功能化介孔材料的合成及其在蛋白质分离中的应用

发布时间：2018-11-25 19:18

【摘要】：蛋白质作为生命的物质基础,在生物体的各项生命活动中起着至关重要的作用,特别是一些低丰度蛋白质(LAP),常常是某些疾病的生物标志物,但是在生物体系中不同种类的蛋白质含量相差很大,在检测的过程中,由于高丰度蛋白质(HAP)信号的压制,经常会导致LAP信息的丢失,而这些LAP往往具有重要的生物学意义。因此,设计具有高选择性能的分离材料,对复杂生物体系中的HAP进行去除,并对LAP进行富集,是复杂生物样品中检测LAP的有效途径。本论文根据蛋白质自身等电点(pI)及裸露氨基酸残基的不同,设计合成了两种功能化的介孔材料,用于生物样品中HAP的去除。1.运用原子转移自由基聚合反应,在Santa Barbara Amorphous 15(SBA 15)表面嫁接了叔氨基及羧基官能团,使材料具有pH敏感的性质,用于分离具有不同pI的蛋白质。红外、热重、X射线衍射、Zeta电势、元素分析、透射电镜、氮吸附等表征结果显示了材料的成功制备。在不同pH条件下,叔氨基和羧基的质子化及去质子化效应使材料表面带有不同的电荷,通过静电作用吸附带有相反电荷的蛋白质,实现了对酸性和碱性蛋白质的特异性分离。此外,还将实验中筛选出的材料应用于实际生物样品中HAP的去除,凝胶电泳半定量结果显示,材料对鸡蛋清中卵清白蛋白和溶菌酶的去除率能够达到99%和95%,对血清中白蛋白的去除率能达到80%,这些数据表明,所设计合成的材料能显著地降低样品的复杂性,有利于样品中其它LAP的检测。2.通过热引发的八乙烯基多面体低聚倍半硅氧烷的自由基聚合反应合成了具有高比表面积的介孔材料,并以此为基质,对其表面进行后修饰和Cu~(2+)的固定,利用Cu~(2+)与蛋白质中裸露组氨酸(His)的亲和作用,选择性吸附样品中富含裸露His的HAP。表征及吸附实验结果表明该材料具有较大的比表面积、较好的吸附选择性和较高的吸附容量(对牛血红蛋白的吸附容量可达3150 mg/g),且重复使用六次后吸附容量基本保持不变。另外,该材料不仅能高效地去除全血中高丰度的血红蛋白和白蛋白,而且能够富集细胞裂解液中的蛋白质。所得的材料有望与其它材料联合使用,获得更多的人类蛋白质组学信息,为疾病的临床诊断及病理研究提供帮助。
[Abstract]:Protein, as the material basis of life, plays an important role in the life activities of organisms, especially some low-abundance protein (LAP), is often the biomarker of some diseases. However, the content of different kinds of proteins varies greatly in biological systems. In the process of detection, the suppression of (HAP) signal of high abundance protein often leads to the loss of LAP information, and these LAP often have important biological significance. Therefore, it is an effective way to detect LAP in complex biological samples by designing separation materials with high selectivity, removing HAP from complex biological systems and enriching LAP. In this paper, two kinds of functional mesoporous materials were designed and synthesized according to the differences of protein isoelectric point (pI) and bare amino acid residues, which were used to remove HAP in biological samples. 1. Tertiary amino groups and carboxyl functional groups were grafted on the surface of Santa Barbara Amorphous SBA 15 by atom transfer radical polymerization, which made the material pH sensitive and used to separate proteins with different pI. Infrared, thermogravimetric, X-ray diffraction, Zeta potential, elemental analysis, transmission electron microscopy, nitrogen adsorption and other characterization results show that the material was successfully prepared. Under different pH conditions, the protonation and deprotonation of tertiary amino and carboxyl groups lead to different charges on the surface of the materials. The specific separation of acidic and basic proteins is achieved by electrostatic adsorption of proteins with opposite charges. In addition, the materials selected in the experiment were applied to the removal of HAP from the actual biological samples. The results of semi-quantitative gel electrophoresis showed that the removal rates of ovalbumin and lysozyme from egg white could reach 99% and 95% respectively. The removal rate of albumin in the serum can reach 80%. These data show that the designed and synthesized materials can significantly reduce the complexity of the sample and facilitate the detection of other LAP in the sample. 2. The mesoporous materials with high specific surface area were synthesized by the free radical polymerization of octavinyl polyhedron oligosilane. The surface of mesoporous materials was modified and fixed by Cu~ (2). By using the affinity of Cu~ (2) to naked histidine (His) in protein, the HAP. rich in naked His was selectively adsorbed from the sample. The results of characterization and adsorption experiments show that the material has a large specific surface area, a better adsorption selectivity and a higher adsorption capacity (the adsorption capacity of bovine hemoglobin can reach 3150 mg/g). The adsorption capacity remained unchanged after repeated use for six times. In addition, this material can not only efficiently remove the high abundance hemoglobin and albumin in the whole blood, but also enrich the protein in the cell lysate. The obtained materials are expected to be used in conjunction with other materials to obtain more information on human proteomics and to provide help for clinical diagnosis and pathological research of diseases.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TB383.4;O658

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