本文關(guān)鍵詞： 蛋白標簽 SNAP-tag 免洗熒光探針 熒光成像　出處：《大連醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：蛋白質(zhì)是細胞的重要組成部分,是生命活動的主要承擔(dān)者。蛋白質(zhì)的種類繁多,至今還有很多蛋白的性質(zhì)未知,因此,對活細胞內(nèi)蛋白質(zhì)進行特異性熒光標記的技術(shù)應(yīng)運而生。蛋白熒光標記技術(shù)因能夠可視化的觀測活細胞內(nèi)蛋白質(zhì)的結(jié)構(gòu)與功能而被科學(xué)家們廣泛應(yīng)用。目前常用的蛋白熒光標記技術(shù)包括基因編碼的熒光蛋白法、非天然氨基酸法和自標記蛋白標簽技術(shù)。但是熒光蛋白存在分子量較大、熒光光譜單一等缺點,而非天然氨基酸法前期基因改造復(fù)雜使其應(yīng)用存在一定的局限性。因此自標記蛋白標簽技術(shù)被廣泛的應(yīng)用于研究活細胞中蛋白質(zhì)的定位和動態(tài)功能。至今,已經(jīng)開發(fā)了各種蛋白質(zhì)標簽以研究活體中的蛋白質(zhì)系統(tǒng),SNAP-tag是其中最優(yōu)秀的融合標簽之一。SNAP-tag是人源DNA烷基轉(zhuǎn)移酶(hAGT)的變體,它能夠?qū)Ｒ恍缘呐cO6-芐基鳥嘌呤(O6-benzylguanine,BG)及其衍生物共價連接。目前,各種連接BG底物的熒光探針已被設(shè)計出來,而且能專一、快速、不可逆地與SNAP tag共價連接,這使其廣泛應(yīng)用于藥物監(jiān)測、蛋白質(zhì)-蛋白質(zhì)相互作用、熒光傳感器、和超分辨顯微鏡。雖然已經(jīng)發(fā)現(xiàn)了很好的熒光探針,但是由于背景光較強這些探針需要在成像之前清除。這不僅耗費時間,而且這種清洗可能會影響實時監(jiān)測一些分子內(nèi)部的活動(例如受體-配體結(jié)合、內(nèi)吞作用、物質(zhì)運輸?shù)?。此外,因為多余的熒光探針容易在各種細胞器中堆積,我們很難做到完全清除。因此,為了克服這些困難,開發(fā)了新的具有開關(guān)效應(yīng)的熒光探針。本文采用SNAP-tag蛋白標簽技術(shù),用新型的熒光探針標記體內(nèi)的蛋白,能夠在免洗的條件下實時監(jiān)控蛋白在細胞內(nèi)的分布。基于此,本論文主要做了以下工作:首先,基于熒光團的環(huán)境敏感與淬滅機制,我們設(shè)計合成了一系列基于1,8-萘酰亞胺熒光團的熒光探針BGAN-R(R=2C、8C、12C、DM)。通過熒光檢測、動力學(xué)檢測等一系列實驗我們發(fā)現(xiàn)BGAN-2C能夠快速、專一的與SNAP-tag共價反應(yīng),并且熒光顯著增強。該探針細胞毒性小,在活細胞成像實驗中,其能夠快速地標記細胞內(nèi)特定的蛋白質(zhì)。隨后,基于光誘導(dǎo)電子轉(zhuǎn)移機制我們設(shè)計合成了BGAN-DPA探針。通過熒光檢測、動力學(xué)等研究,發(fā)現(xiàn)BGAN-DPA與SNAP-tag蛋白結(jié)合后熒光顯著增強。并且結(jié)合后的蛋白-探針復(fù)合物對銅離子有專一性響應(yīng)。最后將BGAN-DPA應(yīng)用到HEK 293細胞的生物成像研究中,在免洗條件下實現(xiàn)了細胞內(nèi)線粒體上蛋白的標記及銅離子的檢測。綜上所述,通過設(shè)計合成的新型熒光探針,實現(xiàn)了SNAP-tag蛋白在細胞內(nèi)的可視化追蹤以及細胞內(nèi)銅離子的檢測,并且不需要任何洗滌過程。
[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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q26

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相關(guān)期刊論文 前1條

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本文編號：1443389