荧光共振能量转移体系的建立及其在核酸和抗体检测中的应用

发布时间：2018-04-21 11:17

本文选题：荧光共振能量转移 + 突变型Kras基因　；参考：《山东大学》2010年硕士论文

【摘要】： Kras基因是一种癌基因,在肿瘤中的突变率较高。它就像体内的一个“开关”,调控着肿瘤细胞生长和血管生成过程中的信号传导,影响着肿瘤的生长和扩散。Kras基因的检测能够为肿瘤的临床诊断,病情评估和预后判断以及基因的靶向治疗提供重要依据。因此,对组成Kras基因及其突变体的寡核苷酸分子进行定量检测,在癌症的诊疗方面具有重要意义。白细胞介素-8(IL-8)是白细胞介素家族中的一员,是具有多效性和重叠性的生物活性因子。IL-8能够调控血细胞的生长发育和分化成熟以及机体的免疫应答,同时与多种癌症有关。人源化的抗IL-8单克隆抗体已经应用于相关疾病的临床研究中。设计出快速高效的检测抗IL-8单克隆抗体的方法,将为各种炎症的治疗和肿瘤的研究提供有利条件。本文对三种新型荧光共振能量转移(FRET)体系进行了系统的研究,并将这些体系应用于核酸检测和免疫分析,定量检测了突变型Kras基因片段和生物素化的抗IL-8单克隆抗体本文第一章为绪论部分,该部分全面总结了FRET的原理,理论计算方法和荧光探针的研究进展,重点介绍了FRET体系在生物分析中的应用,对核酸检测,免疫分析,蛋白质相互作用等方面作了详细的叙述。本文第二章对表面活性剂CPB作用下的FRET体系进行了详细的研究。该体系通过阳离子表面活性剂对DNA的压缩作用来增强FRET信号,在不必分离过剩探针的情况下实现了长间距的供受体之间的FRET,并对含有30个碱基的靶DNA进行了定量检测。此外,利用圆二色谱法初步探讨了CPB与双链DNA的作用机理,对实验现象进行了合理解释。本文第三章对表面活性剂CTAB增敏的FRET体系进行了详细的研究。该体系通过阳离子表面活性剂与量子点和染料标记的二抗分子的相互作用来实现FRET,不必对抗体的特定区域进行标记或使用特殊的抗体片段。基于FRET导致的受体荧光峰面积的增加,对生物素化的IL-8的单克隆抗体实现了定量检测。本文第四章对DNA嵌入型染料JOJO-1作为供体的FRET体系进行了研究。研究发现,利用DNA嵌入染料JOJO-1作为供体构建FRET体系,不必对捕获DNA进行标记,而且可以有效降低空白中的假阳性,提高方法的选择性。基于FRET造成的受体荧光峰面积的增加,成功的对含有30个碱基的靶DNA进行了定量分析。本论文的主要特点：一、研究表面活性剂对FRET体系的影响,并利用静电相互作用,建立起两种新的定量检测长链核酸和抗体分子的方法。二、研究DNA嵌入型染料作为供体的FRET体系,建立了一种不必标记捕获探针,高信噪比,高选择性的核酸检测方法。三、上述研究工作为开创新的FRET体系,选择新型荧光探针提供了一定的理论和实践基础,在核酸,抗体等生物大分子的定量检测方面具有潜在的应用价值。
[Abstract]:Kras gene is a kind of oncogene with high mutation rate in tumor. It is like a "switch" in the body, which regulates the signal transduction during tumor cell growth and angiogenesis, and affects the growth and diffusion of tumor. The detection of Kras gene can be used for the clinical diagnosis of tumor. Evaluation of disease, prognosis and gene targeting therapy provide important evidence. Therefore, quantitative detection of the oligonucleotide molecules of Kras gene and its mutants is of great significance in the diagnosis and treatment of cancer. Interleukin-8 (IL-8) is a member of the interleukin family, which is a bioactive and overlapping bioactive factor. IL-8 can regulate the growth, differentiation, maturation and immune response of blood cells, and is related to many kinds of cancer. Humanized anti-IL-8 monoclonal antibodies have been used in clinical studies of related diseases. A rapid and efficient method for the detection of monoclonal antibodies against IL-8 will provide favorable conditions for the treatment of various kinds of inflammation and the study of tumor. In this paper, three novel fluorescence resonance energy transfer (fret) systems were systematically studied and applied to nucleic acid detection and immunoassay. The mutational Kras gene fragments and biotinylated monoclonal antibodies against IL-8 were quantitatively detected. The first chapter of this paper is the introduction, which summarizes the principle of FRET, the theoretical calculation method and the research progress of fluorescent probe. The application of FRET system in biological analysis, the detection of nucleic acid and immunoassay are introduced. Protein interactions are described in detail. In the second chapter, the FRET system under the action of surfactant CPB is studied in detail. The system enhances the FRET signal by the compression of cationic surfactants to DNA, realizes the fret between the long distance donor receptors without separating the excess probes, and quantitatively detects the target DNA containing 30 bases. In addition, the mechanism of interaction between CPB and double-stranded DNA was preliminarily discussed by circular dichroism chromatography, and the experimental phenomena were explained reasonably. In the third chapter, the system of FRET sensitized by surfactant CTAB is studied in detail. The system realizes fret by the interaction of cationic surfactants with quantum-dot and dyestuff labeled second antibody molecules. It is not necessary to label specific regions of antibodies or to use special antibody fragments. Based on the increase of receptor fluorescence peak area caused by FRET, quantitative detection of monoclonal antibodies against biotinylated IL-8 was carried out. In chapter 4, the FRET system of DNA embedded dye JOJO-1 as donor is studied. It is found that DNA embedded dye JOJO-1 as donor to construct FRET system does not need to label captured DNA, and can effectively reduce false positive in blank and improve the selectivity of the method. Based on the increase of receptor fluorescence peak area caused by FRET, the target DNA containing 30 bases was analyzed quantitatively. The main features of this thesis are as follows: Firstly, the influence of surfactants on FRET system was studied, and two new methods for quantitative detection of long chain nucleic acid and antibody molecules were established by electrostatic interaction. Secondly, the FRET system with DNA embedded dyes as donor was studied, and a new method of nucleic acid detection was established, which was characterized by high signal-to-noise ratio (SNR) and high SNR without labeling capture probe. Third, the above work provides a theoretical and practical basis for the creation of a new FRET system and the selection of novel fluorescent probes, and has potential application value in the quantitative detection of biological macromolecules such as nucleic acids and antibodies.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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