小分子与蛋白相互作用分析的生物传感方法研究

发布时间：2018-01-16 20:41

本文关键词：小分子与蛋白相互作用分析的生物传感方法研究　出处：《湖南大学》2011年硕士论文　论文类型：学位论文

【摘要】：蛋白质的相关信息一直是生命科学研究的重点,其中小分子与蛋白质的相互作用近年来引起了人们的研究兴趣。蛋白质的许多重要功能都有小分子的参与,研究小分子与蛋白质相互作用对促进生命科学的发展有着重要意义。利用化学小分子的多样性,选择适当的活性小分子,设计合成能够高选择性地探测蛋白质的功能、结构以及与活性小分子作用模式的探针——化学小分子探针,可以为重大疾病的诊断和防治提供新的标记物、新的药物作用靶点和新的先导结构,从而为创新药物的发现奠定基础。随着对生命科学的深入研究,发展能测定宽范围亲和力的小分子-蛋白质方法成为生物化学、药物开发及生物传感工作者面临的迫切任务。本研究论文针对当前测量小分子-蛋白质相互作用方法中的一些重点、难点问题展开讨论,其主要内容如下: (1)研制了一种基于蛋白结合调控的灵敏度高、特异性强、通用度好的DNA裂解分子机器,用于小分子与蛋白相互作用的分析。为此,我们设计了一条标记小分子的DNA单链,与另一条长度稍长的DNA单链杂交成异源双链,再与Fok I组装形成分子机器。如果输入一条5′端标记荧光基团(FAM)和猝灭基团四甲基罗丹明(TAMRA)的荧光探针,荧光探针会与异源双链的长链互补,Fok I开始工作即切割探针,荧光基团与淬灭基团分离,荧光信号急剧增强;切割后的探针从长链上脱落,成为原来的分子机器,继续参与下一个反应。若小分子的结合蛋白存在,在荧光探针与长链互补之后,由于空间位阻作用,Fok I不能到达切割位点,荧光探针不会切开,荧光基团与淬灭基团的FRET作用依然存在,不会出现荧光信号,从而达到对目标蛋白的检测。通过分子机器与荧光探针的配对、剪开、脱离、再配对这个循环,实现了对目标蛋白的循环放大检测。在考察了叶酸-叶酸结合蛋白、叶酸-二氢叶酸还原酶、生物素-链霉亲和素、生物素-生物素抗体等不同亲和力的小分子-蛋白结合体系后,发现该方法特异性灵敏度高、特异性好、重现性强,有望成为一个通用的检测小分子与蛋白质相互作用的平台。(第2章) (2)将切刻酶放大技术与血红素核酸适体化学发光相结合,建立了一种基于切刻酶放大技术结合血红素适配体化学发光检测小分子-蛋白相互作用的均相分析方法。设计了一条包含切刻酶Nt.BstNBI酶切位点、血红素适配体的互补序列、分子内折叠区域三个部分的序列,当Bst DNA聚合酶大片段和dNTPs存在的条件时该序列不断被延伸、复制,切刻酶切割下游位点碱基,通过链置换反应,将血红素适配体置换掉,从而可以完成血红素适配体的循环复制。血红素-血红素适配体复合物与鲁米诺-过氧化氢体系共存时,可发出强烈的化学发光信号。当有小分子结合蛋白存在时,由于小分子与其蛋白相互作用,使聚合酶和切刻酶的活性同时受到抑制,化学发光信号减弱,从而实现了对小分子结合蛋白的检测。该方法具有灵敏度高,选择性强,重现性好等优点,有望用于高通量蛋白-小分子相互作用的筛选和检测。(第3章)
[Abstract]:Protein information has been the focus of life science research, the interaction between small molecules and proteins have attracted considerable interest in recent years. Many important functional proteins are small molecules involved in the study, the interaction between small molecules and proteins have an important significance for promoting the development of life science. The use of chemical diversity small molecules, choose appropriate active small molecule design and synthesis, can highly selectively detect the protein structure and function, and the effect of probe active small molecule model -- the chemical molecular probes, provide new markers for diagnosis and treatment for major diseases, novel drug targets and new pilot structure. In order to lay the foundation for discovering new drugs. With the in-depth study of life science, the development of small molecule - Determination of a wide range of protein affinity can become biological methods Chemistry, drug development and the urgent task facing biosensors.
The main content of this paper is to discuss some important and difficult problems in the measurement of small molecule protein interaction.
(1) developed a protein binding regulation based on high sensitivity, specificity, DNA cleavage of good general molecular machines for analysis, small molecules and protein interaction. Therefore, we designed a small molecular marker of DNA single strand, a heteroduplex and DNA single strand hybridization another length a little longer, and then Fok I assembled molecular machine. If the input of a 5 'end labeling (FAM) and four methyl Luo Danming quencher (TAMRA) fluorescence probe, fluorescence probe will complement long chain and heteroduplex, Fok I began to work cutting probe, fluorescent and quenching group separation, the fluorescence signal increases rapidly; probe after cutting off from the long chain, as the original molecular machines, to continue to participate in the next reaction. If there exists a small molecule binding protein, after complementary fluorescence probe with long chain, due to steric effects, Fok I not to As the cleavage site, fluorescent probes do not open, FRET fluorophore and quencher still exists, no fluorescence signal, so as to achieve the detection of the target protein. The molecular machines and fluorescent probe pairs, cut out, match again this cycle, the cycle of amplification of target protein detection. In the study of folic acid - binding protein, folate and dihydrofolate reductase, biotin streptavidin, biotin and biotin antibody affinity of different small molecule protein binding system, found the method with high specificity and sensitivity, specificity, reproducibility, is expected to become a universal detection of small molecules the interaction of protein with platform (chapter second).
(2) will cut enzyme amplification and hemin aptamer chemiluminescence combined with a cut enzyme amplification technique combined with chemiluminescence detection of heme aptamer molecules based on protein interaction phase analysis method. The design contains a cut enzyme Nt.BstNBI endonuclease, complementary sequence of heme aptamer, intramolecular folding sequence of three parts, replication in the presence of Bst DNA polymerase large fragment and dNTPs conditions when the sequence is continuously extended, cut the downstream enzyme cutting site base, through the chain replacement reaction, will replace the heme aptamer, which can complete the replication cycle of heme aptamer. Heme heme ligand complexes with suitable coexistence of Lumino - hydrogen peroxide system, a strong chemiluminescence signal. When there is a small molecule binding protein exists, due to the small molecular and protein interaction With the polymerase and nicking, enzyme activity and inhibited chemiluminescence signal weakened, so as to realize the detection of small molecule binding protein. This method has high sensitivity, high selectivity, good reproducibility, is expected to be used for screening and detecting the interaction of high throughput protein - small molecule (Chapter third).

【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341

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