离子交换电色谱纯化蛋白质的研究-生物化工专业论文.docxVIP

摘要本文将离子交换色谱和横向交变电场耦合，研究了此种色谱柱应用于卵清蛋 摘要 本文将离子交换色谱和横向交变电场耦合，研究了此种色谱柱应用于卵清蛋 白这个实际物系的分离性能，并研究了蛋白质在该电色谱中的保留行为。 首先，利用填充Q SepharoseFastFlow的横向电场色谱柱实现了卵清蛋白这 一实际物系的分离，得到了卵铁传递蛋白和卵清白蛋白产品。前端分析研究表明， 在30mA，1／20Hz的交变电场下，2mL的色谱柱上进行卵清蛋白的上样吸附，对 于相同的穿透浓度，处理量能达到无电场时的2．33倍。用离子强度阶越洗脱实 现不同蛋白的分离得到，加电场时进样7mL时，卵铁传递蛋白的纯度为73．74％， 浓缩率5．72，收率82．96％；卵清白蛋白的纯度为90．14％，浓缩率1．67，收率 91．13％。进样量达到穿透点的21mL时，得到的结果与进样7mL相比，浓缩率 相当，收率略低。与无电场情况下进样量分别为3mL和9mL的分离效果相比， 分离所得样品的浓缩率稍有降低，收率基本一致。当将色谱柱轴向放大三倍至 6mL时，加电场与不加电场仍以7：3的比例进样，仍然能够得到相近并较高的 浓缩率和收率。 在考察横向电色谱柱内蛋白质保留行为的研究中，以pH4．0的条件确保BSA 不会被DEAE Sepharose Fast F]ow介质吸附，通过实验和数据分析得到了柱效、 保留体积、轴向扩散系数等参数。结果显示，由于离子交换介质表面和孔内产生 电渗流的作用，理论板高度随电流强度的增大而减小，文章对电流交变周期对柱 效的影响也做了考察。轴向扩散系数与柱效的变化相关联，它的变化趋势说明横 向电色谱柱内溶质的流型可以趋于活塞流。 关键词：电色谱；电渗流；卵清蛋白；柱效：轴向扩散系数 AB AB STRACT In this article．all anion—exchange chromatography combined wim an oscillatory transverse electric field was used to study the separation characteristic of hen egg white proteins．And retention behavior of protein in the electrochromatography was studied． First，a separation of egg white proteins was carried out using Q Sepharose Fast Flow on this electrochromatographic column and ovotransferrin and ovalbumin were gained．The frontal analysis study shows that，when useing a 2mL volume electrochromatographic column and with an oscillatory current density of 30mA， 1／20Hz，the output was 2．33 times as that without all current density at the sanle extent of proteins breakthrough．After adsorption,the egg white proteins were eluted by stepwise of ionic strength．When 7mL feedstock Was loaded，the separation resulted in one peak of ovotransferrin wit}l a purity of 73．74％and one peak of ovalbumin with a purity of 90．14％．The concentration factors of ovotransferrin and ovalbumin were 5．72 and 1．67，and recoveries 82．96％and 90．14％，respectively． Loading to the breakthrough point，21mL，we got the almost the SalTle concentration factors and a slight lower yield as that 7mL loaded．When compared、Ⅳith that loading 3mL and 9mL respectively without c