SNAP-tag荧光探针与细胞内蛋白标记应用研究

发布时间：2018-01-19 08:19

本文关键词： 蛋白标签 SNAP-tag 免洗荧光探针荧光成像　出处：《大连医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：蛋白质是细胞的重要组成部分,是生命活动的主要承担者。蛋白质的种类繁多,至今还有很多蛋白的性质未知,因此,对活细胞内蛋白质进行特异性荧光标记的技术应运而生。蛋白荧光标记技术因能够可视化的观测活细胞内蛋白质的结构与功能而被科学家们广泛应用。目前常用的蛋白荧光标记技术包括基因编码的荧光蛋白法、非天然氨基酸法和自标记蛋白标签技术。但是荧光蛋白存在分子量较大、荧光光谱单一等缺点,而非天然氨基酸法前期基因改造复杂使其应用存在一定的局限性。因此自标记蛋白标签技术被广泛的应用于研究活细胞中蛋白质的定位和动态功能。至今,已经开发了各种蛋白质标签以研究活体中的蛋白质系统,SNAP-tag是其中最优秀的融合标签之一。SNAP-tag是人源DNA烷基转移酶(hAGT)的变体,它能够专一性的与O6-苄基鸟嘌呤(O6-benzylguanine,BG)及其衍生物共价连接。目前,各种连接BG底物的荧光探针已被设计出来,而且能专一、快速、不可逆地与SNAP tag共价连接,这使其广泛应用于药物监测、蛋白质-蛋白质相互作用、荧光传感器、和超分辨显微镜。虽然已经发现了很好的荧光探针,但是由于背景光较强这些探针需要在成像之前清除。这不仅耗费时间,而且这种清洗可能会影响实时监测一些分子内部的活动(例如受体-配体结合、内吞作用、物质运输等)。此外,因为多余的荧光探针容易在各种细胞器中堆积,我们很难做到完全清除。因此,为了克服这些困难,开发了新的具有开关效应的荧光探针。本文采用SNAP-tag蛋白标签技术,用新型的荧光探针标记体内的蛋白,能够在免洗的条件下实时监控蛋白在细胞内的分布。基于此,本论文主要做了以下工作:首先,基于荧光团的环境敏感与淬灭机制,我们设计合成了一系列基于1,8-萘酰亚胺荧光团的荧光探针BGAN-R(R=2C、8C、12C、DM)。通过荧光检测、动力学检测等一系列实验我们发现BGAN-2C能够快速、专一的与SNAP-tag共价反应,并且荧光显著增强。该探针细胞毒性小,在活细胞成像实验中,其能够快速地标记细胞内特定的蛋白质。随后,基于光诱导电子转移机制我们设计合成了BGAN-DPA探针。通过荧光检测、动力学等研究,发现BGAN-DPA与SNAP-tag蛋白结合后荧光显著增强。并且结合后的蛋白-探针复合物对铜离子有专一性响应。最后将BGAN-DPA应用到HEK 293细胞的生物成像研究中,在免洗条件下实现了细胞内线粒体上蛋白的标记及铜离子的检测。综上所述,通过设计合成的新型荧光探针,实现了SNAP-tag蛋白在细胞内的可视化追踪以及细胞内铜离子的检测,并且不需要任何洗涤过程。
[Abstract]:Protein is an important part of cells and the main carrier of life activities. There are many kinds of proteins, so the properties of many proteins are unknown. The technique of specific fluorescent labeling of proteins in living cells emerges as the times require. Protein fluorescence labeling is widely used by scientists because of its ability to visualize the structure and function of proteins in living cells. The fluorescent labeling techniques include the gene encoding fluorescent protein method. Non-natural amino acid method and self-labeled protein labeling technology, but fluorescent protein has the disadvantages of large molecular weight, single fluorescence spectrum, and so on. However, the genetic modification of non-natural amino acid method has some limitations in its application. Therefore, self-labeled protein labeling technology has been widely used to study the localization and dynamic function of proteins in living cells. Up to now, self-labeling technology has been widely used to study the localization and dynamic function of proteins in living cells. Various protein labels have been developed to study protein systems in vivo. SNAP-tag is one of the most excellent fusion tags. SNAP-tag is a variant of human DNA alkyltransferase hAGT. It can specifically connect with O6-benzylguanine (O6-benzylguanine) and its derivatives. At present, various fluorescent probes have been designed to bind BG substrates. Moreover, it can be used in drug monitoring, protein-protein interaction and fluorescence sensor because of its specificity, fast and irreversible covalent connection with SNAP tag. And superresolution microscopes. Although very good fluorescent probes have been found, these probes need to be cleared before imaging because of the strong background light. This is not only time consuming. Moreover, such cleaning may affect the real-time monitoring of some intramolecular activities (such as receptor-ligand binding, endocytosis, material transport, etc.). Because the excess fluorescent probes tend to accumulate in a variety of organelles, it is difficult to eliminate them completely. A novel fluorescent probe with switching effect was developed. In this paper, a novel fluorescent probe was used to label the protein in vivo using SNAP-tag protein labeling technique. It can monitor the distribution of proteins in cells in real time under the condition of no-washing. Based on this, this paper mainly does the following work: firstly, the mechanism of environmental sensitivity and quenching based on fluorescence groups. We have designed and synthesized a series of fluorescence probes BGAN-RN ~ (2) C ~ ((2)) C ~ (2 +) C ~ (2 +) ~ (12) C ~ ((12)) C ~ (2 +) based on 1 ~ (8) -naphthalimide fluorescence group. We found that BGAN-2C can react with SNAP-tag quickly and specifically, and the fluorescence is significantly enhanced. The cytotoxicity of the probe is small. In the living cell imaging experiment, it can quickly label specific proteins in the cell. Then, based on the photoinduced electron transfer mechanism, we designed and synthesized the BGAN-DPA probe. Dynamics, etc. It was found that the fluorescence of BGAN-DPA was significantly enhanced after binding with SNAP-tag protein, and the binding protein-probe complex had a specific response to copper ions. Finally, BGAN-DPA was applied to. HEK. In the biological imaging of 293 cells. The labeling of mitochondrial proteins and the detection of copper ions in mitochondria were realized under the condition of no-washing. In conclusion, a new fluorescent probe was designed and synthesized. The visual tracing of SNAP-tag protein in cells and the detection of copper ions in cells are realized, and no washing process is required.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q26

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