基于DNA酶和小分子連接DNA末端保護的蛋白質熒光分析
發(fā)布時間：2018-11-26 20:46
【摘要】：蛋白質是生命體的基本組成成分,是維持生命正常生理功能的重要活性物質。蛋白質與營養(yǎng)代謝、物質運輸、機能防御、遺傳等生理活動密切相關。而且有研究表明,在疾病的發(fā)展過程中,一些特異性蛋白質的表達與分布也會發(fā)生改變,因此蛋白質的分析檢測對掌握蛋白質的生理功能,研究蛋白質參與的生理活動以及相關疾病的診斷、治療都具有重要意義�；谛》肿优潴w和蛋白質之間的特異性相互作用的末端保護原理最近引起了蛋白質研究工作者的廣泛興趣。末端保護是指首先將小分子配體連接在DNA上,目標蛋白質與小分子配體結合后,能夠阻止外切酶對連接小分子的DNA鏈進行消解。末端保護對DNA序列的編碼無限制,可以與各種基于核酸的擴增技術和檢測手段相結合,這為蛋白質分子的靈敏檢測提供了新的途徑。本論文基于小分子連接DNA末端保護原理建立了系列蛋白質熒光分析的方法,主要內容如下:方法一:基于DNA酶和小分子連接DNA末端保護的熒光信號降低法檢測蛋白質。在本實驗中,我們設計了一個發(fā)夾結構的DNA探針。探針3'端標記有生物素(biotin)。當目標蛋白質-鏈霉親合素(STV)不存在時,核酸外切酶Ⅲ(ExoⅢ)能夠特異性地從3'端水解DNA探針的雙鏈部分。未被水解的探針的單鏈部分能夠形成DNA酶(DNAzyme)結構。DNAzyme與加入的分子信標結合,在中間的RNA堿基處將分子信標切斷。分子信標上的熒光基團和猝滅基團因此而分離,熒光信號恢復。之后,DNAzyme又與新的分子信標結合,發(fā)生剪切作用,釋放熒光基團。DNAzyme不斷循環(huán)利用,直至全部的分子信標參與反應,熒光信號達到最強。加入目標蛋白質STV后,STV與biotin特異性結合,能夠阻止ExoⅢ從3'端水解DNA探針的雙鏈部分。DNA探針仍然保持發(fā)夾結構,無法形成DNAzyme。沒有DNAzyme的作用,分子信標不能被切斷,保持熒光猝滅狀態(tài)。該方法通過熒光信號的降低對目標蛋白質進行定量測定,可以檢測到低至50 pmol的STV。方法簡單快速,檢測成本低,但靈敏度還有待于進一步提高。方法二:基于DNA酶和小分子連接DNA末端保護的熒光信號放大法檢測蛋白質。在本實驗中,我們將與小分子連接的DNA探針設計成DNAzyme的序列。目標蛋白質與小分子結合后,末端受到蛋白質保護,核酸外切酶Ⅰ(ExoⅠ)不能消解DNA探針。DNA探針保持DNAzyme結構,與加入的分子信標結合,切斷分子信標,釋放熒光信號。DNAzyme不斷循環(huán)利用,與新的分子信標結合,發(fā)生剪切反應,釋放出越來越多的熒光基團,產(chǎn)生越來越強的熒光信號。當沒有目標蛋白質出現(xiàn)時,DNA探針被ExoⅠ水解,DNAzyme結構消失,加入的分子信標保持淬滅狀態(tài),體系熒光信號很弱。通過檢測熒光信號的增長來實現(xiàn)目標蛋白質的定量檢測。我們將該方法分別應用于鏈霉親和素(STV)和葉酸受體(FR)的檢測,取得了良好的效果。該方法可以檢測到低至0.1 pmol的STV和100 ng的FR,靈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.
【學位授予單位】：河北大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O657.3
本文編號：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.
【學位授予單位】：河北大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O657.3
【參考文獻】
相關期刊論文 前1條
1 張松柏;鄭麗英;胡霞;沈廣宇;劉學文;沈國勵;俞汝勤;;基于競爭觸發(fā)滾環(huán)擴增的熒光適配體傳感器高靈敏檢測凝血酶[J];分析化學;2015年11期
相關碩士學位論文 前1條
1 梁長城;功能性核酸8-17 DNAzyme的活性調控[D];中國海洋大學;2012年
，本文編號：2359581
本文鏈接：http://sikaile.net/kejilunwen/huaxue/2359581.html
教材專著
