【作者】 闫巍；

【导师】 贾之慎； 朱岩；

【作者基本信息】 浙江大学 ， 化学， 2007， 硕士

【摘要】 亲和色谱是一种高效简便的蛋白纯化分离技术，高效亲和色谱（HPAC）是在经典亲和色谱技术上结合高效液相色谱（HPLC）的特点发展起来的新技术，它具有分离速度快、分离效果好、回收率高和操作简单等优点，适用于批量样品的分析和制备。8微米左右的表面多孔单分散交联聚甲基丙烯酸环氧丙酯／甲基丙烯酸乙二醇酯P（GMA-EDMA）微球可作为高效液相色谱柱填料中的基质材料。本文将P（GMA-EDMA）微球作为高效液相色谱的基质材料，三嗪染料和金属作为亲和配基，制备一种新型的复合配基亲和色谱填料并将其用于对蛋白质的分离纯化，主要做了以下研究工作：1．以分散聚合法制备的2微米聚苯乙烯微球为种子，GMA、EDMA为单体，用一步种子溶胀法制备了表面多孔的P（GMA-EDMA）微球。该微球7微米左右，粒径单分散，表面多孔，刚性好，适合用做高效色谱柱填料。2．以自制P（GMA-EDMA）微球为基球，通过不同的连接方式，将染料配基PBMX-R和金属Ni²⁺固定在基球上，制备成两种复合配基高效亲和色谱填料Ni²⁺-IDA-PB-P（GMA-EDMA）和Ni²⁺-PB-P（GMA-EDMA）。3．用匀浆法将所制的复合配基亲和色谱填料填装成高效亲和色谱柱，通过比较复合配基亲和色谱与单一亲和配基亲和色谱对蛋白质的保留行为和吸附情况，探究复合配基亲和色谱对蛋白质的保留机理，将含有染料配基和金属配基的Ni²⁺-IDA-PB-P（GMA-EDMA）、Ni²⁺-PB-P（GMA-EDMA）复合配基亲和色谱柱与含有单一配基的亲和色谱柱对蛋白质的吸附情况进行比较，选择最佳色谱柱。4．在10×0.46cm的Ni²⁺-IDA-PB-P（GMA-EDMA）色谱柱上，探讨了pH、流速和固定了不同金属离子的色谱柱对溶菌酶分离的影响，得出溶菌酶的最佳淋洗条件为：流动相A为0.02mol／L pH6.86磷酸盐缓冲液，B为含1.0mol／L NaCl的pH6.86磷酸盐缓冲液，淋洗梯度为15_minB从0到100％，进样量为20μL，流速为0.5_ml／min。在最佳淋洗条件下对不同浓度溶菌酶进行测定，该高效亲和色谱柱具有良好的线性、重现性、稳定性等色谱性能。实际应用中，它能将鸡蛋清样品中的溶菌酶成功分离，并从猪心中分离纯化出高浓度的细胞色素C。更多还原

【Abstract】 High-performance Affinity Chromatography（HPAC） is a method combined with classical affinity chromatography in an HPLC system. It has advantages of rapid analysis, high column efficiency and high recovery. Affinity chromatographic column based on porous particles of 8μm P（GMA-EDMA） is applicable for the analysis and micropreparative separation of proteins. In this paper, we prepared multiple-ligand affinity resin in which P（GMA-EDMA） microspheres were used as the support, and dye ligand and metal ligand were incorporated into these microbeads for the separation and purification of proteins. The main work follows:1. Porous particles, P（GMA-EDMA） micropheres with diameter of 7μm were prepared by a single-step swelling and polymerization method, using polystyrene micropheres prepared by dispersion polymation as seed particles and GMA、EDMA as monomers. The synthesized monodisperse P（GMA-EDMA） resin possesses uniform、porous characteristics and high mechanic intensity.2. Procion blue MX-R and Ni²⁺were coupled with P（GMA-EDMA） microspheres through different ways, thus two novel multiple-ligand affinity resins Ni²⁺-IDA-PB-P（GMA-EDMA）、Ni²⁺-PB-P （GMA-EDMA） were prepared.3. The multipe-ligand immobilized P（GMA-EDMA） beads were slurry packed into stainless steel column.The protein retention machanism on multiple-ligand affinity chromatography was explored by comparing protein retention behavior and adsorption ability on multiple-ligand affinity chromatography with on mono-ligand affinity chromatography. Ni²⁺-IDA-PB-P（GMA-EDMA） column was selected as the best column for protein separation and purification.Ni²⁺-IDA-PB-P（GMA-EDMA）column（10×0.46cm I.D.） were investigated in selectivity, stabilization, protein loading capacity and recovery et. al., using lysozyme as standard protein. On this column,lysozyme was well eluted under the selected experimental conditions. The eluent flow rate was set at 0.5 ml/min with injection volume of 20μL and the mobile phase linear gradient was set in 15 min from 0 to 1 mol/L sodium chloride in 20 mmol/L phosphate buffer at pH 6.86. The column has low column backpressure, good stability, high protein mass recovery, and was factual used for the separation of lysozyme from egg white and purification of cytochrome C from procine heart.更多还原