抗Cyclin D1人源胞内单链抗体的原核表达与鉴定

发布时间：2018-05-24 04:16

  本文选题：Cyclin + D1　； 参考：《吉林大学》2011年硕士论文

【摘要】：细胞周期是细胞生命活动的基本过程,细胞周期的紊乱将导致细胞异常增殖,从而引发肿瘤形成。周期蛋白Cyclins通过其表达量在细胞周期各时相中的变化调节细胞周期的进程,是调控细胞周期网络系统的关键分子。Cyclin Dl是细胞进入增殖周期合成的第一个周期蛋白,它能够特异性的结合CDK4形成Cyclin Dl/CDK4复合物,控制细胞周期进入S期。Cyclin Dl在多种人类恶性肿瘤细胞中呈过度表达,目前人们发现Cyclin Dl还与肿瘤的发生、肿瘤细胞的浸润和转移都密切相关,因此,开展以Cyclin Dl为靶点的肿瘤基因治疗具有巨大的实际意义和应用前景。 胞内抗体技术是近年来刚刚兴起的一种能特异性阻断细胞内重要靶蛋白的生物学技术,因其具有高效、低毒、作用范围广泛、病人不易产生抗药性等优点,该技术已经成为利用细胞内免疫机制来进行基因治疗的新手段,得到了广泛的应用。尤其是胞内单链抗体,因其克服了其他工程抗体分子结构复杂、分子量大、免疫原性强,半衰期短等缺点,已经成为了胞内抗体最主要的形式之一。近年来,人们已经将单链抗体导入多种细胞内实现了靶蛋白特异性灭活、恶性增殖细胞表型发生改变,为肿瘤的基因治疗提供了新的思路。 为此,本研究以p3.1-AD载体为模板,PCR扩增目的基因AD使其获得NotⅠ和BssHⅡ的酶切位点及His标签。将该片段正向插入到PDAN5载体的Not I和BssHⅡ区域。测序鉴定重组质粒,将重组质粒转化到大肠杆菌HB2151。IPTG,37℃诱导AD基因表达,Wstern-blot鉴定目的蛋白,HisTrap HP Kit柱纯化后SDS-PAGE分析纯化效果。本研究成功构建了pAD质粒；并诱导表达了胞内单链抗体AD,但是并没有能够成功的纯化出具有高纯度的AD蛋白,我们推测是由于破碎上清中存在过多的杂蛋白,干扰了目的AD蛋白的结合。综上所述,以Cyclin Dl为靶点的胞内抗体治疗作为一种新型肿瘤基因治疗方法,有潜在的应用价值,但要获得抗人细胞周期蛋白D1胞内单链抗体AD,还需进一步的摸索纯化条件。
[Abstract]:Cell cycle is the basic process of cell life activity. The disorder of cell cycle will lead to abnormal proliferation of cells, which will lead to tumor formation. Cyclin Cyclins regulates the process of cell cycle through the changes of the expression of cyclin Cyclins in different phases of cell cycle. It is the key molecule of cell cycle network system. Cyclin D1 is the first cyclin in cell cycle synthesis. It can specifically bind CDK4 to form Cyclin Dl/CDK4 complex, and control the cell cycle into S phase. Cyclin D1 is overexpressed in various human malignant tumor cells. At present, it has been found that Cyclin DL is also associated with tumor genesis. The invasion and metastasis of tumor cells are closely related. Therefore, the development of tumor gene therapy targeting Cyclin D1 has great practical significance and application prospect. Intracellular antibody technology is a new biological technique which can specifically block the important target proteins in cells in recent years, because of its high efficiency, low toxicity, wide range of action and so on, patients are not easy to develop drug resistance. This technique has become a new method for gene therapy using intracellular immune mechanism and has been widely used. In particular, intracellular single-chain antibodies have become one of the most important forms of intracellular antibodies because they overcome the disadvantages of complex molecular structure, high molecular weight, strong immunogenicity and short half-life of other engineering antibodies. In recent years, the introduction of scFv into a variety of cells has achieved target protein specific inactivation, and the phenotype of malignant proliferative cells has changed, which provides a new idea for gene therapy of tumor. In this study, the target gene AD was amplified by using p3.1-AD vector as template to obtain the restriction sites and His tags of Not 鈪，

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