發(fā)布時(shí)間：2018-05-29 16:54

  本文選題：DNA水凝膠 + 末端脫氧核苷酸轉(zhuǎn)移酶　； 參考：《湖南大學(xué)》2016年碩士論文

【摘要】：功能水凝膠作為一種新型生物材料,由于其物理機(jī)械性能與生物組織的類似性,已經(jīng)成為生物醫(yī)學(xué)領(lǐng)域一種重要的生物材料。近幾年來,DNA水凝膠得到了快速發(fā)展,由于其優(yōu)異的生物相容性、可降解性以及易于功能化等優(yōu)點(diǎn),DNA水凝膠在生物傳感、藥物的運(yùn)載與釋放、蛋白的體外表達(dá)以及單細(xì)胞操控等方面都有著廣泛的應(yīng)用。但是以往DNA水凝膠的合成方法中,往往需要大量的DNA作為原料,極大增大了合成成本,限制了DNA凝膠的發(fā)展。因此,本文中,我們基于末端脫氧核苷酸轉(zhuǎn)移酶(TdT)的催化聚合性能,以二維或者三維DNA納米材料作為基本結(jié)構(gòu)單元,開發(fā)了兩種簡(jiǎn)便、快捷且成本低的方法構(gòu)建DNA水凝膠,并且將其應(yīng)用于物質(zhì)的包裹及可控釋放,蛋白的富集,和多酶反應(yīng)體系的構(gòu)建,具體如下：1.基于TdT無模板擴(kuò)增DNA的性能,以十字交叉型DNA結(jié)構(gòu)(X-DNA)作為基本結(jié)構(gòu)單元,構(gòu)建了一種合成DNA水凝膠的新方法。該方法中,以X-DNA為引物,通過TdT擴(kuò)增將三磷酸脫氧胸苷(dTTP)和三磷酸脫氧腺苷(dATP)添加至X-DNA的3’-羥基末端,合成四端帶有富含胸腺嘧啶(T)的X-DNA-Tn和富含腺嘌呤(A)的X-DNA-An,然后利用A-T堿基互補(bǔ)配對(duì),自組裝形成DNA水凝膠。本方法中,TdT的引入極大的減少了水凝膠合成中初始DNA的用量,由原來的數(shù)十微摩級(jí)別降低到幾個(gè)微摩,從而降低了成膠成本。另外,我們考察了這種凝膠形成的機(jī)理,發(fā)現(xiàn)帶有富A序列或者帶有富T序列X-DNA無法單獨(dú)形成凝膠,且以線性DNA為引物合成的富A序列與富T序列的雜交以及錯(cuò)配堿基(如A/C)的擴(kuò)增產(chǎn)物都無法合成水凝膠。這些結(jié)果表明,該凝膠的合成中以十字交叉型DNA作為中心點(diǎn),而延伸臂作為交聯(lián)劑,二者缺一不可。接著,利用流變儀、掃描電子顯微鏡、原子力顯微鏡分別表征其力學(xué)性能、表面形貌以及內(nèi)部結(jié)構(gòu)。最后,我們探究了該DNA水凝膠在大分子物質(zhì)的封裝以及可控釋放方面的應(yīng)用。2.利用上述通用型的合成方法構(gòu)建了一種功能DNA水凝膠——DNA酶水凝膠(DNAzyme hydrogel).將一段DNAzyme(Dz)序列連接于上述X-DNA結(jié)構(gòu)上,形成Dz-X.DNA。該結(jié)構(gòu)在一些陽離子(K+)存在的情況下,能夠形成鳥嘌呤-四鏈體,與氯化血紅素(hemin)結(jié)合后具有模擬過氧化物酶活性,能夠催化H2O2氧化ABTS2產(chǎn)生綠色產(chǎn)物ABTS-。首先,我們以Dz-X-DNA作為結(jié)構(gòu)單元合成DNAzyme水凝膠,探索了其過氧化物酶活性,并且將其用于H2O2的可視化檢測(cè)。基于這一現(xiàn)象,我們發(fā)展了兩種生物酶串聯(lián)體系：結(jié)合葡萄糖氧化酶(GOx)構(gòu)建了雙酶串聯(lián)體系；將GOx以及半乳糖苷酶(β-Gal)固定于凝膠中即可構(gòu)成三酶串聯(lián)反應(yīng)體系。在這些酶聯(lián)體系中,我們合成的凝膠不僅充當(dāng)生物酶的支架,更為重要的是,它還是串聯(lián)反應(yīng)中的一個(gè)不可或缺的催化反應(yīng)單元,參與整個(gè)酶聯(lián)反應(yīng)的過程。這些有效的酶聯(lián)反應(yīng)為葡萄糖、乳糖提供了一種潛在的檢測(cè)方法。3.以三維DNA納米材料——DNA納米管(DNA-nanotube)為基本結(jié)構(gòu)單元,構(gòu)建了一種結(jié)構(gòu)更為穩(wěn)定,機(jī)械性能更強(qiáng)的DNA水凝膠。該方法中,DNA-nanotube在TdT的催化下延四周擴(kuò)增產(chǎn)生多條富T或者富A側(cè)鏈,這些側(cè)鏈的雜交即可形成DNA水凝膠。流變性能測(cè)試證實(shí)了其凝膠的形成,且發(fā)現(xiàn)以DNA-nanotube為單體合成的凝膠,其機(jī)械強(qiáng)度更大,需要的初始DNA量更少。此外,我們初步探索了該凝膠的應(yīng)用范圍,實(shí)驗(yàn)發(fā)現(xiàn)其可用于蛋白以及多種生物酶的富集,并且能夠保持其生物活性,實(shí)現(xiàn)多酶串聯(lián)反應(yīng)。
[Abstract]:As a new biological material, functional hydrogels have become an important biological material in the field of biomedicine because of their physical and mechanical properties similar to biological tissues. In recent years, DNA hydrogels have developed rapidly. Due to their excellent biocompatibility, biodegradability and ease of functionalization, DNA hydrogels are in the field. Biosensors, drug delivery and release, protein expression in vitro and single cell manipulation are widely used. However, in the previous synthesis methods of DNA hydrogels, a large number of DNA were often needed as raw materials, which greatly increased the cost of synthesis and limited the development of DNA gel. Therefore, in this paper, we based on the terminal deoxy nucleoside. The catalytic polymerization of acid transferase (TdT), using two or three dimensional DNA nanomaterials as the basic structural units, developed two simple, fast and low cost methods to construct DNA hydrogels, and applied it to the encapsulation and controlled release of material, the accumulation of protein and the construction of the multi enzyme reaction system, as follows: 1. based on the TdT model The performance of DNA, using cross type DNA structure (X-DNA) as the basic structural unit, is a new method to synthesize DNA hydrogels. In this method, X-DNA is used as primers to add TdT three deoxylated thymidine (dTTP) and three phosphoric acid deoxy adenosine (dATP) to the 3 '- hydroxyl terminal of X-DNA, and the four ends are rich in thymus rich gland. The X-DNA-Tn of pyrimidine (T) and X-DNA-An rich in adenine (A) and then the complementary pairing of A-T bases and self-assembly to form DNA hydrogels. In this method, the introduction of TdT greatly reduced the amount of initial DNA in the hydrogel synthesis, reduced to a few micro frictional levels from the original tens of micro frictional levels, thus reducing the cost of the gelation. In addition, we examined it. The formation of this gel shows that a rich A sequence or a rich T sequence X-DNA can not form a gel alone, and the rich A sequence synthesized by the linear DNA primers and the rich T sequence and the mismatched base (such as A/C) can not synthesize the hydrogel. These results show that the synthesis of the gel is made of cross type DNA in the synthesis of the gel. As the center point and the extension of the arm as a crosslinker, the two are indispensable. Then, using rheometer, scanning electron microscope and atomic force microscope to characterize their mechanical properties, surface morphology and internal structure. Finally, we explored the application of the DNA hydrogel in the encapsulation and controlled release of macromolecules by.2.. A functional DNA hydrogel, DNA hydrogel (DNAzyme hydrogel), is constructed with a functional synthesis method. A DNAzyme (Dz) sequence is connected to the above X-DNA structure to form a Dz-X.DNA. that can form guanine four chain in the presence of some cations (K+), which is combined with hemin (hemin) to simulate peroxises. Enzyme activity of chemical compounds can catalyze H2O2 oxidation of ABTS2 to produce green product ABTS-. first. We synthesized DNAzyme hydrogel with Dz-X-DNA as a structural unit, and explored its peroxidase activity and used it for the visual detection of H2O2. Based on this phenomenon, we developed two kinds of biological enzyme series system: combined with glucose oxidase (GOx). A double enzyme series system was constructed, and GOx and galactosidase (beta -Gal) were immobilized in the gel to form a three enzyme series reaction system. In these enzymes, our synthesized gel not only acts as a scaffold for biological enzymes, but also, more importantly, is an indispensable catalytic unit in the series reaction and participates in the whole enzyme. These effective enzyme reactions are glucose, and lactose provides a potential detection method,.3., with a three-dimensional DNA nanomaterial, DNA nanotube (DNA-nanotube) as the basic structural unit. A more stable and more mechanical DNA hydrogel has been constructed. In this method, DNA-nanotube extends four under the catalysis of TdT. A number of rich T or A rich side chains were produced by week amplification. The hybridization of these side chains could form DNA hydrogels. The rheological properties test confirmed the formation of its gel and found that the gel with DNA-nanotube as a monomer was more mechanical and needed less initial DNA. In addition, we initially explored the application range of the gel. Experimental discovery It can be used for protein and enzyme enrichment, and can maintain its biological activity and achieve multiple enzyme tandem reactions.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q523;O648.17
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本文編號(hào)：1951675