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Clonagem, expressão, purificação e ensaio biológico da proteína GM-CSF: fator estimulador de colônias de granulócitos e macrófagos

机译:GM-CSF蛋白的克隆,表达,纯化和生物学分析:粒细胞和巨噬细胞集落的刺激因子

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摘要

The granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that belongs to a group of glycoprotein that regulates the proliferation and differentiation of hematopoietic cells, more specifically granulocytes and macrophages. The human GM-CSF is a 14. 4 kDa protein consisting of 127 amino acid polypeptide chain and shares 52% of similarities with murine protein. The human protein has 4 cysteine residues that forms two disulphide bonds; only the Cysteine 54 and 96 are required for the biologic activity of it. This biopharmaceutical has been widely used in neutropenic patients who receive high-dose chemotherapy or were transplanted. Therefore, GM-CSF is used to restore hematopoietic dysfunctions, to stimulate the hyper-production of functionally primed effectors cells and to augment host defense against infection and malignant diseases. Its use is associated with significant decreases in chemotherapy-associated infections, antibiotic use, length of hospital day and mortality. The international patent of the biopharmaceutical Molgramostim (generic name) expired in 2006, and thus became an interesting product to pharmaceutical industries, including those settled in Brazil. Presently, Molgramostim is sold in Brazil as an imported biopharmaceutical, which, in turn becomes very costly to the Brazilian government. Therefore, the aim of this work is to develop a methodology for subsequent production of a national Molgramostim. In this work the granulocyte-macrophage colony-stimulating factor gene was assembled by PCR. It was cloned into pET30a(+) expression vector and the best condition for expression of the protein was in the BL21(DE3) strain from Escherichia coli. To the isolation of inclusion bodies an efficient protocol was developed using multi step washing procedure and purification method of the recombinant protein from inclusion bodies using only a cationic and then an anionic exchange column. The immunoassay and N-terminal sequencing confirmed the identity of rhGM-CSF. The result of the biological activity assay, in vitro, showed that the rhGM-CSF produced has an equivalent biological potential to the standard reference. The protein rhGM-CSF was produced through simple, cost effective and economically feasible process and is extremely important to the industrial procedure and healthcare community.
机译:粒细胞巨噬细胞集落刺激因子(GM-CSF)是一种细胞因子,属于一组糖蛋白,可调节造血细胞,特别是粒细胞和巨噬细胞的增殖和分化。人GM-CSF是一种14个4 kDa的蛋白质,由127个氨基酸的多肽链组成,与鼠类蛋白质具有52%的相似性。人类蛋白质具有4个半胱氨酸残基,形成两个二硫键;半胱氨酸54和96仅需要其生物活性。该生物制药已广泛用于接受大剂量化疗或被移植的中性粒细胞减少症患者。因此,GM-CSF可用于恢复造血功能障碍,刺激功能性启动子效应细胞的过度产生并增强宿主抵抗感染和恶性疾病的防御能力。它的使用与化疗相关的感染,抗生素的使用,住院天数的长短和死亡率的显着降低有关。生物制药Molgramostim的国际专利(通用名称)于2006年到期,因此成为制药行业(包括定居在巴西的制药行业)的一种有趣产品。目前,Molgramostim在巴西以进口生物制药的形式出售,这反过来对巴西政府而言是非常昂贵的。因此,这项工作的目的是开发一种用于随后生产国家莫尔莫古斯丁的方法。在这项工作中,通过PCR组装了粒细胞-巨噬细胞集落刺激因子基因。将其克隆到pET30a(+)表达载体中,表达该蛋白的最佳条件是大肠杆菌的BL21(DE3)菌株。为了分离包涵体,使用多步洗涤程序和仅使用阳离子交换柱然后使用阴离子交换柱从包涵体纯化重组蛋白的方法开发了一种有效的方案。免疫测定和N端测序证实了rhGM-CSF的身份。体外生物活性测定的结果表明,所产生的rhGM-CSF具有与标准参照品相当的生物潜能。 rhGM-CSF蛋白是通过简单,经济高效且经济可行的方法生产的,对工业程序和医疗保健界极为重要。

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    Schwanke Raquel Cristina;

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  • 年度 2008
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  • 正文语种 Português
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