盐析萃取分离纯化血浆蛋白和重组人胰岛素

发布时间：2018-01-20 09:45

本文关键词： 盐析萃取血浆蛋白重组人胰岛素离子交换层析疏水层析　出处：《大连理工大学》2015年硕士论文　论文类型：学位论文

【摘要】：IgG和白蛋白是血浆中主要的功能蛋白质,临床上用于抗病毒、治疗烧伤和失血过多引起的血流动力学疾病等。胰岛素是体内唯一一个降低血糖的激素,临床上多用来治疗糖尿病。当前,工业上蛋白质的分离纯化主要采用有机溶剂沉淀、多步离心等初分离的方法以及柱层析的方法进行分离纯化,这些方法存在着操作繁琐、周期长、蛋白的回收率低和易失活等问题,本论文开展了亲水性有机溶剂盐析萃取偶联柱层析分离纯化血浆中IgG和白蛋白的研究和盐析萃取分离纯化重组人胰岛素的研究。首先,在前人的结果基础上,验证了500g体系中17.5%乙醇/20% K2HPO4体系和14%乙醇/20% K2HPO4盐析萃取分离效果,发现14%乙醇和20% K2HPO4体系放大稳定,在pH7.0下,白蛋白和IgG分布在上相且得率分别为90%和65%。17.5%乙醇/20% K2HPO4体系则易使IgG沉淀。对14%乙醇/20% K2HPO4体系的萃取相,进行了疏水层析和离子交换层析条件的优化,在优化的条件下,可以得到电泳纯的IgG,但白蛋白的纯度较低。其次考察了血浆蛋白在叔丁醇/盐体系中的分配情况。结果表明,在15%叔丁醇/14% K2HPO4,pH 7.0和添加7.5% NaCl的条件下,血浆中的白蛋白主要分布在下相,收率为98%,而IgG以沉淀的方式分布在上下相界面中间,收率为77%,有利于两种蛋白的分离。将叔丁醇／K2HPO4体系的的萃取相和沉淀相,使用优化的疏水层析和离子交换层析进行纯化,可以得到电泳纯度为100%的IgG和纯度为87%的白蛋白,该方法操作简便、收率高,具有较好的应用前景。最后研究了重组人胰岛素在盐析萃取体系中的分配情况,并通过对盐析萃取体系的初筛及条件的优化,筛选出了最优的盐析萃取体系。在18%乙醇/22% K2HPO4, pH9.0条件下,重组人胰岛素分布在上相,回收率在98%以上。
[Abstract]:IgG and albumin are the main functional proteins in plasma, which are used clinically to treat hemodynamic diseases caused by burn and blood loss, etc. Insulin is the only hormone that lowers blood sugar. At present, the separation and purification of proteins in industry are mainly carried out by organic solvent precipitation, multi-step centrifugation and column chromatography. These methods have some problems, such as complicated operation, long period, low protein recovery and easy inactivation. In this paper, the separation and purification of IgG and albumin in plasma by hydrophilic organic solvent salt-out extraction coupled column chromatography and salt-out extraction and purification of recombinant human insulin were carried out. Based on the previous results, the extraction and separation effects of 17.5% ethanol / 20% K _ 2HPO _ 4 and 14% ethanol / 20% K _ 2HPO _ 4 were verified. It was found that the system of 14% ethanol and 20% K2HPO4 was stable and amplified under pH7.0. Albumin and IgG were distributed in the upper phase and the yields were 90% and 65.17.5% ethanol / 20% K2HPO4 system and IgG precipitated easily. For 14% ethanol / 20%. Extraction phase of K2HPO4 system. The conditions of hydrophobic chromatography and ion exchange chromatography were optimized. Under the optimized conditions, pure IgG could be obtained. However, the purity of albumin was low. Secondly, the distribution of plasma protein in tert-butanol / salt system was investigated. The results showed that 15% tert butanol / 14% K2HPO4 was obtained. Under the conditions of pH 7.0 and 7.5% NaCl, the albumin in plasma was mainly distributed in the lower phase, and the yield was 98. The IgG was distributed in the middle of the upper and lower phase interface by precipitation. The extraction and precipitation phases of tert-butanol / K2HPO4 system were purified by optimized hydrophobic chromatography and ion exchange chromatography. IgG with a purity of 100% and albumin with a purity of 87% can be obtained. The method is easy to operate and has a high yield. Finally, the distribution of recombinant human insulin in salting-out extraction system was studied, and the initial screening and conditions of salt-out extraction system were optimized. Under the conditions of 18% ethanol / 22% K2HPO4 and pH9.0, the recombinant human insulin was distributed in the upper phase and the recovery rate was over 98%.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6

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