抗CDK4人源单链抗体作用机制的研究
发布时间：2018-07-14 13:41
【摘要】：研究表明肿瘤细胞恶性增殖主要是由于细胞周期的失调所导致,Cyclin D-CDK4/CDK6是调控细胞周期由G1向S期的过渡关键因子。CDK4基因己被证实是一种癌基因,在多种肿瘤细胞和癌变组织中都发现伴随有CDK4的过度表达和过度活化现象,因此CDK4可做为肿瘤基因治疗中另一个潜在的靶点。本课题组在前期的工作中,已获得抗CDK4单链抗体并将其转入肿瘤细胞中,成功的逆转了肿瘤细胞恶性增殖的表型,但是我们对于该单链抗体究竟以何种方式识别并作用于抗原分子,抑制靶蛋白功能,进而发挥其抗肿瘤作用的机制等基因水平上的问题还不清楚。 表位代表了抗原分子上的一个免疫活性区,是抗原抗体识别的基础,随着生物学技术的发展,对于表位的预测已经发展了多种方法,其中噬菌体筛选技术因其高通量、操作简单、能同时预测构象型表位和线性表位等优点,现已经被广泛的应用于各种研究中,特别是以单链抗体为靶蛋白筛选噬菌体库来分析其所对应抗原的表位己成为目前预测抗原蛋白表位的重要方法。因为每一种表位预测方法都有一定的局限性,因此采用计算预测(如同源建模、分子对接等)和生物实验数据结合的方法进行抗原蛋白构象表位分析和定位已经成了目前的研究热点,并呈现出了快速发展的趋势。 本研究首先分别制备了具有生物活性的CDK4抗原蛋白和抗CDK4的人源单链抗体(scFv)蛋白,进而以scFv为靶点,从噬菌体随机环七肽库中筛选得到一组特异性结合抗CDK4的人源单链抗体的环七肽基因,并用生物学软件对这组环七肽的序列进行了比对分析。为了能够获得更准确的数据支持和更直观的了解蛋白相互作用的方式,我们接着同源模建了scFv的空间结构,用Builter软件绘制了环七肽的三维结构,然后用分子对接的计算机方法模拟了小肽与scFv相互作用的空间结构。根据噬菌体筛选和计算机模拟的结果,我们推测经噬菌体筛选的小肽分析获得的表位可能为CDK4表位的组成部分或者对CDK4的表位活性空间构象的形成起着非常重要的作用。
[Abstract]:The results show that the malignant proliferation of tumor cells is mainly due to the maladjustment of cell cycle. CDK4 gene has been proved to be a kind of oncogene, which is the key factor to regulate the transition from G1 to S phase of cell cycle. The overexpression and over-activation of CDK4 have been found in many kinds of tumor cells and cancerous tissues, so CDK4 can be used as another potential target in tumor gene therapy. In our previous work, anti-CDK4 scFv was obtained and transferred into tumor cells, which successfully reversed the malignant proliferation phenotype of tumor cells. However, it is not clear how the scFv can recognize and act on antigen molecules, inhibit the function of target proteins, and then exert the mechanism of anti-tumor effect. Epitopes represent an immunoreactive region on antigen molecules and are the basis of antigen and antibody recognition. With the development of biological technology, many methods have been developed for epitope prediction, among which phage screening technology has high throughput. It is easy to operate and can predict conformational epitopes and linear epitopes at the same time. In particular, it has become an important method to predict the epitope of antigen by screening phage library with scFv as target protein to analyze the epitopes of its corresponding antigen. Because of the limitations of each epitope prediction method, computational prediction, such as homologous modeling, is used. Molecular docking) and biological experimental data for the analysis and localization of antigen protein epitopes have become the current research focus, and have shown a rapid development trend. In this study, biologically active CDK4 antigen proteins and human scFv anti-CDK4 protein were prepared, and then scFv was used as the target. A group of cyclopeptide genes with specific binding to human single-chain antibody against CDK4 was obtained from phage random cyclic heptapeptide library. The sequence of this group of cyclic heptapeptides was compared and analyzed with biological software. In order to obtain more accurate data support and more intuitively understand the way of protein interaction, we then build the spatial structure of scFv by homologous model, and draw the three-dimensional structure of cycloheptapeptide with build software. Then the spatial structure of the interaction between small peptide and scFv was simulated by the computer method of molecular docking. Based on the results of phage screening and computer simulation, we speculate that the epitopes obtained by phage screening may be part of CDK4 epitopes or play an important role in the formation of epitope active space conformation of CDK4.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
本文编号：2121829
[Abstract]:The results show that the malignant proliferation of tumor cells is mainly due to the maladjustment of cell cycle. CDK4 gene has been proved to be a kind of oncogene, which is the key factor to regulate the transition from G1 to S phase of cell cycle. The overexpression and over-activation of CDK4 have been found in many kinds of tumor cells and cancerous tissues, so CDK4 can be used as another potential target in tumor gene therapy. In our previous work, anti-CDK4 scFv was obtained and transferred into tumor cells, which successfully reversed the malignant proliferation phenotype of tumor cells. However, it is not clear how the scFv can recognize and act on antigen molecules, inhibit the function of target proteins, and then exert the mechanism of anti-tumor effect. Epitopes represent an immunoreactive region on antigen molecules and are the basis of antigen and antibody recognition. With the development of biological technology, many methods have been developed for epitope prediction, among which phage screening technology has high throughput. It is easy to operate and can predict conformational epitopes and linear epitopes at the same time. In particular, it has become an important method to predict the epitope of antigen by screening phage library with scFv as target protein to analyze the epitopes of its corresponding antigen. Because of the limitations of each epitope prediction method, computational prediction, such as homologous modeling, is used. Molecular docking) and biological experimental data for the analysis and localization of antigen protein epitopes have become the current research focus, and have shown a rapid development trend. In this study, biologically active CDK4 antigen proteins and human scFv anti-CDK4 protein were prepared, and then scFv was used as the target. A group of cyclopeptide genes with specific binding to human single-chain antibody against CDK4 was obtained from phage random cyclic heptapeptide library. The sequence of this group of cyclic heptapeptides was compared and analyzed with biological software. In order to obtain more accurate data support and more intuitively understand the way of protein interaction, we then build the spatial structure of scFv by homologous model, and draw the three-dimensional structure of cycloheptapeptide with build software. Then the spatial structure of the interaction between small peptide and scFv was simulated by the computer method of molecular docking. Based on the results of phage screening and computer simulation, we speculate that the epitopes obtained by phage screening may be part of CDK4 epitopes or play an important role in the formation of epitope active space conformation of CDK4.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
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