本文關鍵詞：小分子與蛋白相互作用分析的生物傳感方法研究　出處：《湖南大學》2011年碩士論文　論文類型：學位論文

  更多相關文章： 光學生物傳感器 DNA裂解 小分子 蛋白質 相互作用

【摘要】：蛋白質的相關信息一直是生命科學研究的重點,其中小分子與蛋白質的相互作用近年來引起了人們的研究興趣。蛋白質的許多重要功能都有小分子的參與,研究小分子與蛋白質相互作用對促進生命科學的發(fā)展有著重要意義。利用化學小分子的多樣性,選擇適當?shù)幕钚孕》肿?設計合成能夠高選擇性地探測蛋白質的功能、結構以及與活性小分子作用模式的探針——化學小分子探針,可以為重大疾病的診斷和防治提供新的標記物、新的藥物作用靶點和新的先導結構,從而為創(chuàng)新藥物的發(fā)現(xiàn)奠定基礎。隨著對生命科學的深入研究,發(fā)展能測定寬范圍親和力的小分子-蛋白質方法成為生物化學、藥物開發(fā)及生物傳感工作者面臨的迫切任務。 本研究論文針對當前測量小分子-蛋白質相互作用方法中的一些重點、難點問題展開討論,其主要內(nèi)容如下: (1)研制了一種基于蛋白結合調(diào)控的靈敏度高、特異性強、通用度好的DNA裂解分子機器,用于小分子與蛋白相互作用的分析。為此,我們設計了一條標記小分子的DNA單鏈,與另一條長度稍長的DNA單鏈雜交成異源雙鏈,再與Fok I組裝形成分子機器。如果輸入一條5′端標記熒光基團(FAM)和猝滅基團四甲基羅丹明(TAMRA)的熒光探針,熒光探針會與異源雙鏈的長鏈互補,Fok I開始工作即切割探針,熒光基團與淬滅基團分離,熒光信號急劇增強;切割后的探針從長鏈上脫落,成為原來的分子機器,繼續(xù)參與下一個反應。若小分子的結合蛋白存在,在熒光探針與長鏈互補之后,由于空間位阻作用,Fok I不能到達切割位點,熒光探針不會切開,熒光基團與淬滅基團的FRET作用依然存在,不會出現(xiàn)熒光信號,從而達到對目標蛋白的檢測。通過分子機器與熒光探針的配對、剪開、脫離、再配對這個循環(huán),實現(xiàn)了對目標蛋白的循環(huán)放大檢測。在考察了葉酸-葉酸結合蛋白、葉酸-二氫葉酸還原酶、生物素-鏈霉親和素、生物素-生物素抗體等不同親和力的小分子-蛋白結合體系后,發(fā)現(xiàn)該方法特異性靈敏度高、特異性好、重現(xiàn)性強,有望成為一個通用的檢測小分子與蛋白質相互作用的平臺。(第2章) (2)將切刻酶放大技術與血紅素核酸適體化學發(fā)光相結合,建立了一種基于切刻酶放大技術結合血紅素適配體化學發(fā)光檢測小分子-蛋白相互作用的均相分析方法。設計了一條包含切刻酶Nt.BstNBI酶切位點、血紅素適配體的互補序列、分子內(nèi)折疊區(qū)域三個部分的序列,當Bst DNA聚合酶大片段和dNTPs存在的條件時該序列不斷被延伸、復制,切刻酶切割下游位點堿基,通過鏈置換反應,將血紅素適配體置換掉,從而可以完成血紅素適配體的循環(huán)復制。血紅素-血紅素適配體復合物與魯米諾-過氧化氫體系共存時,可發(fā)出強烈的化學發(fā)光信號。當有小分子結合蛋白存在時,由于小分子與其蛋白相互作用,使聚合酶和切刻酶的活性同時受到抑制,化學發(fā)光信號減弱,從而實現(xiàn)了對小分子結合蛋白的檢測。該方法具有靈敏度高,選擇性強,重現(xiàn)性好等優(yōu)點,有望用于高通量蛋白-小分子相互作用的篩選和檢測。(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).

【學位授予單位】：湖南大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R341

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