破伤风毒素及其片段的纯化

本文报道了破伤风毒素及其片段AB、BC、A和C的纯化方法。应用硫酸铵沉淀和Ultrogel AcA_(34)凝胶过滤制备的纯化毒素,于SDS-PAGE和PAGE中均形成单一的蛋白带,分子量为15万,pI为5.95,毒力为1～3×10~4MLD/μg。毒素经DTT还原、尿素处理、再用Ultrogel AcA_(44)凝胶过滤制备A和BC片段。A片段的分子量为54000,pI为4.8。BC片段的分子量为98 000,pI为7.2。木瓜酶处理毒素后用高压液相层析制备AB和C片段。纯化的AB片段分子量为98 000,于等电聚焦中为2条主要区带,pI为5.3和5.5,另有数条小区带;纯化的C片段分子量为51000,于PAGE中形成5条蛋白带,于等电聚焦中主要区带的等电点为7.3,另有几条小区带。

Purification of Tetanus Toxin and Its Fragments

The purification methods for tetanus toxin and its fragments AB, BC, A and C were described. Tetanus toxin was purified by ammonium sulfate fractionation and gel filtration on an Ultrogel AcA ~(34)column. Purified toxin migrated as a single protein band in PAGE and SDS-PAGE with a toxicity of 1～3×10~4 MLD/μg and a molecular weight of 150000. The pI was 5.95. Fragments A and BC were isolated by gel filtration on a column of Ultrogel ACA_(44) from DTT reduced and urea treated toxin. Fragments A and BC showed single protein bands in PAGE and SDS-PAGE with molecular weights of 54000 and 98000 and pIs 4.8 and 7.2 respectively. Fragments AB and C were purified by HPLC from papain digested toxin. Fragments AB and C also showed single protein bands in SDS-PAGE with molecular weights of 98000 and 51000 respectively. Fragment AB was focused as two major bands with pIs of 5.3 and 5.5 and several faint bands. Fragment C showed 5 protein bands and several faint bands, and pI of the major band in isoelectricfocusing was 7.3.