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题名	血浆抗凝血酶-III及其它微量蛋白的组合层析纯化
作者	金晓霞
学位类别	硕士
答辩日期	2004
授予单位	中国科学院过程工程研究所
授予地点	中国科学院过程工程研究所
导师	苏志国
关键词	血液制品 血浆微量蛋白 Cohn组分IV 低温乙醇沉淀 分离纯化 组合 层析
其他题名	Integrated Chromatographic Purification of Antithromibin-III and Other Trace Proteins from Plasma
学位专业	生物化工
中文摘要	从血浆中分离纯化各种药用蛋白质仍然是生物技术的重要产业，而高纯度微量血 浆蛋白制品的开发是血液制品发展的一个不容忽视的趋势，层析法以其特有的选择性 和灵活性在血浆蛋白的分离纯化中起着举足轻重的作用，它已成为获得某些较高纯度 微量血浆蛋白不可或缺的方法。本文首先研究了血浆中抗凝血酶-III（Alltithrombin-III，AT-III）的分离纯化，通 过正交实验优化了常温下肝素一琼脂糖亲和层析过程，并将其应用于低温4℃条件，显 著提高了从人血浆中提取戌plll的分离效果，为批量制备临床治疗用AFllJ浓缩剂提 供依据。另外，采用国产的发色底物测定AT-III的生物活性，探索了自制凝胶等电聚 焦电泳检测纯化的AT-III产品的方法，操作简便、价格低廉和准确性高。接下来重点对低温乙醇废弃组分IV中的微量蛋白的利用进行了研究。用聚乙二醇（Polvethylelle glycol，PEG）沉淀处理组分IV悬液，在优化的亲和层析条件下，从组 分W中成功提纯了AT-III，比较了二步PEG沉淀和一步PEG沉淀预处理对亲和层析 过程的影响，结果表明一步PEG沉淀预处理更利于工业放大。在分离纯化AT-III的基础上，本文进一步探索了对组分IV中其它微量蛋白的综合 利用。在肝素一琼脂糖亲和层析过程中，发现由低盐淋洗得到的淋洗液的主要成分为 a1-抗胰蛋白酶（AIpha-1 AntitryPsin，al-AT），仅采用一步凝胶过滤层析将其提纯， 并建立了测定其生物活性的微量发色底物法。另外，将亲和层析过程中未被吸附的组 分进行疏水层析和离子交换层析，获得了人血清白蛋白（Human Serum Albtlmin，HSA） 和运铁蛋白。其中，疏水层析采用了Octyl SePharose 4 FF，pH7.0，缓冲溶液中 （NH4）2SO4浓度为1.5mol／L，获取了组分IV中近乎100％的人血清白蛋白和34.2％的 电泳纯运铁蛋白。离子交换层析分离该未被吸附组分时，利用电泳滴定曲线确定了层 析条件：采用DEAE一SePharose FF阴离子交换剂，缓冲液的pH为7.5，初始盐浓度 为0，获取了组分IV中48.2％的电泳纯的运铁蛋白和27.3％的白蛋白。比较两者结果 表明，疏水层析更利于组分IV中白蛋白的提取，离子交换层析更利于运铁蛋白的纯化。最后，对于层析法和低温乙醇法相结合实现废弃组分W的综合利用，本文确定了以下工艺路线。
英文摘要	Fractionation purification of therapeutic proteins from human plasma, plays an important role in biopharmaceutical industry. Chromatography has been widely used in the last few years for plasma fractionation and become an indispensable tool for purification of trace plasma proteins because of its high specificity and selectivity. Heparin affinity chromatography was used in this study to isolate Antithrombin-III (AT-III) from human plasma. Orthogonal test was designed to investigate the influence of operation parameters in the affinity chromatography process. High activity recovery of AT-III was obtained through process optimization. In addition, AT-III biological activity was measured with a chromo genie substrate(S 131450) and the purity of AT-III was determined by isoelectric focusing (IEF) using the self-made gel, which was more convenient, cheap and promising. Separation of trace plasma proteins from Colin IV was investigated chiefly. According to the above method, two PEG precipitation steps was adopted to pretreat the Cohn IV suspension. AT-III was isolated successfully by optimized affinity chromatpgraphic condition. One step PEG precipitation was suggested in the scale up preparation. Furthermore, gel filtration chromatography (GFC), hydrophobic interaction chromatography(HIC) and ion-exchange chromatography (IEC) were investigated to isolate other trace proteins from the Cohn IV. al-AT, which was washed out by 0.65mol/L NaCl during the heparin affinity chromatography, was purified by GFC. And a micromethod was established to determine its biological activity. Octyl Sepharose 4 FF, with mild hydrophobic interaction condition (1.5mol/L (NH4)2SO4, O.lmol/L PBS, pH7.0) was screened out for HIC. About 100% albumin with purity of 80% and 34.2% of purified transferrin were obtained from pass through fraction of the affinity chromatography. Basing on the analysis of titration curve electrophoresis, anion exchange chromatography medium, DEAE—Sepharose FF was suggested for further use. Also 48.2% of purified transferrin was obtained by IEC at pH7.5. In conclusion, chromatography technology and traditional precipitation methods of plasma was integrated to establish a novel method to separate proteins from Cohn IV: (1) IV suspension was pretreated by one PEG precipitation step; (2) Extraction of AT-III by heparin affinity chromatography and more than 50% of purified AT-III was obtained; (3)cd-AT was further purified by GFC; (4)other proteins such as albumin, transferrin was separated from Cohn IV by HIC or IEC.
语种	中文
公开日期	2013-09-16
页码	98
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1429]
专题	过程工程研究所_研究所（批量导入）
推荐引用方式 GB/T 7714	金晓霞. 血浆抗凝血酶-III及其它微量蛋白的组合层析纯化[D]. 中国科学院过程工程研究所. 中国科学院过程工程研究所. 2004.

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