载体蛋白质破伤风类毒素两种精制方法的研究  

Comparison of two methods for purification of tetanus toxoid as a carrier protein

作　　者：吴兵[1] 范锋锋[1] 刘方蕾[1] 于旭博 王欣茹[1] 罗树权[1] 方红春[1] 李燕婷[1] 陈作江[1] 谢贵林[1] 赵志强[1] 

机构地区：[1]兰州生物制品研究所有限责任公司甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《中国生物制品学杂志》2014年第12期1588-1594,共7页Chinese Journal of Biologicals

基　　金：国家科技支撑计划项目(2008BAI66B01);国家科技重大专项疫苗综合性技术研究开发大平台建设(2013ZX09402-302-215);国家科技部"重大新药创制"科技重大专项课题之国药集团技术创新产学研联盟子课题13价肺炎球菌结合疫苗临床前研究(2011ZX09401-403-012)

摘　　要：目的比较两种柱层析方法纯化破伤风类毒素(tetanus toxoid,TT或TTd)化学特性和免疫原性。方法以硫酸铵盐析一步纯化TT原液,再分别以Superdex 200和Sephacryl S-300 HR柱层析进一步纯化,并对纯化TT进行各项化学特性及小鼠免疫原性检测。结果经硫酸铵盐析纯化,TT纯度可达85%左右,再经柱层析纯化后,两种方法测定(SDS-PAGE和HPLC)TT单体含量(纯度)均可达95%以上,盐析和柱层析两步纯化后,蛋白质回收率均在45%以上,各项检定指标均符合《中国药典》三部(2005年版)中《吸附破伤风疫苗制造及检定规程》的要求;纯化TT在小鼠体内产生了较好的免疫原性。结论经一步或多步纯化可显著提高TT的纯度,并最大限度地减少TT二聚体和多聚体含量;采用两种柱层析方法制备的纯化TT具有更好的化学特性和良好的免疫原性。本实验为多糖-蛋白质结合疫苗及其他以TT为联合疫苗组分的疫苗提供了更加安全有效的蛋白质。Objective To compare the chemical character and immunogenicity of tetanus toxoids（TT）purified by two kinds of column chromatography. Methods Bulk of TT was purified by salting out with ammonium sulfate,and further purified by Superdex 200 and Sephacryl S-300 HR column chromatography respectively,then analyzed for chemical character,and determined for immunogenicity in mice. Results The purity of TT after salting out with ammonium sulfate reached about85%. However,after column chromatography,the TT monomer contents（purities）determined by SDS-PAGE and HPLC were more than 95%. Both the recovery rates of protein after salting out and column chromatography were more than 45%.All the quality indexes met the requirements for tetanus vaccine in Chinese Pharmacopoeia（Volume Ⅲ,2005 edition）. The purified TT showed good immunogenicity in mice. Conclusion One-step and several steps of chromatography increased the purity of TT significantly and minimized dimer and polymer contents. The purified TT prepared by two chromatographic methods showed good chemical character and good immunogenicity in mice. The study provided more safe and effective protein for polysaccharide-protein conjugate vaccine and other TT-containing combined vaccines.

关 键 词：破伤风类毒素 载体蛋白质 柱层析 特性 免疫原性 

分 类 号：R378.81[医药卫生—病原生物学] R392-33[医药卫生—基础医学]