一种连续蔗糖密度梯度离心分离哺乳动物细胞核糖体前体和核糖体的技术优化

摘要

Objective To purify pre-ribosome and ribosome of mammalian ceils using continuous sucrose density gradient ultracentrifugation.Methods Continuous sucrose density gradient was established by ultracentrifugation,and the continuous sucrose density gradient of 10％-30％ and 10％-45％ were used to extract the pre-ribosome and ribosome in mammalian cells,respectively.The mammalian cell lysis buffer was added to the established continuous sucrose density gradient.Pre-ribosome and ribosome with different sedimentation coefficients were collected and the A260 absorbance of each sample was measured.Proteins of each sample were extracted to detect the large subunit protein,RPL15 by Western Blot.Results Large subunit ribosomal protein RPL15 exists on 60S of the pre-ribosome,and also on 60S,80S and polyribosome of mature ribosome.Conclusions The continuous sucrose density gradient,which is established by the swing-out rotor,can be used to isolate the pre-ribosome and ribosome of mammalian cells rapidly.This method has the advantages of good separation effect and simple operation,which provides a good method for rapid and large amount preparation and separation of various kinds of ribosomes.%目的 利用连续蔗糖密度梯度的方法分离提取哺乳动物细胞的核糖体前体和核糖体.方法 采用超速离心法建立连续蔗糖密度梯度,分别用梯度为10％～30％和10％～45％的连续蔗糖密度梯度提取哺乳动物细胞内的核糖体前体和核糖体;然后将哺乳动物细胞裂解液加入到已建立好的连续蔗糖密度梯度上进行超速离心;再将不同沉降系数的核糖体前体和核糖体收集到不同的1.5 ml离心管中,测量每一个样品的吸光度值A260并提取其中的蛋白质,利用Western Blot检测核糖体大亚基蛋白RPL15的定位.结果 核糖体大亚基蛋白RPL15位于核糖体前体的60S上和成熟核糖体的60S、80S和多聚核糖体上.结论 利用水平转头建立的连续蔗糖密度梯度可快捷分离哺乳动物细胞内的核糖体前体和核糖体.该法分离效果好,操作简单,为快速和大量制备、分离多种核糖体提供了一种良好的方法.