三步法纯化雪旺细胞源神经营养蛋白

The purification of Schwann cell derived neurotrophic proteins by three steps method

目的探索三步法纯化及获得雪旺细胞源神经营养蛋白的方法，为获得纯化蛋白进行单克隆抗体研究奠定基础。方法收集预损伤大鼠坐骨神经２００条，用酶消化结合差速粘附法纯化并收集其中雪旺细胞，将细胞超声粉碎、离心，收集上清液超滤，再经ｓｅｐｈａｄｅｘＧ１００层析，得到两个主峰，分段收集，将其中活性部分过高效液相色谱柱。结果得到三个主峰，相对分子质量分别为６８×１０３、５４×１０３和３０×１０３，用ＭＴＴ法测定生物活性，５４×１０３蛋白生物活性最大，经ＳＤＳＰＡＧＥ电泳证实基本为单一蛋白带。结论应用三步法同时各步采用合适方案是纯化雪旺细胞源神经营养蛋白的较好方法。

Objective To study the methods of purification and collection of Schwann cell derived neurotrophic proteins and set the foundation for the McAb production of the purified protein. Methods Schwann cells (SCs) were purified and collected by the methods of enzyme digesting and speed difference adhesion from 200 sciatic nerves of adult DS rats after two weeks′injury. The SCs were ultrasonicated and centrifuged and the supernate collected. Before passing through the Sephadex G 100 gel filtrated, the supernate were ultrafiltrated. Two peaks were obtained and separated to eight parts. After neurotrophic activity was assayed, the best active part was collected and passed through high pressure liquid chromatography (HPLC) molecular column. Result Three protein peaks were obtained with the molecular weight being 68 kD, 54 kD, and 30 kD respectively. The neurotrophic activity, assayed by MTT, of 54 kD protein was the highest, and analyzed on SDS PAGE, it was one band of protein. Conclusion Three steps method with appropriate schemes is a good way to purif SC derived neurotrophic proteins.