基于DNA酶和小分子连接DNA末端保护的蛋白质荧光分析
发布时间：2018-11-26 20:46
【摘要】：蛋白质是生命体的基本组成成分,是维持生命正常生理功能的重要活性物质。蛋白质与营养代谢、物质运输、机能防御、遗传等生理活动密切相关。而且有研究表明,在疾病的发展过程中,一些特异性蛋白质的表达与分布也会发生改变,因此蛋白质的分析检测对掌握蛋白质的生理功能,研究蛋白质参与的生理活动以及相关疾病的诊断、治疗都具有重要意义。基于小分子配体和蛋白质之间的特异性相互作用的末端保护原理最近引起了蛋白质研究工作者的广泛兴趣。末端保护是指首先将小分子配体连接在DNA上,目标蛋白质与小分子配体结合后,能够阻止外切酶对连接小分子的DNA链进行消解。末端保护对DNA序列的编码无限制,可以与各种基于核酸的扩增技术和检测手段相结合,这为蛋白质分子的灵敏检测提供了新的途径。本论文基于小分子连接DNA末端保护原理建立了系列蛋白质荧光分析的方法,主要内容如下:方法一:基于DNA酶和小分子连接DNA末端保护的荧光信号降低法检测蛋白质。在本实验中,我们设计了一个发夹结构的DNA探针。探针3'端标记有生物素(biotin)。当目标蛋白质-链霉亲合素(STV)不存在时,核酸外切酶Ⅲ(ExoⅢ)能够特异性地从3'端水解DNA探针的双链部分。未被水解的探针的单链部分能够形成DNA酶(DNAzyme)结构。DNAzyme与加入的分子信标结合,在中间的RNA碱基处将分子信标切断。分子信标上的荧光基团和猝灭基团因此而分离,荧光信号恢复。之后,DNAzyme又与新的分子信标结合,发生剪切作用,释放荧光基团。DNAzyme不断循环利用,直至全部的分子信标参与反应,荧光信号达到最强。加入目标蛋白质STV后,STV与biotin特异性结合,能够阻止ExoⅢ从3'端水解DNA探针的双链部分。DNA探针仍然保持发夹结构,无法形成DNAzyme。没有DNAzyme的作用,分子信标不能被切断,保持荧光猝灭状态。该方法通过荧光信号的降低对目标蛋白质进行定量测定,可以检测到低至50 pmol的STV。方法简单快速,检测成本低,但灵敏度还有待于进一步提高。方法二:基于DNA酶和小分子连接DNA末端保护的荧光信号放大法检测蛋白质。在本实验中,我们将与小分子连接的DNA探针设计成DNAzyme的序列。目标蛋白质与小分子结合后,末端受到蛋白质保护,核酸外切酶Ⅰ(ExoⅠ)不能消解DNA探针。DNA探针保持DNAzyme结构,与加入的分子信标结合,切断分子信标,释放荧光信号。DNAzyme不断循环利用,与新的分子信标结合,发生剪切反应,释放出越来越多的荧光基团,产生越来越强的荧光信号。当没有目标蛋白质出现时,DNA探针被ExoⅠ水解,DNAzyme结构消失,加入的分子信标保持淬灭状态,体系荧光信号很弱。通过检测荧光信号的增长来实现目标蛋白质的定量检测。我们将该方法分别应用于链霉亲和素(STV)和叶酸受体(FR)的检测,取得了良好的效果。该方法可以检测到低至0.1 pmol的STV和100 ng的FR,灵敏度比方法一提高了500倍。
[Abstract]:The protein is the basic component of a living body, and is an important active substance for maintaining the normal physiological function of the life. The protein is closely related to the physiological activities such as nutrition metabolism, material transportation, functional defense, and inheritance. and the research shows that the expression and distribution of some specific proteins can also change during the course of the development of the disease, so the protein analysis and detection can be used for grasping the physiological function of the protein, researching the physiological activity of the protein participation and the diagnosis of the related diseases, The treatment is of great significance. The principle of the terminal protection based on the specific interaction between the small molecule ligand and the protein has recently led to the extensive interest of the protein researchers. The end protection means that the small molecule ligand is first attached to the DNA, and after the target protein and the small molecule ligand are combined, the exonuclease can be prevented from digesting the DNA chain connecting the small molecule. The terminal protection has no restriction on the coding of the DNA sequence, and can be combined with various nucleic acid-based amplification technology and detection means, which provides a new way for sensitive detection of the protein molecules. In this paper, a series of protein fluorescence analysis methods are established based on the principle of the terminal protection of small molecule-linked DNA, and the main contents are as follows: Method 1: The protein is detected by the method of reducing the fluorescence signal of the DNA end protection based on the DNA enzyme and the small molecule. In this experiment, we designed a DNA probe with a hairpin structure. The probe 3 'end is labeled with biotin. When the target protein-streptavidin (STV) is not present, the exonuclease III (Exo III) can specifically hydrolyze the double-stranded portion of the DNA probe from the 3' end. The single-stranded portion of the unhydrolyzed probe can form a DNA enzyme structure. The DNAzyme binds to the added molecular beacon, and the molecular beacon is cut off at the intermediate RNA base. The fluorescent and photoout groups on the molecular beacon are thus separated, and the fluorescence signal is recovered. Then, the DNAzyme is combined with the new molecular beacon, and the shearing action is taken to release the fluorescent group. DNAzyme is continuously recycled until all the molecular beacons participate in the reaction, and the fluorescence signal reaches the strongest. After addition of the target protein STV, the STV specifically binds to the biotin to prevent the Exo III from hydrolyzing the double-stranded portion of the DNA probe from the 3 'end. The DNA probe still maintains the hairpin structure and cannot form a DNAzyme. Without the function of DNAzyme, the molecular beacon can not be cut off and the fluorescence quenching state is maintained. The method is capable of detecting a STV of low to 50 pmol by quantitative determination of the target protein by the reduction of the fluorescence signal. The method is simple and rapid, the detection cost is low, but the sensitivity needs to be further improved. method 2: a protein is detected by a fluorescence signal amplification method based on the DNA end protection of a DNA enzyme and a small molecule. In this experiment, we designed a DNA probe linked to a small molecule into a sequence of DNAzyme. After the target protein and the small molecule are combined, the terminal is protected by the protein, and the exonuclease I (Exo I) cannot dissolve the DNA probe. The DNA probe maintains the DNAzyme structure, binds to the added molecular beacon, cuts off the molecular beacon, and releases the fluorescence signal. DNAzyme is continuously recycled and combined with the new molecular beacon to produce a more and more fluorescent signal, and more and more fluorescent groups are released. When the target protein is not present, the DNA probe is hydrolyzed by the Exo I, the DNAzyme structure disappears, the added molecular beacon is kept in a quenching state, and the fluorescence signal of the system is weak. The quantitative detection of the target protein is achieved by detecting the growth of the fluorescent signal. We applied the method to the detection of the streptavidin (STV) and the folate receptor (FR), respectively, and achieved good results. The method can detect the STV and 100ng FR of low to 0.1 pmol, and the sensitivity is 500 times higher than that of the method.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3
本文编号：2359581
[Abstract]:The protein is the basic component of a living body, and is an important active substance for maintaining the normal physiological function of the life. The protein is closely related to the physiological activities such as nutrition metabolism, material transportation, functional defense, and inheritance. and the research shows that the expression and distribution of some specific proteins can also change during the course of the development of the disease, so the protein analysis and detection can be used for grasping the physiological function of the protein, researching the physiological activity of the protein participation and the diagnosis of the related diseases, The treatment is of great significance. The principle of the terminal protection based on the specific interaction between the small molecule ligand and the protein has recently led to the extensive interest of the protein researchers. The end protection means that the small molecule ligand is first attached to the DNA, and after the target protein and the small molecule ligand are combined, the exonuclease can be prevented from digesting the DNA chain connecting the small molecule. The terminal protection has no restriction on the coding of the DNA sequence, and can be combined with various nucleic acid-based amplification technology and detection means, which provides a new way for sensitive detection of the protein molecules. In this paper, a series of protein fluorescence analysis methods are established based on the principle of the terminal protection of small molecule-linked DNA, and the main contents are as follows: Method 1: The protein is detected by the method of reducing the fluorescence signal of the DNA end protection based on the DNA enzyme and the small molecule. In this experiment, we designed a DNA probe with a hairpin structure. The probe 3 'end is labeled with biotin. When the target protein-streptavidin (STV) is not present, the exonuclease III (Exo III) can specifically hydrolyze the double-stranded portion of the DNA probe from the 3' end. The single-stranded portion of the unhydrolyzed probe can form a DNA enzyme structure. The DNAzyme binds to the added molecular beacon, and the molecular beacon is cut off at the intermediate RNA base. The fluorescent and photoout groups on the molecular beacon are thus separated, and the fluorescence signal is recovered. Then, the DNAzyme is combined with the new molecular beacon, and the shearing action is taken to release the fluorescent group. DNAzyme is continuously recycled until all the molecular beacons participate in the reaction, and the fluorescence signal reaches the strongest. After addition of the target protein STV, the STV specifically binds to the biotin to prevent the Exo III from hydrolyzing the double-stranded portion of the DNA probe from the 3 'end. The DNA probe still maintains the hairpin structure and cannot form a DNAzyme. Without the function of DNAzyme, the molecular beacon can not be cut off and the fluorescence quenching state is maintained. The method is capable of detecting a STV of low to 50 pmol by quantitative determination of the target protein by the reduction of the fluorescence signal. The method is simple and rapid, the detection cost is low, but the sensitivity needs to be further improved. method 2: a protein is detected by a fluorescence signal amplification method based on the DNA end protection of a DNA enzyme and a small molecule. In this experiment, we designed a DNA probe linked to a small molecule into a sequence of DNAzyme. After the target protein and the small molecule are combined, the terminal is protected by the protein, and the exonuclease I (Exo I) cannot dissolve the DNA probe. The DNA probe maintains the DNAzyme structure, binds to the added molecular beacon, cuts off the molecular beacon, and releases the fluorescence signal. DNAzyme is continuously recycled and combined with the new molecular beacon to produce a more and more fluorescent signal, and more and more fluorescent groups are released. When the target protein is not present, the DNA probe is hydrolyzed by the Exo I, the DNAzyme structure disappears, the added molecular beacon is kept in a quenching state, and the fluorescence signal of the system is weak. The quantitative detection of the target protein is achieved by detecting the growth of the fluorescent signal. We applied the method to the detection of the streptavidin (STV) and the folate receptor (FR), respectively, and achieved good results. The method can detect the STV and 100ng FR of low to 0.1 pmol, and the sensitivity is 500 times higher than that of the method.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3
【参考文献】
相关期刊论文 前1条
1 张松柏;郑丽英;胡霞;沈广宇;刘学文;沈国励;俞汝勤;;基于竞争触发滚环扩增的荧光适配体传感器高灵敏检测凝血酶[J];分析化学;2015年11期
相关硕士学位论文 前1条
1 梁长城;功能性核酸8-17 DNAzyme的活性调控[D];中国海洋大学;2012年
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