循环自由流等电聚焦方法的建立及应用研究

等电聚焦是利用蛋白质分子或其它两性电解质分子的等电点不同,在一个连续、稳定、线性的pH梯度中进行蛋白质分析分离的技术。薄层凝胶等电聚焦具有高分辨率,但上样量小,聚焦后样品分析处理不便,仅局限于分析。循环自由流液相等电聚焦以液体循环流动代替固相载体,上样量大,聚焦后样品分析处理方便,有较大的制造潜力。Bier等已开展这项技术的研究。该技术应用到遗传工程上可对干扰素等进行精制纯化^3]。本实验应用循环自由流等电聚焦仪(Recycling free-flow isoelectric fo-cusing,RIEF)进行了pH梯度的建立、模型化合物的分离、相关参数的比较，并被步应用于生物活性蛋白的精制和活性回收，旨在为遗传工程产品下游工艺提供一种新型而行之有效的方法。

Recycling Free-flow Isoelectric Focusing and its Application

A new approach of preparative eiectrophoresis (Recycling free -flow Ison-electric focusing, RIFE) was established. Some parameters of this method (flow rate, loading, voltage and temperature) were compared. A mixture consisting of model compounds (Hb,Cyt. C and BSA) was separated. Crude RNase A was purified and its purity was improved to above 99%. Some harmful effects in RIEF,such as diffusion,convection and electroomosis, were discussed. High protein loading and high acativity recovery were achived.