hTFPI重组蛋白表达条件的优化

发布时间：2018-01-17 21:06

本文关键词：hTFPI重组蛋白表达条件的优化　出处：《中国协和医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 在多种疾病进程中，包括癌症、心脑血管疾病和炎症等，由组织因子(tissue factor，TF)引发的外源性凝血系统的激活扮演重要角色，一些患者因此致死。组织因子途径抑制因子(tissue factor pathway inhibitor，TFPI)是抑制外源凝血的一种重要蛋白因子。大量临床实验和动物实验证明，重组人组织因子途径抑制因子(human recombinant tissue factor pathway inhibitor，rhTFPI)对血栓形成、感染性休克、血管再狭窄、内毒素血症和DIC等具有明显防治作用，在临床上有广泛的应用前景。目前，，用多种表达系统表达rhTFPI的效果很不理想，存在的主要问题是表达量较低，以哺乳动物细胞表达系统的表达量最高(约2-5mg／L)，但成本也最高。我们应用巴斯德毕赤酵母表达系统进行了rhTFPI的表达。通过优化表达条件，使摇瓶培养下的rhTFPI的表达量达到了1.6mg／L；应用发酵罐进行高密度培养后，表达量比摇瓶培养方式提高了近2倍，达到了4.6mg／L。随后，我们建立了相应的纯化方法，应用了硫酸铵分级沉淀，阴离子交换层析和凝胶过滤层析，纯化收率为43.1±0.38％，蛋白纯度大于95％。实验结果显示，利用巴斯德毕赤酵母表达rhTFPI与其他表达系统相比具有以下优势：与哺乳动物细胞表达系统相比具有成本优势，无论是细胞培养成本和还是蛋白纯化成本，前者都比后者明显低廉；与大肠杆菌表达体系相比，具有表达量高，纯化简单，且重组蛋白翻译后修饰更接近于天然的TFPI。
[Abstract]:In many disease processes, including cancer, cardio-cerebrovascular disease and inflammation, the activation of exogenous coagulation system induced by tissue factor-TFF plays an important role. Some patients died as a result. Tissue factor pathway inhibitor. Tissue factor pathway inhibitor. TFPI is an important protein factor to inhibit exogenous coagulation, which has been proved by a large number of clinical and animal experiments. Recombinant human recombinant tissue factor pathway inhibitor. RhTFPI has significant preventive and therapeutic effects on thrombosis, septic shock, vascular restenosis, endotoxemia and DIC. At present, the expression of rhTFPI by multiple expression systems is not satisfactory. The main problem is that the expression of rhTFPI is relatively low, and the expression of mammalian cells is the highest (about 2-5 mg / L). But the cost is also the highest. We used Pasteurus pastoris expression system for the expression of rhTFPI. Through the optimization of expression conditions. The expression of rhTFPI reached 1.6 mg / L in shaking flask culture. After high density culture in fermenter, the expression level was nearly 2 times higher than that in shaking flask culture, reaching 4.6 mg / L. Subsequently, we established the corresponding purification method and applied ammonium sulfate fractionation. The yield of anion exchange chromatography and gel filtration chromatography was 43.1 卤0.38 and the purity of protein was higher than 95%. Using Pichia pastoris to express rhTFPI has the following advantages over other expression systems: it has a cost advantage over mammalian cell expression system. Both the cost of cell culture and the cost of protein purification were significantly lower in the former than in the latter. Compared with the Escherichia coli expression system, the recombinant protein has the advantages of high expression, simple purification, and post-translational modification of the recombinant protein is more similar to the natural TFPI.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R341

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