线粒体铁蛋白及β-乳球蛋白金纳米簇的制备及表征

发布时间：2018-04-10 08:58

本文选题：金纳米簇　切入点：人线粒体铁蛋白　出处：《中国农业大学》2017年博士论文

【摘要】：纳米材料的特性与其尺寸大小密切相关。纳米簇是指直径小于2 nm的纳米颗粒,由特定属性的金属制备而成。特殊的外侧电子排布使金属纳米簇在激发光的作用下可以发射出荧光。其中,金纳米簇由于低毒性、制备简便、高荧光性及化学稳定性等优异的性质而受到广泛关注。蛋白质—金纳米簇复合物不仅提高了金纳米簇荧光的稳定性,同时借助于蛋白质自身的功能特性扩大了金纳米簇的应用范围。但是在目前已有的研究中,对蛋白质—金纳米簇的结构报道较少,蛋白质的金离子结合位点尚不清楚,同时,蛋白质—金纳米簇在复杂基质中的应用也鲜有报道。本文分别采用人线粒体铁蛋白及β-乳球蛋白作为模板制备金纳米簇,对蛋白质—金纳米簇的制备条件及功能应用进行了探究,主要结果如下:1.线粒体铁蛋白是动物体内天然存在的具有重要功能的笼形蛋白,由24个相同亚基构成,每个亚基的分子量约为21 kD。其外经为12 nm,内径为8 nm,中空环境可以包埋一些药物及活性小分子。在线粒体铁蛋白的氨基酸序列中有两个半胱氨酸残基,通过利用X-射线晶体衍射以及蛋白质定点突变技术研究发现:对于野生型人线粒体铁蛋白,半胱氨酸残基是金离子的结合位点,而位于线粒体铁蛋白130位的半胱氨酸残基还可以在结合金的基础上进一步形成金纳米簇;为了保留铁蛋白内部空腔的包埋功能以及外表面的可修饰特性,论文首先将野生型人线粒体铁蛋白半胱氨酸残基突变成丙氨酸,然后对位于两纳米蛋白壳内的铁氧化还原中心进行了改造,将天然谷氨酸残基突变为半胱氨酸残基,通过X-射线晶体衍射以及荧光表征证实纳米金原子簇可以定点在改造后的亚铁氧化还原中心形成。这些研究说明了半胱氨酸残基是蛋白质—金纳米簇制备的重要氨基酸,而纳米金原子簇在蛋白质的形成位点是可以调控的,这些发现不但丰富并拓展了蛋白纳米金的制备方法,而且为把铁蛋白开发成具有成像功能的新型纳米平台打下了坚实的基础。2.β-乳球蛋白来源丰富,水溶性好。通过对该蛋白的氨基酸序列进行分析,我们发现它同样具有半胱氨酸残基,根据上述结果,其具备形成纳米金原子簇的潜力。为了证实这个想法,论文优化了 β-乳球蛋白制备金纳米簇的条件,得到了荧光强且稳定的蛋白金纳米簇。同时发现β-乳球蛋白—金纳米簇的荧光具有可以被重金属汞离子特异性淬灭、灵敏度高、且不受复杂基质干扰的特点,因而可以应用到复杂的食品及生物样晶体系中检测汞离子。论文进一步研究发现:由于β-乳球蛋白可以和小肠上皮细胞膜上的受体结合,所以β-乳球蛋白—金纳米簇可以作为稳定的Caco-2细胞膜成像试剂;更重要的是β-乳球蛋白一金纳米簇在动物体内依然保持稳定的荧光,并能通过肾脏代谢,对机体无破坏作用,所以这些新制备的金纳米簇不但可以应用于细胞成像还可以应用于小动物活体成像中。综上所述,这些研究成果有助于我们对金纳米簇与蛋白质结合位点以及其形成机制的理解;同时,这些研究拓宽了金纳米簇蛋白质复合物的应用范围,即在复杂食品基质中的应用。
[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.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O614.123;TB383.1

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