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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 properties and immunogenicity of purified tetanus toxoid (TT or TTd) by two column chromatographic methods. Methods TT liquid was further purified by ammonium sulfate salinization and further purified by Superdex 200 and Sephacryl S-300 HR respectively. The chemical properties of purified TT and the immunogenicity of mouse were also tested. The results of purification by ammonium sulfate, TT purity of up to 85%, and then purified by column chromatography, the two methods (SDS-PAGE and HPLC) TT monomer content (purity) can reach more than 95%, salt After two steps of purification and column chromatography, the recovery rate of protein was over 45%. All the test indexes were in line with the requirements of “manufacturing and testing of tetanus vaccine of adsorption” in the three parts of Chinese Pharmacopoeia (2005 edition). Purification TT produced better immunogenicity in mice. Conclusion The purity of TT can be significantly increased by one or more steps of purification, and the TT dimer and multimer content can be reduced to a minimum. The purified TT prepared by two column chromatographic methods has better chemical properties and good immunity Original. This experiment provides a more safe and effective protein for the polysaccharide-protein conjugate vaccine and other vaccines that use TT as a combined vaccine component.