【摘要】： EDAG是一種與造血細胞分化調(diào)控密切相關(guān)的核蛋白質(zhì),對造血系統(tǒng)發(fā)育分化起重要調(diào)控作用,但對其作用機制尚缺乏深入的了解。因此,將EDAG蛋白質(zhì)轉(zhuǎn)導(dǎo)入造血干細胞內(nèi),研究其對造血干細胞增殖、分化、凋亡調(diào)控的影響,將對闡明其作用機制及應(yīng)用前景具有重要意義。 為將EDAG蛋白質(zhì)有效導(dǎo)入造血干細胞,我們采用了基于蛋白質(zhì)轉(zhuǎn)導(dǎo)結(jié)構(gòu)域(protein transduction domain, PTD)的蛋白質(zhì)跨膜遞送技術(shù)。它能將與之融合的蛋白質(zhì)以一種濃度依賴的方式跨膜導(dǎo)入細胞,轉(zhuǎn)導(dǎo)效率高,每個細胞內(nèi)的蛋白質(zhì)濃度近乎相同,且對細胞損傷較小。目前,其在醫(yī)學(xué)研究領(lǐng)域中的應(yīng)用已受到廣泛的關(guān)注。 為優(yōu)化跨膜遞送技術(shù)各環(huán)節(jié)的條件,我們選擇綠色熒光蛋白質(zhì)(GFP)為研究對象,系統(tǒng)摸索了表達、純化、轉(zhuǎn)導(dǎo)等條件。我們首先采用DNA重組技術(shù)構(gòu)建了PTD-GFP表達載體,共構(gòu)建VP22-GFP、Tat-GFP、Tat-GFP-Tat、Tat-GFP-9R 4種融合蛋白質(zhì)原核表達載體,轉(zhuǎn)染大腸桿菌BL21,經(jīng)過摸索誘導(dǎo)劑濃度、菌密度、誘導(dǎo)時間等條件,得到各融合蛋白質(zhì)的最佳誘導(dǎo)表達條件。在采用Ni-NTA親和純化柱純化融合蛋白質(zhì)時,我們嘗試了兩種純化途徑:一是天然蛋白質(zhì)直接上柱純化(Native條件)、二是先對表達蛋白質(zhì)變性,然后純化并復(fù)性(Hybrid條件)。比較兩種純化路線所得蛋白質(zhì)的跨膜轉(zhuǎn)導(dǎo)能力發(fā)現(xiàn),Hybrid途徑純化的融合蛋白質(zhì)具有較強的跨膜轉(zhuǎn)導(dǎo)能力,其中Tat-GFP-Tat蛋白質(zhì)具有最強的活性。 隨后,我們分別構(gòu)建了VP22-EDAG、Tat-EDAG、Tat-EDAG-Tat和Tat-EDAG-9R的原核表達載體,轉(zhuǎn)染大腸桿菌BL21,經(jīng)誘導(dǎo)均可表達目的蛋白質(zhì),除由于VP22-EDAG表達量很低,未獲得純化蛋白質(zhì)外,其余得到相應(yīng)的蛋白質(zhì),并得到Western-bolt的驗證。 本研究系統(tǒng)摸索了PTD融合蛋白質(zhì)表達、純化、轉(zhuǎn)導(dǎo)等條件,并構(gòu)建了多種PTD-EDAG表達載體,經(jīng)初步純化獲得Tat-EDAG-Tat融合蛋白質(zhì),為進一步研究奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R341

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