本文選題：熒光共振能量轉(zhuǎn)移 + 突變型Kras基因��； 參考：《山東大學(xué)》2010年碩士論文

【摘要】： Kras基因是一種癌基因,在腫瘤中的突變率較高。它就像體內(nèi)的一個(gè)“開(kāi)關(guān)”,調(diào)控著腫瘤細(xì)胞生長(zhǎng)和血管生成過(guò)程中的信號(hào)傳導(dǎo),影響著腫瘤的生長(zhǎng)和擴(kuò)散。Kras基因的檢測(cè)能夠?yàn)槟[瘤的臨床診斷,病情評(píng)估和預(yù)后判斷以及基因的靶向治療提供重要依據(jù)。因此,對(duì)組成Kras基因及其突變體的寡核苷酸分子進(jìn)行定量檢測(cè),在癌癥的診療方面具有重要意義。 白細(xì)胞介素-8(IL-8)是白細(xì)胞介素家族中的一員,是具有多效性和重疊性的生物活性因子。IL-8能夠調(diào)控血細(xì)胞的生長(zhǎng)發(fā)育和分化成熟以及機(jī)體的免疫應(yīng)答,同時(shí)與多種癌癥有關(guān)。人源化的抗IL-8單克隆抗體已經(jīng)應(yīng)用于相關(guān)疾病的臨床研究中。設(shè)計(jì)出快速高效的檢測(cè)抗IL-8單克隆抗體的方法,將為各種炎癥的治療和腫瘤的研究提供有利條件。 本文對(duì)三種新型熒光共振能量轉(zhuǎn)移(FRET)體系進(jìn)行了系統(tǒng)的研究,并將這些體系應(yīng)用于核酸檢測(cè)和免疫分析,定量檢測(cè)了突變型Kras基因片段和生物素化的抗IL-8單克隆抗體 本文第一章為緒論部分,該部分全面總結(jié)了FRET的原理,理論計(jì)算方法和熒光探針的研究進(jìn)展,重點(diǎn)介紹了FRET體系在生物分析中的應(yīng)用,對(duì)核酸檢測(cè),免疫分析,蛋白質(zhì)相互作用等方面作了詳細(xì)的敘述。 本文第二章對(duì)表面活性劑CPB作用下的FRET體系進(jìn)行了詳細(xì)的研究。該體系通過(guò)陽(yáng)離子表面活性劑對(duì)DNA的壓縮作用來(lái)增強(qiáng)FRET信號(hào),在不必分離過(guò)剩探針的情況下實(shí)現(xiàn)了長(zhǎng)間距的供受體之間的FRET,并對(duì)含有30個(gè)堿基的靶DNA進(jìn)行了定量檢測(cè)。此外,利用圓二色譜法初步探討了CPB與雙鏈DNA的作用機(jī)理,對(duì)實(shí)驗(yàn)現(xiàn)象進(jìn)行了合理解釋。 本文第三章對(duì)表面活性劑CTAB增敏的FRET體系進(jìn)行了詳細(xì)的研究。該體系通過(guò)陽(yáng)離子表面活性劑與量子點(diǎn)和染料標(biāo)記的二抗分子的相互作用來(lái)實(shí)現(xiàn)FRET,不必對(duì)抗體的特定區(qū)域進(jìn)行標(biāo)記或使用特殊的抗體片段。基于FRET導(dǎo)致的受體熒光峰面積的增加,對(duì)生物素化的IL-8的單克隆抗體實(shí)現(xiàn)了定量檢測(cè)。 本文第四章對(duì)DNA嵌入型染料JOJO-1作為供體的FRET體系進(jìn)行了研究。研究發(fā)現(xiàn),利用DNA嵌入染料JOJO-1作為供體構(gòu)建FRET體系,不必對(duì)捕獲DNA進(jìn)行標(biāo)記,而且可以有效降低空白中的假陽(yáng)性,提高方法的選擇性�；贔RET造成的受體熒光峰面積的增加,成功的對(duì)含有30個(gè)堿基的靶DNA進(jìn)行了定量分析。 本論文的主要特點(diǎn)： 一、研究表面活性劑對(duì)FRET體系的影響,并利用靜電相互作用,建立起兩種新的定量檢測(cè)長(zhǎng)鏈核酸和抗體分子的方法。 二、研究DNA嵌入型染料作為供體的FRET體系,建立了一種不必標(biāo)記捕獲探針,高信噪比,高選擇性的核酸檢測(cè)方法。 三、上述研究工作為開(kāi)創(chuàng)新的FRET體系,選擇新型熒光探針提供了一定的理論和實(shí)踐基礎(chǔ),在核酸,抗體等生物大分子的定量檢測(cè)方面具有潛在的應(yīng)用價(jià)值。
[Abstract]:Kras gene is a kind of oncogene with high mutation rate in tumor. It is like a "switch" in the body, which regulates the signal transduction during tumor cell growth and angiogenesis, and affects the growth and diffusion of tumor. The detection of Kras gene can be used for the clinical diagnosis of tumor. Evaluation of disease, prognosis and gene targeting therapy provide important evidence. Therefore, quantitative detection of the oligonucleotide molecules of Kras gene and its mutants is of great significance in the diagnosis and treatment of cancer. Interleukin-8 (IL-8) is a member of the interleukin family, which is a bioactive and overlapping bioactive factor. IL-8 can regulate the growth, differentiation, maturation and immune response of blood cells, and is related to many kinds of cancer. Humanized anti-IL-8 monoclonal antibodies have been used in clinical studies of related diseases. A rapid and efficient method for the detection of monoclonal antibodies against IL-8 will provide favorable conditions for the treatment of various kinds of inflammation and the study of tumor. In this paper, three novel fluorescence resonance energy transfer (fret) systems were systematically studied and applied to nucleic acid detection and immunoassay. The mutational Kras gene fragments and biotinylated monoclonal antibodies against IL-8 were quantitatively detected. The first chapter of this paper is the introduction, which summarizes the principle of FRET, the theoretical calculation method and the research progress of fluorescent probe. The application of FRET system in biological analysis, the detection of nucleic acid and immunoassay are introduced. Protein interactions are described in detail. In the second chapter, the FRET system under the action of surfactant CPB is studied in detail. The system enhances the FRET signal by the compression of cationic surfactants to DNA, realizes the fret between the long distance donor receptors without separating the excess probes, and quantitatively detects the target DNA containing 30 bases. In addition, the mechanism of interaction between CPB and double-stranded DNA was preliminarily discussed by circular dichroism chromatography, and the experimental phenomena were explained reasonably. In the third chapter, the system of FRET sensitized by surfactant CTAB is studied in detail. The system realizes fret by the interaction of cationic surfactants with quantum-dot and dyestuff labeled second antibody molecules. It is not necessary to label specific regions of antibodies or to use special antibody fragments. Based on the increase of receptor fluorescence peak area caused by FRET, quantitative detection of monoclonal antibodies against biotinylated IL-8 was carried out. In chapter 4, the FRET system of DNA embedded dye JOJO-1 as donor is studied. It is found that DNA embedded dye JOJO-1 as donor to construct FRET system does not need to label captured DNA, and can effectively reduce false positive in blank and improve the selectivity of the method. Based on the increase of receptor fluorescence peak area caused by FRET, the target DNA containing 30 bases was analyzed quantitatively. The main features of this thesis are as follows: Firstly, the influence of surfactants on FRET system was studied, and two new methods for quantitative detection of long chain nucleic acid and antibody molecules were established by electrostatic interaction. Secondly, the FRET system with DNA embedded dyes as donor was studied, and a new method of nucleic acid detection was established, which was characterized by high signal-to-noise ratio (SNR) and high SNR without labeling capture probe. Third, the above work provides a theoretical and practical basis for the creation of a new FRET system and the selection of novel fluorescent probes, and has potential application value in the quantitative detection of biological macromolecules such as nucleic acids and antibodies.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R346

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本文編號(hào)：1782222