本文選題：金納米簇　切入點：人線粒體鐵蛋白　出處：《中國農(nóng)業(yè)大學》2017年博士論文

【摘要】：納米材料的特性與其尺寸大小密切相關。納米簇是指直徑小于2 nm的納米顆粒,由特定屬性的金屬制備而成。特殊的外側電子排布使金屬納米簇在激發(fā)光的作用下可以發(fā)射出熒光。其中,金納米簇由于低毒性、制備簡便、高熒光性及化學穩(wěn)定性等優(yōu)異的性質而受到廣泛關注。蛋白質—金納米簇復合物不僅提高了金納米簇熒光的穩(wěn)定性,同時借助于蛋白質自身的功能特性擴大了金納米簇的應用范圍。但是在目前已有的研究中,對蛋白質—金納米簇的結構報道較少,蛋白質的金離子結合位點尚不清楚,同時,蛋白質—金納米簇在復雜基質中的應用也鮮有報道。本文分別采用人線粒體鐵蛋白及β-乳球蛋白作為模板制備金納米簇,對蛋白質—金納米簇的制備條件及功能應用進行了探究,主要結果如下:1.線粒體鐵蛋白是動物體內(nèi)天然存在的具有重要功能的籠形蛋白,由24個相同亞基構成,每個亞基的分子量約為21 kD。其外經(jīng)為12 nm,內(nèi)徑為8 nm,中空環(huán)境可以包埋一些藥物及活性小分子。在線粒體鐵蛋白的氨基酸序列中有兩個半胱氨酸殘基,通過利用X-射線晶體衍射以及蛋白質定點突變技術研究發(fā)現(xiàn):對于野生型人線粒體鐵蛋白,半胱氨酸殘基是金離子的結合位點,而位于線粒體鐵蛋白130位的半胱氨酸殘基還可以在結合金的基礎上進一步形成金納米簇;為了保留鐵蛋白內(nèi)部空腔的包埋功能以及外表面的可修飾特性,論文首先將野生型人線粒體鐵蛋白半胱氨酸殘基突變成丙氨酸,然后對位于兩納米蛋白殼內(nèi)的鐵氧化還原中心進行了改造,將天然谷氨酸殘基突變?yōu)榘腚装彼釟埢?通過X-射線晶體衍射以及熒光表征證實納米金原子簇可以定點在改造后的亞鐵氧化還原中心形成。這些研究說明了半胱氨酸殘基是蛋白質—金納米簇制備的重要氨基酸,而納米金原子簇在蛋白質的形成位點是可以調控的,這些發(fā)現(xiàn)不但豐富并拓展了蛋白納米金的制備方法,而且為把鐵蛋白開發(fā)成具有成像功能的新型納米平臺打下了堅實的基礎。2.β-乳球蛋白來源豐富,水溶性好。通過對該蛋白的氨基酸序列進行分析,我們發(fā)現(xiàn)它同樣具有半胱氨酸殘基,根據(jù)上述結果,其具備形成納米金原子簇的潛力。為了證實這個想法,論文優(yōu)化了 β-乳球蛋白制備金納米簇的條件,得到了熒光強且穩(wěn)定的蛋白金納米簇。同時發(fā)現(xiàn)β-乳球蛋白—金納米簇的熒光具有可以被重金屬汞離子特異性淬滅、靈敏度高、且不受復雜基質干擾的特點,因而可以應用到復雜的食品及生物樣晶體系中檢測汞離子。論文進一步研究發(fā)現(xiàn):由于β-乳球蛋白可以和小腸上皮細胞膜上的受體結合,所以β-乳球蛋白—金納米簇可以作為穩(wěn)定的Caco-2細胞膜成像試劑;更重要的是β-乳球蛋白一金納米簇在動物體內(nèi)依然保持穩(wěn)定的熒光,并能通過腎臟代謝,對機體無破壞作用,所以這些新制備的金納米簇不但可以應用于細胞成像還可以應用于小動物活體成像中。綜上所述,這些研究成果有助于我們對金納米簇與蛋白質結合位點以及其形成機制的理解;同時,這些研究拓寬了金納米簇蛋白質復合物的應用范圍,即在復雜食品基質中的應用。
[Abstract]:The characteristics and size of nano materials is closely related to the nano cluster refers to the nanoparticles diameter less than 2 nm, the metal specific properties is prepared. The special electronic configuration of lateral metal nanoclusters in excitation light can emit fluorescence. The gold nanoclusters due to low toxicity, preparation simple, high fluorescence properties and excellent chemical stability and attracted widespread attention. Protein gold nanoclusters composite not only improves the stability of fluorescent gold nanoclusters, functional properties and by the protein itself to expand the range of application of the gold nanoclusters. But in the present study, reports on protein structure gold nanoclusters are gold ion binding sites of the protein is not clear, at the same time, the application of protein - gold nanoclusters in complex matrix is also rarely reported. This paper uses human mitochondrial ferritin And beta lactoglobulin as a template for the preparation of gold nanoclusters, condition and function application for preparation of protein - gold nanoclusters were studied, the main results are as follows: 1. mitochondrial ferritin is clathrin has an important function in the presence of natural animal body, composed of 24 identical subunits, the molecular weight of each the subunits of approximately 21 kD. outside after 12 nm, 8 nm in diameter and hollow environment can be embedded some drugs and active small molecules. There are two cysteine residues in the amino acid sequence of mitochondrial ferritin, by using X- ray diffraction and protein mutagenesis study revealed that for the wild type human mitochondrial ferritin, cysteine residues is the binding site of gold ions, cysteine residues located in mitochondrial ferritin 130 further formation of gold nanoclusters in base alloy on the node in order to keep the iron egg white inside; The cavity embedding function and the outer surface can be modified, firstly the wild-type mitochondrial ferritin cysteine residues were mutated into alanine, and then reconstruct the iron oxide in the two nanometer protein shell Reduction Center, natural glutamic acid residues mutated to cysteine residues by X- ray crystal diffraction and fluorescence characterization confirmed that gold nanoparticles clusters can be formed in the center point reduction of ferrous oxidation after transformation. These studies indicate that cysteine residues is an important amino acid protein - gold nanoclusters prepared, and nano gold clusters in the formation of protein loci can be regulated, these findings not only enrich and expand the method the preparation of gold nanoparticles for protein, and the ferritin in developing new nano imaging platform has the function to lay a solid foundation for.2. beta lactoglobulin rich source, water soluble Good. Through the analysis of the amino acid sequence of this protein, we found that it has the same cysteine residues, according to the above results, the formation of nano gold cluster potential. In order to confirm this idea, the paper optimized the beta lactoglobulin preparation of gold nanoparticles, obtained gold nanoclusters strong fluorescent protein and stable. At the same time that the fluorescence of beta lactoglobulin - gold nanoclusters can be used by mercury ion specific quenching, high sensitivity, and is not affected by the characteristics of complex matrix interference, which can be applied to complex food and biological samples in crystal detection of mercury ions. The further research shows that due to the combination of beta lactoglobulin and intestinal epithelial cell membrane receptor, so beta lactoglobulin - gold nanoclusters can be used as the cell membrane of Caco-2 imaging reagent stability; more important is the beta lactoglobulin a gold nanoclusters Continue to maintain a steady fluorescence in the animal body, and through the kidneys, no damage to the body, so these new gold nanoclusters prepared can be used not only in cell imaging can also be used in small animal imaging. In summary, the results of these studies contribute to our binding sites and the formation mechanism of gold nanoparticles with the understanding of protein clusters; at the same time, the study broadens the scope of application of gold nanoclusters protein complexes, namely the application in complex food matrices.

【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：O614.123;TB383.1

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相關期刊論文 前1條

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本文編號：1730541