两种人骨形态发生蛋白的分离和高度纯化

Isolation and Highly Purification of Two Kinds of Natural hBMPs

以新鲜人截肢肢体长管骨的皮质骨为材料,首先提取初步纯化的hBMP,经用40%、30%饱和度硫酸铵在4℃下进行盐析,获得含23.3KD、17.15KD和11.8KD 3种分子量蛋白的盐析产物,经制备性电泳洗脱,将3种分子量的蛋白分开,对3种蛋白进行活性检验发现,分子量为11.8KD和23.3KD的蛋白具有hBMP活性,且不与十二烷基磺酸钠(SDS)复合沉淀。但17.15KD蛋白无hBMP活性,并可与SDS复合沉淀。用二巯苏糖醇(DTT)进行还原不会改变11.8KD和23.3KD hBMP的电泳特性。初步认为,天然hBMP分子具有多样性,盐析结合电泳洗脱是获取纯化hBMP的较好方法。

To find out whether natural hBMP was manifold and to inquire into an simple and convenient way of purifying hBMP,so that resistant nonunions in sports trauma could be supplied with hBMP,fresh human cortical bones from diaphyses of amputated quadruples were used to isolate partially purified hBMP.Sediments of 17.15KD,11.8KD,and 23.3KD proteins were got by salting out sith(NH_4)SO_4 of 30%and 40%saturation at 4℃.Electrophoresis was used to separate them from each other and then elute each of them from the gel separately.Their bioassay showed that 11.8KD and 23.3KD proteins appeared hBMP activity, but 17.15KD protein did not.In addition,17.15KD protein could coprecipitated with SDS,but 11.8KD and 23.3KD hBMP could not.The electrophoresis characteristics of 11. 8KD and 23.3KD hBMPs were not changed with or without DTT reduction.It was believed that natural hBMP molecules were manifold,and salting-out combined with electrophoresis elution was a simple and convenient way of preparing highly purified hBMP.