抗cyclin D1人源单链抗体AD9的原核表达、纯化及活性研究

发布时间：2018-03-05 05:08

本文选题：cyclin　切入点：D1　出处：《吉林大学》2008年硕士论文　论文类型：学位论文

【摘要】： 细胞周期蛋白D1(cyclin D1)作为一种重要的周期蛋白,与肿瘤的发生、发展及患者的预后密切相关,并在多种肿瘤中存在着过度表达。通过反义核酸、导入抗体和降解肿瘤细胞内cyclin D1蛋白均能有效地阻碍肿瘤细胞的增殖。因此cyclin D1被认为是肿瘤基因治疗的重要靶点。单链抗体(single chain variable fragment, scFv)作为具有抗原结合活性的最小的抗体片段,具有分子量小,易于侵入实体瘤,免疫原性较小,能通过基因工程技术大量生产等特点,具有良好的应用前景。为了有效的灭活肿瘤细胞内过度活化的cyclin D1/CDK4的活性,抑制肿瘤细胞的增殖,本研究通过PCR方法对从人源噬菌体抗体库中筛选得到抗cyclin D1单链抗体基因wtAD9中的linker区的终止密码子实行了定点突变,并在原核细胞中进行了诱导表达、纯化,并通过ELISA、Western blot、竞争性抑制、亲和力测定、免疫荧光和免疫共沉淀等实验方法对AD9进行了活性表征。结果表明AD9能够特异性结合重组cyclin D1蛋白和肿瘤细胞内源性的cyclin D1蛋白,并具有较高的亲和力。本实验得到的结果对后续开展抗cyclin D1胞内抗体肿瘤基因治疗的研究奠定了基础。
[Abstract]:Cyclin D1), as an important cyclin, is closely related to the occurrence, development and prognosis of tumor, and is overexpressed in many kinds of tumors. Both the introduction of antibodies and the degradation of cyclin D1 protein in tumor cells can effectively inhibit the proliferation of tumor cells, so cyclin D1 is considered as an important target of tumor gene therapy. Single chain variable fragment (scFV), as the smallest antibody fragment with antigen-binding activity, has the characteristics of small molecular weight, easy invading solid tumor, low immunogenicity, and can be produced in large quantities by genetic engineering technology. It has good application prospect. In order to effectively inactivate the activity of cyclin D1 / CDK4 and inhibit the proliferation of tumor cells, In this study, the terminal codon of the linker region of cyclin D1 single chain antibody gene wtAD9 was screened from human phage phage antibody library by PCR, and was induced and purified in prokaryotic cells. The activity of AD9 was characterized by Elisa Western blot, competitive inhibition, affinity assay, immunofluorescence and immunoprecipitation. The results showed that AD9 could specifically bind to recombinant cyclin D1 protein and endogenous cyclin D1 protein in tumor cells. And has high affinity. The results of this study laid a foundation for further research on tumor gene therapy of anti cyclin D1 antibodies.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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