抗cyclin D1人源单链抗体的原核表达、纯化及活性研究

发布时间：2018-06-03 05:04

本文选题：噬菌体抗体 + cyclin　；参考：《吉林大学》2006年硕士论文

【摘要】： 细胞周期调节紊乱是导致肿瘤发生的重要原因,细胞周期蛋白D1(cyclin D1)作为细胞进入增殖周期后的第一个周期蛋白,在多种肿瘤细胞中存在过度表达,与肿瘤的发生,发展和预后都有着极为密切的关系,因此cyclin D1蛋白被认为是肿瘤治疗中一个重要靶点。单链抗体(single chain variable fragment, scFv)是具有抗原结合活性的最小的抗体片段,具有分子小,易于穿入组织细胞,免疫原性小,能通过基因工程技术大量生产等特点,具有较好的应用前景,尤其是在肿瘤诊断和治疗中具有独特的优势。本研究采用ELISA、Westernblot、Dot-blot、竞争性抑制、亲和力测定、免疫荧光染色等实验方法,对以重组cyclin D1蛋白为抗原,从人源噬菌体抗体库中筛选出来的抗cyclin D1噬菌体单链抗体进行了可溶性表达、纯化及活性分析。发现筛选到的噬菌体单链抗体能够特异性结合重组cyclin D1蛋白并且具有较强的亲和力,同时,噬菌体单链抗体还能够结合细胞内源性的cyclin D1蛋白。本实验获得的结果对后续开展抗cyclin D1胞内抗体肿瘤治疗的研究奠定了基础。
[Abstract]:The disorder of cell cycle regulation is an important cause of tumorigenesis. As the first cyclin of cell into the proliferative cycle, cyclin D1(cyclin D1 is overexpressed in many kinds of tumor cells, and it is associated with tumorigenesis. Development and prognosis are closely related, so cyclin D1 protein is considered as an important target in tumor therapy. Single chain variable fragment, scFv) is the smallest antibody fragment with antigen-binding activity. It has the advantages of small molecule, easy penetration into tissue cells, low immunogenicity, and can be produced in large quantities by genetic engineering. Especially in the diagnosis and treatment of cancer has a unique advantage. In this study, the recombinant cyclin D1 protein was used as antigen to express the single chain antibody against cyclin D1 phage selected from human phage antibody library by the methods of competitive inhibition, affinity assay and immunofluorescence staining. Purification and activity analysis. It was found that the screened phage scFv could specifically bind to recombinant cyclin D1 protein and had strong affinity. At the same time, phage scFv could bind to intracellular cyclin D1 protein. The results of this study laid a foundation for the further study of anti-cyclin D 1 anti-tumor therapy.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392;Q78

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