亲和微滤载体的制备及其在抗hCG抗体分离纯化中的应用

发布时间：2018-05-18 14:19

本文选题：亲和微滤 + 亲和膜　；参考：《浙江工业大学》2012年硕士论文

【摘要】：目前，抗体已被广泛应用于疾病的诊断、治疗以及靶向载体等方面，但是其价格昂贵，造成其昂贵的主要原因之一在于其纯化成本高，虽然目前有许多方法用于纯化抗体，但都存在着诸如分辨率不高、处理量不大或分离介质昂贵等缺陷，因此开发更廉价、更简便的纯化方法十分必要。本课题以抗HCG抗体为模型蛋白，选用廉价的小分子亲和配基（磺胺二甲基嘧啶），并结合膜分离处理量大的优点，进行抗体纯化新方法的研究，研究取得的结果如下：首先，以葡聚糖凝胶微球为载体，磺胺二甲基嘧啶为亲和配基，通过环氧接枝，制备了亲和微滤载体，并对制备条件进行了优化，所制得的载体配基密度为85.5μmol/g（湿），而且重复利用率高，耐酸碱性及机械强度良好；将其应用于亲和微滤分离抗体，最佳吸附pH值为6.0，吸附容量为8.1mg/g（湿），一步分离抗体纯度大于95%，回收率69.8%；将其用于柱层析，虽然在酸性条件下洗脱不如蛋白A柱，但在高盐条件下洗脱效果与蛋白A柱相当，活性保持良好。选用不同的膜基质，通过环氧接枝，制备了磺胺二甲基嘧啶亲和膜，并对制备条件进行了优化，结果表明，醋酸纤维素膜为膜基质的效果较好，最终配基密度为114.6μmol/g，将其应用于抗体分离，最佳吸附为pH6.0，，吸附容量为5.7mg/g，一步纯化抗体的纯度大于95%，回收率37.6%。
[Abstract]:At present, antibody has been widely used in the diagnosis, treatment and targeting vector of disease, but it is expensive, one of the main reasons for its expensive is the high cost of purification, although there are many methods to purify antibody, However, there are some disadvantages such as low resolution, low processing capacity or expensive separation medium. Therefore, it is necessary to develop cheaper and simpler purification methods. In this paper, we use anti-HCG antibody as model protein, select cheap small molecular affinity ligand (sulfadimethylpyrimidine), and combine the advantages of large membrane separation and treatment capacity to study the new method of antibody purification. The results are as follows: Firstly, using dextran gel microspheres as carrier and sulfadimethylpyrimidine as affinity ligand, the affinity microfiltration carrier was prepared by epoxy grafting, and the preparation conditions were optimized. The ligand density of the carrier was 85.5 渭 mol / g (wet), and its reutilization rate was high, acid and alkali resistance and mechanical strength were good, and it was used to isolate antibody by affinity microfiltration, and the ligand density of the carrier was 85.5 渭 mol / g. The optimum adsorption pH value was 6.0, the adsorption capacity was 8.1 mg / g (wet), the purity of one-step separation antibody was greater than 95, and the recovery was 69.8%. It was used in column chromatography, although it was better than protein A column in acid condition, but the elution effect was equivalent to that of protein A column under high salt condition. The activity remains good. The sulfadimethylpyrimidine affinity membrane was prepared by epoxy grafting with different membrane substrates. The preparation conditions were optimized. The results showed that cellulose acetate membrane was a good membrane matrix. The final ligand density was 114.6 渭 mol / g. The best adsorption was pH6.0, the adsorption capacity was 5.7 mg / g, the purity of one-step purified antibody was greater than 95%, and the recovery was 37.6%.
【学位授予单位】：浙江工业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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