重组人白细胞介素-15的基因克

发布时间：2018-03-01 08:40

本文关键词： 人白介素15 构建表达纯化纯度活性　出处：《吉林大学》2006年硕士论文　论文类型：学位论文

【摘要】：重组人白介素-15具有广泛的应用前景,本论文研究了它的制备工艺。目前人们认为hIL-15至少可以有以下三个用途:作为疫苗的佐剂;治疗血小板减少;治疗实体瘤。研究制备工艺意味着要在提高产率的同时降低成本。这里主要从原核表达和蛋白纯化两个方面进行了工艺的改进。本论文的研究主要包括工程菌的构建,蛋白的表达和纯化。从外周血单核细胞提取总RNA,RT-PCR之后,获得了人白介素-15的基因序列。为了筛选获得一种高效表达的工程菌株,构建了四种类型的表达质粒,从而比较他们的表达效率。通过优化表达条件,提高了蛋白的表达量。然后通过三步纯化,经过包涵体的处理,离子交换层析,凝胶过滤层析,使蛋白的纯度达到要求,初步建立起了重组人白介素-15的纯化工艺。也考虑了不同复性条件和保存方法对蛋白活性的影响。通过本论文的实验,找到了一种可行的制备工艺。使蛋白的纯度可以达到95.3%,比活能达到1.1×107IU/mg。实验结果达到了预期的目标,为以后工艺放大打下了坚实的基础。
[Abstract]:Recombinant human interleukin-15 has a wide application prospect. In this paper, the preparation process of recombinant human interleukin-15 has been studied. At present, it is believed that hIL-15 can be used as adjuvant for vaccine, as adjuvant for treatment of thrombocytopenia; Treatment of solid tumor. The study of preparation technology means to increase the yield while reducing the cost. This paper mainly from the prokaryotic expression and protein purification two aspects of the improvement of the process. In this paper, the construction of engineering bacteria, the expression and purification of protein were studied. The gene sequence of human interleukin-15 was obtained after total RNA-RT-PCR was extracted from peripheral blood monocytes, and a highly expressed engineering strain was obtained. Four types of expression plasmids were constructed to compare their expression efficiency. By optimizing the expression conditions, the expression level of protein was improved. After three steps of purification, inclusion body treatment, ion exchange chromatography, gel filtration chromatography, The purification process of recombinant human interleukin-15 was preliminarily established, and the effects of different renaturation conditions and preservation methods on the protein activity were also considered. Through the experiments in this paper, a feasible preparation process was found. The purity of the protein can reach 95.3 and the specific activity can reach 1.1 脳 107 IU / mg. The experimental results have reached the expected goal and laid a solid foundation for the later process amplification.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392;Q78

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