摘要
目的:探讨凝胶排阻层析纯化破伤风抗毒素的条件。方法:使用Sephadex G-100装柱,以紫外检测蛋白质特征峰A280nm绘制的散点图为评价指标,采用单因素试验考察不同流速、洗脱剂、样品浓度对破伤风抗毒素蛋白分离的影响;经非还原型SDS-PAGE凝胶电泳后对染色结果进行扫描对比;按照《中国药典》异常毒性检查法对纯化后的组分进行安全性检查。结果:凝胶排阻层析纯化破伤风抗毒素的最佳条件为以磷酸盐缓冲液(pH6.5)为洗脱液,样品浓度为100%,流速为0.3 mL/min;SDS-PAGE凝胶电泳结果表明在该条件下可以较好地纯化破伤风抗毒素有效成分F(ab')2;异常毒性检查结果为小鼠及豚鼠均正常存活,无异常毒性。结论:优化的凝胶排阻层析条件可以较好地纯化破伤风抗毒素有效成分,且安全无毒,为进一步推动该类生物制品的质量提高提供参考。
Objective:To investigate the conditions of purification of tetanus antitoxin by gel exclusion chromatography.Methods:The Sephadex G-100 column was used to detect the protein characteristic peak A280nm and the scatter plot was used to evaluate the evaluation index.The influence of different flow rates,eluent and sample concentration on the protein separation of tetanus antitoxin was investigated by single factor test.The staining results were scanned and compared by non-reduced SDSPAGE gel electrophoresis.The purified components were examined for safety according to the abnormal toxicity test in Chinese Pharmacopoeia.Results:The optimum conditions for the purification of tetanus antitoxin by gel isolation chromatography were phosphate buffer(pH6.5)as eluent,the sample concentration was 100%and the flow rate was 0.3 mL/min.SDS-PAGE gel electrophoresis results showed that the effective component F(ab')2 of tetanus antitoxin could be well purified under these conditions.The results of abnormal toxicity test showed that both mice and guinea pigs survived normally without abnormal toxicity.Conclusion:The optimized gel exclusion chromatography conditions can purify the effective components of tetanus antitoxin well,and it is safe and non-toxic,providing reference for further improving the quality of this kind of biological products.
作者
周年
王安华
洪燕
郑洋滨
刘宁
ZHOU Nian;WANG Anhua;HONG Yan;ZHENG Yangbin;LIU Ning(Jiangxi Institute for Drug Control,NMPA Key Laboratory of Quality Evaluation of Traditional Chinese Patent Medicine,Jiangxi Province Engineering Research Center of Drug and Medical Device Quality,Nanchang Jiangxi 330029,China;Nanchang Rail Transit Group Limited Corporation,Nanchang Jiangxi 330038,China.)
出处
《药品评价》
CAS
2020年第20期11-14,共4页
Drug Evaluation
基金
江西省食品药品监督管理局科研项目(2017YX13)。
