艰难梭状杆菌重组肠毒素A和B与对应天然肠毒素蛋白的功能活性比较

目的：肠毒素A（TcdA）和肠毒素B（TcdB）是艰难芽孢梭状杆菌主要的毒力因子，我们对已经获得的纯化的重组蛋白TcdA和TcdB与对应的天然肠毒素蛋白的活性和功能进行了比较。方法通过光学显微镜观察重组肠毒素和对应的天然肠毒素对肠上皮细胞的细胞病变作用、蛋白印迹实验观察对Rac1蛋白的糖基化作用，以及激光共聚焦显微镜观察肠毒素对肠上皮细胞骨架的破坏作用。结果纯化得到重组蛋白肠毒素TcdA和rTcdB其分子量与天然的TcdA和TcdB蛋白大小接近，生物活性与天然的TcdB蛋白相似，TcdA和极微量TcdB具有使肠上皮细胞病变、Rac1蛋白的糖基、细胞骨架塌陷。结论艰难梭状芽孢杆菌肠毒素A和B重组蛋白具有良好的对应天然肠毒素相似的生物学功能活性。

Comparison of functions and bioactivity of recombinant clostridium difficile toxin A and B with their native counterparts

Objective Major Clostridium difficile virulence factors are the exotoxins TcdA and TcdB. We compared the func-tions and bioactivity of recombinant TcdA and TcdB with their counterparts. Methods Cytopathic effects of the recombinant toxins and their native counterparts on intestinal epithelium were observed on a reverse light microscopy ,and the effects of these toxins on glucosylation of Rac1 was analyzed by western blotting,with toxins disruption of cytoskeleton of intestinal epithelium by a con-focal microscopy. Results The recombinant toxins （rTcdA and rTcdB） had similar molecular masse to their native toxins ,and their biological activity were found to be similar to their native counterparts after an extensive examination. The recombinant toxins could lead to cytopathic effects,glucosylation of Rac1,and disruption of cytoskeleton of intestinal epithelium in consistent with their native counterparts in biological activities. Conclusion The recombinant rTcdA and TcdB are equivalent to their native counterparts in their biological activity.