雪旺细胞胞浆神经营养蛋白的分离纯化和理化性质测定

EXTRACTION AND PURIFICATION OF SCHWANN CELL CYTOPLASMIC NEUROTROPHIC PROTEINS AND DETERMINATION OF ITS PHYSIOCHEMICAL CHARACTOR

目的：分离纯化雪旺细胞胞浆神经营养蛋白并测定其等电点。方法：培养收集新生１－３天ＳＤ乳鼠四肢神经雪旺细胞，超声粉碎，超速离心，取上清胞浆成份超滤浓缩，收集分子量＞１０ＫＤａ浓缩蛋白液，通过ＤＥＡＥ－Ｓｅｐｈａｃｅｌ离子交换层析，ＳｅｐｈａｄｅｘＧ－１００凝胶过滤层析和Ｄｉｏｌ－１５０高压液相分子筛层析进行分离纯化，等电聚焦电泳测定纯化的活性蛋白质等电点和ＳＤＳ－聚丙烯酰胺凝胶电泳测定其亚基分子量。结果：成功地分离纯化出一种活性蛋白质，分子量约５８ＫＤａ，等电点４．５５，结论：联合应用超滤浓缩、离子交换层析。

Objective: Extraction and purification of Schwann cell cytoplasmic neurotrophic proteins and determination of its isoelectric piont Method: Schwann cells of SD newborn 1 3 day mice were cultured, collected, ultrasonicated, and ultraspeed centrifugalized, then the supernate (containing SC cytoplasmic components) was ultrafiltrated and concentrated The concentrated proteins with molecular weight (MW) >10 KDa were purified through ion exchange chromatography on DEAE Sephacel, gel filtration chromatography on Sephadex G 100 and HPLC on Diol 150 The isoelectric piont(pI) of a purified active protein was detected by isoelectric focusing electrophoresis (IEF), and its subunit molecular weight was determined by SDS PAGE Result: An active protein with MW 58 KDa and pI 4 55 was successfully purified Conclusion: SC cytoplasmic proteins could be purified satisfactorily by Combination of ultrafiltration, ion exchange chromatography, gel filtration chromatography and HPLC