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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。
Objective To optimize the purification of typhoid Vi polysaccharide column chromatography to replace the traditional phenol extraction, ethanol precipitation. Methods One DEAE ion exchange column was used to purify one batch of crude polysaccharide of typhoid Vi polysaccharide in the buffer (20 mmol / L Tris-Cl) at the pH values of 6.0, 7.0 and 8.0, respectively. Compared with traditional phenol extraction, Precipitation method for comparison. The protein content, nucleic acid content, O-acetyl content, molecular size and endotoxin content of the purified samples were determined according to the requirements of the third edition of Chinese Pharmacopoeia (2010 edition), and the polysaccharide recovery of the samples was calculated. Three batches of crude polysaccharide of typhoid Vi were purified by the optimized DEAE column chromatography purification process to verify the stability of the process. Results When the pH value of DEAE ion exchange column buffer (20 mmol / L Tris-Cl) was 7.0 and 8.0, the impurities of the purified product were effectively separated from the purified product of polysaccharide. The polysaccharide recoveries were 36.52% and 38.35%, respectively, which were significantly higher The polysaccharide recovery rate (15.52%) in the traditional process and the endotoxin content (10 EU / μg) of the purified product was significantly lower than that of the conventional process (200 EU / μg). Six batches of refined sugar from three batches of crude Vi polysaccharide were purified by the optimized process (pH 7.0), and the differences of protein content, nucleic acid content, O-acetyl content, molecular size, endotoxin content and polysaccharide recovery were small with standard deviations less than 5%. Conclusion The purification of typhoid Vi polysaccharide column chromatography is optimized. The stability of the process is good. It can replace the traditional phenol extraction and ethanol fractionation precipitation method and is applied to the purification of Vi polysaccharide in typhoid fever.