论文部分内容阅读
目的纯化艰难梭菌(Clostridium difficile,CD)B毒素,并鉴定其生物学特性。方法将艰难梭菌VPI10463株经透析培养,50%饱和硫酸铵盐析,酸沉淀,收集上清液浓缩后,先采用分子筛Sephacryl S-300层析,收集目标蛋白峰,再进行DEAE Sepharose FF和DEAE Sephadex A-25离子交换层析,并对纯化产物进行各项生物学特性鉴定。结果最终纯化的艰难梭菌B毒素蛋白浓度为1.25 mg/ml;Vero细胞毒性为8.0×109 CU/mg;经非变性聚丙烯酰胺凝胶电泳分析呈单一条带,相对分子质量约为220 000,纯度为99.9%;免疫双扩散试验显示单一沉淀线。结论成功纯化了艰难梭菌B毒素,获得的B毒素纯度和细胞毒性较高,免疫反应性良好,为艰难梭菌毒素及其相关疾病的研究和防治奠定了基础。
Objective To purify Clostridium difficile (CD) B toxin and identify its biological characteristics. Methods The Clostridium difficile VPI10463 strain was dialyzed and cultured in 50% saturated ammonium sulfate for acid precipitation. The supernatant was collected and concentrated. Sephacryl S-300 chromatography was used to collect the target protein peaks. Then the protein was separated by DEAE Sepharose FF and DEAE Sephadex A-25 ion exchange chromatography, and the purification of the product for a variety of biological characteristics. Results The final purified C. difficile B toxin protein concentration was 1.25 mg / ml. The Vero cytotoxicity was 8.0 × 109 CU / mg. A single band was detected by non-denaturing polyacrylamide gel electrophoresis. The relative molecular mass was about 220 000 , Purity of 99.9%; immune double diffusion test showed a single precipitation line. Conclusion Clostridium difficile B toxin was successfully purified, and the purity and cytotoxicity of B toxin were high and the immunoreactivity was good. It laid the foundation for the research and prevention of Clostridium difficile toxin and its related diseases.