质含量、核酸含量、O-乙酰基含量、分子大小及内毒素含量,并计算样品的多糖回收率。采用优化的DEAE柱层析纯化工艺纯化3批伤寒Vi多糖粗糖,验证该工艺的稳定性。结果 DEAE离子交换柱缓冲液(20 mmol/L Tris-Cl)pH值为7.0和8.0时,可有效将伤寒Vi多糖的杂质与纯化产物分离,多糖回收率分别为36.52%和38.35%,明显高于传统工艺的多糖回收率(15.52%),且纯化产物的内毒素含量(10 EU/μg)明显低于传统工艺(200 EU/μg)。以优化工艺(pH 7.0)纯化3批伤寒Vi多糖粗糖获得的6批精糖的蛋白质含量、核酸含量、O-乙酰基含量、分子大小、内毒素含量及多糖回收率差异较小,标准偏差均小于5%。结论优化了伤寒Vi多糖柱层析的纯化工艺,该工艺稳定性良好,可替代传统苯酚抽提、乙醇分级沉淀法,应用于伤寒Vi多糖的纯化。

Optimization of procedure for purification of typhoid Vi polysaccharide by column chromatography

Objective To develop a column chromatographic method for purification of typhoid Vi polysaccharide instead of traditional phenol extraction combined with ethanol fractionation.Methods One batch of crude typhoid Vi polysaccharide was purified by DEAE ion exchange chromatography using 20 mmol / L Tris-Cl as buffer at pH 6.0,7.0 and 8.0 respectively,serving traditional phenol extraction combined with ethanol fractionation as control.The purified Vi polysaccharide was determined for protein,nucleic acid,O-acetyl contents as well as molecular size and endotoxin content,based on which the recovery rate of samples was calculated.The optimized procedure was verified for stability by purification of three batches of crude typhoid Vi polysaccharide.Results Foreign matters were removed effectively from the purified Vi polysaccharide by using 20 mmol / L Tris-Cl as buffer at pH 7.0 or 8.0,while the recovery rates of polysaccharide were 36.52% and 38.35% respectively,which were significantly higher than those by traditional method(15.52%).However,the endotoxin content(10 EU / μg) was significantly lower than that by traditional method(200 EU / μg).Six batches of purified Vi polysaccharide were obtained by the optimized procedure(pH 7.0),of which the standard deviations of protein,nucleic acid,O-acetyl contents,molecular size,endotoxin content and polysaccharide recovery rate were less than 5%.Conclusion A procedure of column chromatography was optimized,which showed high stability and might be used for purification of typhoid Vi polysaccharide instead of traditional phenol extraction combined with ethanol fractionation