【作者】 宋磊；

【导师】 陈劲春；

【作者基本信息】 北京化工大学 ， 生物化工， 2010， 博士

【摘要】 通过基于组合生物化学原理的噬菌体或细胞展示技术筛选得到的无机结合肽(inorganic binding peptide)序列,是一类存在于生物体内对相应无机物(金属单质、氧化物、无机盐等)具有特异吸附结合能力的小肽。纳米技术和分子生物技术的最新发展使得无机结合肽可以作为连接分子或模板通过基因设计组装到生物分子表面来促进新型材料的合成,这为材料科学和工程领域提供了一个崭新的思路。作为生物仿生应用的一个例证研究,在这里报道了关于无机结合肽在新兴的纳米生物交叉学科领域的功能性研究,包括它在生物-无机合成中的促进作用以及介导无机纳米粒子在生物分子如病毒表面的吸附以形成生物-无机复合结构。首先,在原核表达系统体系下进行了生物-无机相互作用的研究。根据Klemm研究小组利用细胞表面展示技术筛选得到的一个小肽序列pJKS9,设计引物合成了编码氧化锌特异结合多肽的基因片段用于蛋白-材料之间的相互作用研究。利用大肠杆菌表达载体pET-28a (+)和pGEX-4T-3, ZnO结合肽与His及GST标签融合实现重组表达,并通过Ni-NTA系统进行纯化得到GST-His-ZnO重组蛋白。经过SDS-PAGE蛋白电泳分析和Western免疫印迹检测,最后由(LC-MS/MS)质谱分析对产品进行了证实。通过简易便利的生物合成方法得到的这个双标签、捆绑式重组蛋白既有较好的可溶性,又因为His标签可经济便捷地进行纯化。生物-无机合成方法制备ZnO粉体的实验结果表明,这一新蛋白在利用氢氧化锌溶胶体系形成超细氧化锌前驱粉体过程中,具有促进颗粒细化和加速沉降的积极作用。通过锻烧粉体对最终产品进行X射线衍射分析的结果说明,得到了六方纤锌矿的氧化锌晶体颗粒。新式氧化锌结合蛋白的获得便于开展在许多领域的进一步研究,例如生物传感器、生物微阵列芯片、温和液相环境下生物-无机材料合成、重金属污染水质的生物修复等。在制备复合结构体的探索性实验中,进行了将真核的杆状病毒展示系统和无机结合肽介导的固定化技术联合应用的原理循证研究。所采取的策略是构建两个重组的病毒载体,其中编码AcNPV囊膜糖蛋白gp64的基因与编码主要衣壳蛋白vp39的基因拷贝各自引入到杆状病毒基因组中对病毒体外增殖非必需的polh或者p10的基因位点并进行外源基因的融合。检定筛选出的阳性重组穿梭质粒经过转染昆虫细胞得到囊膜或衣壳经过修饰的重组病毒,从而实现外源基因的融合表达以及展示在出芽病毒粒子的囊膜表面或衣壳上。随后,重组的病毒用于和纳米粒结合以组建生物-无机复合结构。结果表明,利用Bac-to-Bac系统将带有His标签的ZnO无机结合肽成功重组到AcNPV杆状病毒的囊膜表面或衣壳上。重组杆状病毒既保持了病毒的感染能力,又体现出插入的外源蛋白片段特异性结合的功能。重组转座质粒的构建由聚合酶链式反应(PCR)和酶切检定验明,并测序证实。融合肽的表面及衣壳展示由病毒粒子核壳离解分析中的Western blot鉴定以及胶体金免疫电镜观察等分析方法所证实。透射电镜的观察结果清楚表明,纳米粒子在无机结合肽介导下粘附到重组病毒的表面。这项工作表明了开发杆状病毒表面展示与无机肽介导的固定化方法联用技术的可行性,将在生物-无机合成工程领域的研究方面是一项有价值的补充内容。它有助于研究纳米粒子在生物医学探测与示踪方面的应用以及功能性纳米复合器件的制备。基于同样的策略,金特异结合的短肽也通过基因重组工程设计到杆状病毒的囊膜表面,同时利用Tschopp等发明的硼氢化钠还原法制备的胶体金进行了肽介导下的病毒-纳米金结合作用的探索实验。胶体金颗粒吸附到金结合肽修饰的重组杆状病毒表面的观察结果将可能有助于开发以金结合肽为标签的表达体系,类似于商业化的His标签应用。这样,重组蛋白的免疫电镜检测或许可用无需偶联抗体的胶体金更便捷、更经济地进行分析。本文同时报道了由双标签组成的重组蛋白及其结合ZnO的生物活性。更多还原

【Abstract】 Inorganic binding peptides often termed as genetically engineered polypeptides for inorganics (GEPIs) are small peptide sequences selected via combinatorial biology-based protocols of phage or cell surface display technologies. These short peptides exsit in vivo and can specifically bind to inorganics including metal and oxides etc. Recent advances in nanotechnology and molecular biology allow the genetical engineering of these peptides, used as molecular linkers or assemblers, to facilitate novel materials synthesis, which provides a new insight into the material science and engineering field. As a case study on this biomimetic application, here the functional research of inorganic binding peptides in the field of emerging interdiscipline of nanobiotechnology is reported, including its synthesis promotion in the bio-inorganic synthesis of inorganics and mediating the immobilization of inorganics on the biomolecular such as baculovirus to fabricate bio-inorganic compound structure.At first, study on the bio-inorganic interactions was conducted under the background of the prokaryotic expression system. According to the pJKS9 sequence screened by Klemm’s group using a cell-surface display system, the gene encoding a Zinc Oxides-binding peptide was synthesized for a case study of protein-material interactions. It was genetically fused with His6-tag and GST-tag using E.coli expression vector pET-28a(+) and pGEX-4T-3 for the recombinant expression. The recombinant protein GST-His-ZnO was expressed, purified with NTA-Ni system, identified by SDS-PAGE electrophoresis and Western blot analysis and confirmed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. The fusion protein GST-His-ZnO was just acquired by a facile and convenient biosynthesis method. This double-tagged bundled-up protein had a high solubility conferred by GST fusion and can be purified economically because of His tag. Results from the bio-inorganic synthesis experiment indicated that the new protein played a promoting part in grain refinement and accelerated precipitation during the formation of the ultra-fine precursor powders in the Zn(OH)2 sol solution, while the well-made intermediates are ever so vital in two-step liquid phase fabrication of ZnO nanomaterials. X-ray diffraction (XRD)analysis on the final products after calcining the precursor precipitates showed that hexagonal wurtzite ZnO crystals were obtained. The availability of the new ZnO binding protein may allow further exploration in the fields such as biosensors, biomicroarray chips, bio-inorganic material synthesis in mild aqueous environment and bioremediation of the heavy metal polluted water.In the exploratory trial of constructing organic-inorganic composites, a proof-of-principle study on combining eukaryotic baculoviral display technology with peptide-mediated immobilization of inorganic materials was conducted. The strategy was first to construct two recombinant viral vectors in which a second copy of gp64 gene and vp39 gene, respectively encoding the AcNPV(Autographa californica nuclear polyhedrosis virus) major envelope glycoprotein gp64 and major capsid protein vp39 were each introduced at the polh or p10 locus of baculovirus genome dispensable for viral propagation in vitro for the fusion with foreign genes. Then surface and capsid-modified recombinant baculovirus were respectively harvested after transfection of the identified positive recombinant bacmids into insect cells, and then led to fusion protein’s expression and its display on the surface and capsid of the budded virions upon infection. Recombinant viruses were then used to fabricate bio-inorganic composites with nanoparticles. Our results showed using Bac-to-Bac expression system, His-tagged ZnO GEPI was successfully recombinated into the surface and the capsid of baculovirus respectively. Recombinant baculovirus maintained both the viral infectivity and specific binding activity of the insert protein segment. The gene construction of recombinant transfer plasmid was examined by polymerase chain reaction (PCR) analysis and enzymatic digestion identification, and verified by gene sequencing. Surface and display of the fused peptide were revealed by Western blot analysis in dissolution studies and determined by immunogold electron microscopy. Peptide-midiated adherence of the nanoparticles to the recombinant baculovirus was visualized by transmission electron microscopy (TEM) analysis. Here we demonstrated the feasibilities of combining peptide-mediated immobilization with baculovirus display technology. This work would be a valuable addition to the field of bio-inorganic composite engineering. It can facilitate the study on nanoparticles’application in biomedical detection and tracking as well as fabrication of functional complex nanodevices in the future. Base on the same stategy, Au-specific binding peptide was also engineered into the baculovirus envelope to bind to the colloid gold nanoparticles prepared according to NaHB reduction method by Tschopp et al (1982).The adsorption of colloid Au with Au-binding peptide modified recombinant baculovirus may be helpful for the development of Au-binding peptide tagged expression system, then the immunogold microscopy detection of recombinant protein can be performed more facilely and economically with the non antibody-conjugated nanogold particles.GST and His double-tagged protein and its ZnO-binding bioactivity were also reported in our work.更多还原