【作者】 陈英杰；

【导师】 秦松；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋药物学， 2011， 博士

【摘要】 作为藻类的天线色素蛋白,藻胆蛋白以其独特的分子结构和光谱特性在生物医学领域得到了广泛的应用。藻胆蛋白的生物合成和组装改造,不仅为藻胆蛋白的光学研究提供了新的线索,而且为藻胆蛋白荧光探针的开发和应用提供了新的途径。另外,生物纳米技术的快速发展为生物细胞和生命活性物质的富集和分离提供了新的技术和手段。其中,磁性纳米颗粒基于其独特的磁响应特性为核酸、蛋白质和细胞的快速分离检测带来一种全新的纳米介质。本论文充分结合基因重组技术和生物纳米技术,开展了重组荧光藻胆蛋白的组装、分离和应用研究,并利用磁性纳米颗粒的分离特性,进行了新型生物分离介质的制备和研发。1、重组荧光藻胆蛋白的磁性纳米分离介质研究制备了锌离子修饰硅壳磁性纳米颗粒,并将其应用于菌体破碎液中组氨酸标签藻胆蛋白的分离。首次证明锌离子修饰硅壳磁性纳米颗粒对组氨酸标签蛋白的高选择性吸附作用。常温下,该颗粒对组氨酸标签藻胆蛋白的等温吸附曲线满足Langmuir吸附定律,饱和吸附容量与蛋白质的分子质量及空间阻力均有关系。基于这种锌离子修饰硅壳磁性纳米颗粒作为吸附分离介质,可以在不影响藻胆蛋白光谱特性的情况下,实现菌体破碎液中组氨酸标签藻胆蛋白的快速、高效分离。该研究不仅为组氨酸标签藻胆蛋白提供了一种新型亲和吸附分离介质,而且为活性藻胆蛋白的载送提供了一种新的靶向载体。2、重组荧光别藻蓝蛋白的组装研究鉴于组氨酸标签别藻蓝蛋白在层析分离中表现出的独特光谱变化,通过紫外可见和荧光分光光度检测的方法,考察了不同因素对天然别藻蓝蛋白和重组别藻蓝蛋白的光谱特性影响。在一定的浓度范围内,咪唑、EDTA、锌离子和镍离子等四种因素对天然别藻蓝蛋白的光谱基本没有影响,而对组氨酸标签重组别藻蓝蛋白的光谱特性却有明显影响。咪唑和EDTA均对组氨酸标签重组别藻蓝蛋白表现出明显的解聚作用,并且该解聚作用与咪唑和EDTA的浓度呈现很好的线性关系;锌离子和镍离子均对组氨酸标签重组别藻蓝蛋白表现出明显的促聚合作用,该促聚合作用与咪唑和EDTA表现出的解聚作用具有一定的可逆效果。该研究通过检测别藻蓝蛋白光谱变化,探索了重组组氨酸标签别藻蓝蛋白自组装三聚体的稳定性,为重组别藻蓝蛋白三聚体的组装研究提供了新的线索。3、功能化荧光别藻蓝蛋白的生物医学应用研究基于双标签设计法对聚球藻Synechocystis sp. PCC6803别藻蓝蛋白进行分子设计和异源表达,通过大肠杆菌发酵表达的方法制备出具有生物亲和功能的组氨酸标签荧光别藻蓝蛋白;基于金属离子螯合作用将双标签别藻蓝蛋白固定在锌离子修饰磁性纳米颗粒表面,制备出一种粒径为20±5 nm的红色荧光超顺磁性球状材料,该荧光磁性纳米颗粒表现链酶亲和素结合功能;以凋亡Hela细胞的分离目标,对基因重组技术制备的功能化荧光磁性纳米颗粒在生物分离中的应用进行了初步研究,结果表明该颗粒可以通过免疫结合反应对细胞进行快速识别和分离。该研究充分结合基因重组技术和生物纳米技术,不仅为生物检测提供了一种新的分离介质,同时也拓宽了重组藻胆蛋白在生物医学研究中的应用。更多还原

【Abstract】 As light-harvesting chromoproteins, phycobiliproteins have received more and more attentions in the field biomedicine for their unique molecular structures and spectral characterizations. The biosynthesis and reconstruction of phycobiliproteins not only provided novel clews for the research on their spectra, but also supplied a new way for the development and application of fluorescent probes based on phycobiliproteins. In addition, the fast development of bio-nanotechnology provided alternative techniques and means for the collection and separation of cells and bioactive molecules. Based on the particular magnetic property, magnetic nanoparticles were utilized as adsorbents in the fast separation and detection of nucleic acid, protein and cells. Combined with both DNA recombinant technology and bio-nanotechnology, this study investigated the reconstruction, separation and application of recombinant fluorescent phycobiliproteins, and also explored the preparation and application of novel magnetic adsorbents with magnetic nanoparticles.1. Research on the magnetic nanoadsorbents for the separation of recombinant holo-phycobiliproteins with nanoparticlesZinc decorated silica coated magnetic nanoparticles (ZnSiMNPs) were prepared and utilized in the rapid extraction of His-tagged holo-phycobiliproteins from Escherichia coli lysates. It provided the first evidence for the high selective adsorption between ZnSiMNPs and His-tagged protein. The equilibrium adsorption isotherms for His-tagged holo-phycobiliproteins on ZnSiMNPs could be described by the Langmuir adsorption equation, the maximum adsorption amounts of which were relevant to the molecular weights and dimensional resistances of proteins. With this kind of ZnSiMNPs nanoadsorbent, these His-tagged holo-phycobiliproteins were rapidly and efficiently separated from their lysates without spectral variation. Consequently, ZnSiMNPs served as a useful tool for the magnetic separation and delivery of the His-tagged phycobiliprotein.2. Research on the assembly of recombinant holo-allophycocyaninBased on the interesting spectra change of recombinant His-tagged holo-allphycocyanin during the separation process, UV–visible and fluorescence spectroscopy were employed to research effects of some different factors on the spectra of holo-allophycocyanin. It was unexpected that different responses were detected for native and recombinant holo-allophycocyanin. As for the recombinant holo-allophycocyanin, imidazole, EDTA showed obvious disassembly ability linear with their concentrations, while Zn(II) and Ni(II) showed assembly-promoting ability, reversely. However, these factors showed no obvious influence for the spectra of native holo-allophycocyanin. Thus, the stability of the recombinant His-tagged holo-allophycocyanin self-assembly trimer was studied through the detection of spectra changes, providing novel clews for the reconstruction of allophycocyanin trimer.3. Research on the biomedical application of functionlized holo-allphycocyaninAllophycocyanin from Synechocystis sp. PCC6803 was biosynthesized with both Strep II-tag and His-tag at N terminal in Escherichia coli, resulting in the preparation of His-tagged holo-allophycocyanin with streptavidin-binding ability for its Strep II-tag. Metal chelating immobilization of this recombinant protein on zinc decrated silica coated magnetic nanoparticles made a novel kind of multifunctional nanoparticles, characterized as a superparamagnetic and spherical material with a diameter of 20±5 nm, streptavidin-binding ability and red fluorescence. Primary utilization of these nanoparticles on the separation of apoptotic Hela cells indicated its availability in the rapid immuno-recognition and separation of cells. Therefore, this study utilized both DNA recombinant technology and bio-nanotechnolgy to provide novel adsorbents for biological analysis and exploit the application of recombinant phycobiliprotein in biomedicine.更多还原