重组凋亡融合蛋白TRAIL-Fc在CHO中的表达、纯化及生物学活性研究

发布时间：2018-04-08 12:46

  本文选题：CHO细胞　切入点：TRAIL-Fc融合蛋白　出处：《重庆理工大学》2011年硕士论文

【摘要】：肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor related apoptosis inducing ligand,TRAIL)是由Wiley于1995年首次克隆并鉴定的一个能够诱导肿瘤细胞凋亡的TNF家族新成员。TRAIL对正常细胞无毒害作用,能够选择性杀伤肿瘤细胞,因此成为肿瘤治疗研究的热点,也有望成为一种新型的抗肿瘤药物。 目的: 本论文通过高密度培养CHO工程细胞株获得重组TRAIL-Fc融合蛋白,建立TRAIL-Fc纯化中试工艺并对其理化性质和生物学活性进行研究,为TRAIL-Fc最终应用于临床进行肿瘤治疗奠定实验基础。 方法: (1) CHO工程菌无血清驯化:通过逐步降低血清和直接降血清两种驯化方法获得可在无血清培养基中稳定生长的细胞株。为提高细胞株生长密度和活率对无血清培养基进行添加物优化。 (2) 5L反应器培养:采用分批补料式培养,研究温度、pH、溶氧控制及细胞生长代谢情况,探索CHO-TFc在5L反应器中的发酵工艺。TRAIL-Fc融合蛋白纯化工艺:利用Protein A亲和层析柱、CM离子交换层析柱纯化目的蛋白,并对目的蛋白进行理化性质研究。 (3) TRAIL-Fc融合蛋白生物学活性:采用MTT法检测TRAIL-Fc融合蛋白体外对多株肿瘤细胞的生长抑制作用。建立H22小鼠肝癌模型,检测TRAIL-Fc融合蛋白对体内肿瘤细胞生长抑制作用。 结果: (1)最终确定以逐步降血清法对CHO-TFc细胞株进行悬浮驯化,获得能在无血清培养基中稳定悬浮生长的细胞株。 (2)对无血清培养基进行优化,选择在无血清培养基中添加20ng/mlIGF-1。 (3) 5L反应器发酵工艺研究,以葡萄糖浓度及细胞密度为参数来确定补料策略。细胞以2.0×10~6cell/ml的密度接种,生长期葡萄糖浓度控制为2g/L,当细胞密度达到6-8×10~6cell/ml时,降温至32℃进行表达,控制残糖量为0.5g/L,最终发酵表达量为80-100mg/L。 (4)建立稳定的纯化工艺,纯度可达90%以上。质谱及Western blotting结果显示,TRAIL-Fc融合蛋白结构正确。体内外活性测定结果显示,TRAIL-Fc融合蛋白具有较强的肿瘤生长抑制的作用,且其在体内的半衰期明显延长。 结论: 成功完成CHO-TFc工程菌株悬浮驯化,建立5L发酵及纯化工艺,获得高纯度的TRAIL-Fc融合蛋白,并对制备的目的蛋白进行了体内外活性检测,证实了其潜在的肿瘤治疗价值。
[Abstract]:Tumor necrosis factor related apoptosis inducing trail, a novel member of the TNF family, which was first cloned and identified by Wiley in 1995, has no toxic effect on normal cells.It can selectively kill tumor cells, so it has become a hot spot in tumor therapy, and it is also expected to be a new anti-tumor drug.Objective:In this paper, the recombinant TRAIL-Fc fusion protein was obtained by high density culture of CHO engineering cell lines, and a pilot process of TRAIL-Fc purification was established, and its physicochemical properties and biological activities were studied, which laid the experimental foundation for the final application of TRAIL-Fc in clinical tumor therapy.Methods:(1) serum-free acclimation of CHO engineering bacteria: two acclimation methods: gradually reducing serum and direct lowering serum were used to obtain stable cell lines which could grow in serum-free medium.In order to improve cell growth density and viability, the serum-free medium was optimized.(2) 5L reactor culture: batch feeding culture was used to study temperature and pH, dissolved oxygen control and cell growth and metabolism.To explore the fermentation process of CHO-TFc in 5L reactor. The purification process of TRAIL-Fc fusion protein. The aim protein was purified by Protein A affinity chromatography column CM ion exchange chromatography, and the physicochemical properties of the target protein were studied.Biological activity of TRAIL-Fc fusion protein: MTT assay was used to detect the inhibitory effect of TRAIL-Fc fusion protein on the growth of many tumor cells in vitro.The H 22 mouse hepatoma model was established to detect the inhibitory effect of TRAIL-Fc fusion protein on the growth of tumor cells in vivo.Results:Finally, the suspension acclimation of CHO-TFc cell line was carried out by the method of decreasing serum gradually, and the cell line which could grow stably in serum-free medium was obtained.The serum-free medium was optimized by adding 20ng / ml IGF-1 to the serum-free medium.The fermentation technology of 5 L reactor was studied. The feeding strategy was determined by the parameters of glucose concentration and cell density.The cells were inoculated with 2.0 脳 10~6cell/ml density and glucose concentration was controlled to 2 g / L in the growing period. When the cell density reached 6-8 脳 10~6cell/ml, the cells were expressed at 32 鈩，

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