Arimitsu Hideyuki, Sasaki Keiko, Kojima Hiroe, Yanaka Tadashi, Tsuji Takao
Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.
PLoS One. 2013 Dec 10;8(12):e83577. doi: 10.1371/journal.pone.0083577. eCollection 2013.
Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease.
在此，我们描述了一种针对志贺毒素2e（Stx2e）的简单亲和纯化方法，Stx2e是猪水肿病的主要致病因素。用表达质粒pBSK-Stx2e转化的大肠杆菌菌株MV1184在含有林可霉素的CAYE肉汤中培养时会产生Stx2e。Stx2e与商业D-半乳糖凝胶结合，该凝胶含有通过二乙烯砜连接子固定在琼脂糖树脂上的α-D-半乳糖，并用含有4.5 M MgCl2的磷酸盐缓冲盐水洗脱。少量Stx2e与另一种商业α-半乳糖固定化琼脂糖树脂结合，但不与β-半乳糖固定化树脂结合。此外，Stx2e与含有通过二乙烯砜固定在琼脂糖树脂上的β-巯基乙醇的嗜硫吸附树脂结合，并以与从D-半乳糖凝胶中纯化相同的方式进行纯化，但Stx2e样品含有一些污染物。这些结果表明，Stx2e主要通过树脂上的二乙烯砜基团与D-半乳糖凝胶结合，在较小程度上通过α-D-半乳糖结合。通过这些方法，从1 L培养物中获得的Stx2e和减毒突变型Stx2e（mStx2e）的产量分别约为36 mg和27.7 mg，D-半乳糖凝胶和嗜硫吸附树脂对Stx2e的结合能力至少为每1 ml树脂20 mg。此外，使用与原型Stx2不与嗜硫吸附树脂结合以及一些在B亚基中含有插入突变的突变型Stx2e和Stx2的嵌合毒素，我们发现，至少天冬酰胺（B亚基的第17位氨基酸）与Stx2e与二乙烯砜基团的结合有关。分离得到的mStx2e在ICR小鼠中表现出疫苗效果，表明这些方法有利于大规模制备Stx2e类毒素，从而保护猪免受水肿病的侵害。
