肠出血性大肠埃希菌O157：H7Ⅱ型志贺毒素的纯化及功能鉴定

Purification and functional characterization of enterohemorrhagic Escherichia coli O157：H7 Shiga toxin Ⅱ

目的亲和层析纯化肠出血性大肠埃希菌（EHEC）O157：H7Ⅱ型志贺毒素，并鉴定其生物学功能。方法用抗-Ⅱ型志贺毒素分子A亚单位的抗体S1D8耦联至柱填料Sepharose 4B，制备亲和层析柱。纯化EHEC O157：H7菌体分泌的毒素分子，分别用聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western印迹法鉴定毒素分子纯度和特异度，将纯化毒素倍比稀释，观察其对Vero细胞和小鼠的毒性作用，计算其对细胞半数致死量（CD50）和对小鼠的全数致死量（LD100）；观察抗毒素血清对小鼠进行毒素攻击的保护作用。结果通过亲和层析从EHEC O157：H7培养物中成功纯化Ⅱ型志贺毒素。SDS-PAGE显示，其A、B亚单位的相对分子质量分别为32000和7500，纯化的毒素分子可分别与Ⅱ型志贺毒素A、B亚单位特异性的单抗结合；对Vero细胞和小鼠均存在致死作用，其CD50和LD100分别为20ng／L和5ng，小鼠体内抗毒素血清对毒素可有效中和。结论成功纯化Ⅱ型志贺毒素，并证实其在细胞和动物模型中的毒性作用。

Objective To purify Shiga toxin Ⅱ（STX Ⅱ） of enterohaemorrhagic Escherichia coli （EHEC） O157：H7 by affinity chromatography, and characterize its biological function. Methods The immno-affinity chromatography column was prepared by STX 11 A subunit-specific antibody $1D8 coupling to Sepharose 4B matrix. The purity and specificity of STX Ⅱ molecule secreted by EHEC O157：H7 were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot, respectively. The purified toxin was serially diluted and the toxic activities to Vero cell line and mice were observed. The 50% cytotoxic dose （CD50） for Vero cell line and 100% lethal dose （LD100） for mice were calculated. The protection effect of anti-STX Ⅱ polysera to the mice against the purified toxin challenge was also observed. Results STX Ⅱ was successfully purified from culture supernatant of EHEC O157 ： H7 using affinity chromatography scheme. The relative molecular weights of STX Ⅱ A and B subunits were 32000 and 7500 confirmed by SDS-PAGE, respectively. The purified toxin could react with monoclonal antibodies against STX Ⅱ A and B subunits, respectively. The toxin was cytotoxic to Vero cell with CD50 of 20 ng/L and lethal to mice with LD100 of 5ng.The toxin could be neutralized by anti-STX Ⅱ polysera in vivo. Conclusion STX Ⅱ is successfully purified and its toxic effects are confirmed in both cell line and mouse model.