抗CDK4人源单链抗体的原核表达、纯化及活性分析

发布时间：2018-05-03 08:28

本文选题：CDK4 + 单链抗体　；参考：《吉林大学》2009年硕士论文

【摘要】： 细胞周期调节失控是造成肿瘤细胞恶性增殖的主要原因之一。周期蛋白依赖蛋白激酶(cyclin-dependent kinase, CDK)的时相性激活是细胞周期调控机制的核心。CDK4作为细胞进入增殖周期第一个被激活的周期蛋白依赖蛋白激酶,是细胞周期进程中G1/S期转换的限速因子。研究发现在许多肿瘤细胞和组织中都存在着CDK4的过度表达和过度活化,与肿瘤的发生和发展有着密切的关系。作为研究和治疗肿瘤的重要靶点,如何下调过度表达和活化的CDK4已经成为研究热点。单链抗体(single chain antibody, scFv)以其分子量小,免疫原性低等优点,在肿瘤诊断和治疗中具有独特的优势,目前已经有多种基于单链抗体的抗肿瘤药物进入临床试验。本研究构建了pET28a-CDK4表达载体,获得有活性的重组人CDK4蛋白后,以其为抗原,从噬菌体库中筛选得到抗CDK4人源单链抗体基因,将其转化进原核细胞中进行了诱导、表达和纯化,并通过ELISA、Western blot、竞争性抑制、亲和力测定、免疫荧光和免疫沉淀等实验方法对单链抗体进行了活性表征。结果表明纯化的单链抗体不仅能与重组人CDK4蛋白特异性结合并具有较高亲和力,而且也能与细胞内源性的CDK4蛋白特异性结合。本实验得到的结果对后续开展抗CDK4胞内抗体肿瘤基因治疗的研究奠定了基础。
[Abstract]:Uncontrolled cell cycle regulation is one of the main causes of malignant proliferation of tumor cells. The chronological activation of cyclin dependent kinases (CDKs) is the core of cell cycle regulation. CDK4 acts as the first cyclin dependent kinase to be activated in cell cycle. It is the limiting factor of G 1 / S phase transition in cell cycle process. It has been found that overexpression and over-activation of CDK4 exist in many tumor cells and tissues, which is closely related to the occurrence and development of tumor. As an important target of tumor research and treatment, how to down-regulate overexpression and activation of CDK4 has become a hot topic. Single chain antibody, scFv) has unique advantages in tumor diagnosis and treatment due to its small molecular weight and low immunogenicity. At present, a variety of anti-tumor drugs based on scFv have been introduced into clinical trials. In this study, the pET28a-CDK4 expression vector was constructed. After the recombinant human CDK4 protein was obtained, the human single-chain antibody gene against CDK4 was screened from the phage library and transformed into prokaryotic cells for induction, expression and purification. The activity of scFv was characterized by Western blot, competitive inhibition, affinity assay, immunofluorescence and immunoprecipitation. The results showed that the purified scFv could not only bind to the recombinant human CDK4 protein with high affinity, but also specifically bind to the intracellular CDK4 protein. The results of this study laid a foundation for further research on tumor gene therapy of anti-CDK4 antibodies.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【引证文献】