PTD-GFP、PTD-EDAG融合蛋白质的表达、纯化和初步应用
发布时间：2018-08-28 20:29
【摘要】： EDAG是一种与造血细胞分化调控密切相关的核蛋白质,对造血系统发育分化起重要调控作用,但对其作用机制尚缺乏深入的了解。因此,将EDAG蛋白质转导入造血干细胞内,研究其对造血干细胞增殖、分化、凋亡调控的影响,将对阐明其作用机制及应用前景具有重要意义。 为将EDAG蛋白质有效导入造血干细胞,我们采用了基于蛋白质转导结构域(protein transduction domain, PTD)的蛋白质跨膜递送技术。它能将与之融合的蛋白质以一种浓度依赖的方式跨膜导入细胞,转导效率高,每个细胞内的蛋白质浓度近乎相同,且对细胞损伤较小。目前,其在医学研究领域中的应用已受到广泛的关注。 为优化跨膜递送技术各环节的条件,我们选择绿色荧光蛋白质(GFP)为研究对象,系统摸索了表达、纯化、转导等条件。我们首先采用DNA重组技术构建了PTD-GFP表达载体,共构建VP22-GFP、Tat-GFP、Tat-GFP-Tat、Tat-GFP-9R 4种融合蛋白质原核表达载体,转染大肠杆菌BL21,经过摸索诱导剂浓度、菌密度、诱导时间等条件,得到各融合蛋白质的最佳诱导表达条件。在采用Ni-NTA亲和纯化柱纯化融合蛋白质时,我们尝试了两种纯化途径:一是天然蛋白质直接上柱纯化(Native条件)、二是先对表达蛋白质变性,然后纯化并复性(Hybrid条件)。比较两种纯化路线所得蛋白质的跨膜转导能力发现,Hybrid途径纯化的融合蛋白质具有较强的跨膜转导能力,其中Tat-GFP-Tat蛋白质具有最强的活性。 随后,我们分别构建了VP22-EDAG、Tat-EDAG、Tat-EDAG-Tat和Tat-EDAG-9R的原核表达载体,转染大肠杆菌BL21,经诱导均可表达目的蛋白质,除由于VP22-EDAG表达量很低,未获得纯化蛋白质外,其余得到相应的蛋白质,并得到Western-bolt的验证。 本研究系统摸索了PTD融合蛋白质表达、纯化、转导等条件,并构建了多种PTD-EDAG表达载体,经初步纯化获得Tat-EDAG-Tat融合蛋白质,为进一步研究奠定了基础。
[Abstract]:EDAG is a nuclear protein closely related to the regulation of hematopoietic cell differentiation. It plays an important role in the regulation of hematopoietic system development and differentiation, but its mechanism is still not well understood. The mechanism and application prospects are of great significance.
In order to effectively transfer EDAG protein into hematopoietic stem cells, we adopted a protein transduction domain (PTD) based protein transmembrane delivery technique, which can transfer the fusion protein into cells in a concentration-dependent manner, with high transduction efficiency and nearly phase protein concentration in each cell. In addition, it has little damage to cells. At present, its application in medical research has attracted wide attention.
In order to optimize the conditions of transmembrane delivery, green fluorescent protein (GFP) was selected as the research object, and the expression, purification and transduction conditions were explored systematically. When the fusion protein was purified by Ni-NTA affinity purification column, we tried two purification methods: first, the natural protein was purified directly on the column (Native condition), and second, the expressed protein was purified first. Comparing the transmembrane transduction ability of the two purification routes, it was found that the fusion protein purified by Hybrid pathway had strong transmembrane transduction ability, and the Tat-GFP-Tat protein had the strongest activity.
Subsequently, we constructed the prokaryotic expression vectors of VP22-EDAG, Tat-EDAG, Tat-EDAG-Tat and Tat-EDAG-9R respectively, and transfected E.
In this study, the expression, purification and transduction conditions of PTD fusion protein were explored systematically, and a variety of PTD-EDAG expression vectors were constructed. Tat-EDAG-Tat fusion protein was obtained by preliminary purification, which laid a foundation for further research.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R341
本文编号：2210529
[Abstract]:EDAG is a nuclear protein closely related to the regulation of hematopoietic cell differentiation. It plays an important role in the regulation of hematopoietic system development and differentiation, but its mechanism is still not well understood. The mechanism and application prospects are of great significance.
In order to effectively transfer EDAG protein into hematopoietic stem cells, we adopted a protein transduction domain (PTD) based protein transmembrane delivery technique, which can transfer the fusion protein into cells in a concentration-dependent manner, with high transduction efficiency and nearly phase protein concentration in each cell. In addition, it has little damage to cells. At present, its application in medical research has attracted wide attention.
In order to optimize the conditions of transmembrane delivery, green fluorescent protein (GFP) was selected as the research object, and the expression, purification and transduction conditions were explored systematically. When the fusion protein was purified by Ni-NTA affinity purification column, we tried two purification methods: first, the natural protein was purified directly on the column (Native condition), and second, the expressed protein was purified first. Comparing the transmembrane transduction ability of the two purification routes, it was found that the fusion protein purified by Hybrid pathway had strong transmembrane transduction ability, and the Tat-GFP-Tat protein had the strongest activity.
Subsequently, we constructed the prokaryotic expression vectors of VP22-EDAG, Tat-EDAG, Tat-EDAG-Tat and Tat-EDAG-9R respectively, and transfected E.
In this study, the expression, purification and transduction conditions of PTD fusion protein were explored systematically, and a variety of PTD-EDAG expression vectors were constructed. Tat-EDAG-Tat fusion protein was obtained by preliminary purification, which laid a foundation for further research.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R341
【参考文献】
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1 严世荣,丁爱玲,朱明磊,张红梅;HIV TAT——一种生物大分子的运载体[J];药物生物技术;2002年04期
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