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类型 基础研究 预答辩日期 2017-12-02
开始(开题)日期 2015-11-09 论文结束日期 2017-10-23
地点 化工楼小会议室319 论文选题来源 973、863项目     论文字数 5.2 (万字)
题目 蛋白质协同DNA构建超分子自组装的研究
主题词 蛋白质,DNA,自组装,超分子,脱铁蛋白
摘要 第一章:介绍了分子自组装、超分子化学的相关内容。综述脱氧核糖核酸(DNA)及蛋白质自组装研究进展及其应用。提出了本论文设计的思路及意义。 第二章:本章研究内容为运用脱铁蛋白辅助DNA指导金纳米粒子组装成花一样结构的组装体。本章选用的蛋白质为具有pH值相应的脱铁蛋白,当将脱铁蛋白溶液的pH值调节到2的时候,脱铁蛋白的球型结构会发生解离,得到24个亚基。然后将该亚基溶液与制备的粒径为5纳米的金颗粒混合,再把混合液的pH值缓慢调节至中性,脱铁蛋白的亚基会重组恢复至蛋白原来的球型结构,在重组的过程中将金纳米粒子包覆。将该溶液中多余的金纳米粒子除去并浓缩溶液后,将溶液平均分为两份,各加入巯基DNA溶液,而且加入的巯基DNA的碱基序列互补配对。巯基DNA通过脱铁蛋白的亲水通道结合其腔内的金纳米粒子。随后,分别将上述两份溶液中多余的巯基DNA除去之后,再进行浓缩,然后把浓缩的这两份溶液混合并对其进行PCR程序退火。从而使体系组装得到花一样结构的微米球。随后采用紫外-可见吸收光谱、透射电子显微镜、场发射扫描电子显微镜、聚丙烯酰胺凝胶电泳、原子力显微镜及热重分析仪等多种表征手段对组装的花进行表征。多种检测手段的测试结果证明了花的组成成分,初步证实了蛋白质、DNA以及金纳米粒子组装得到这种像花的组装体,推测组装的机理是通过螺旋位错组装而成。 第三章:本章研究的内容为采用一种两性交联剂Sulfo-SMCC将牛血清白蛋白与DNA交联,然后通过DNA的碱基互补配对将牛血清白蛋白连接起来,随后随着DNA进一步的杂化,牛血清白蛋白进一步组装成微米级的纤维。首先,在pH=7.4的水溶液中,交联剂Sulfo-SMCC结构中的羟基琥珀酰亚胺(NHS酯)与牛血清白蛋白表面游离的氨基(-NH2)结合,形成稳定的酰胺键,从而得到交联剂与BSA的复合物BSA-SMCC。然后除去BSA-SMCC复合物溶液中多余的交联剂,并分为两等分,分别加入等份的巯基修饰的DNA溶液(两份加入的巯基DNA碱基序列互补),巯基DNA上的巯基与BSA-SMCC复合物上的马来酰亚胺基团通过加成反应形成稳定的硫醚键,从而实现了将蛋白质牛血清白蛋白与巯基DNA的交联,得到复合物BSA-DNA。随后,将上述两份牛血清白蛋白与DNA的复合物溶液BSA-DNA分别浓缩后混合,利用DNA严格的碱基互补配对功能,使蛋白质与蛋白质连接起来。最后随着DNA杂化,更多的蛋白质通过DNA碱基互补配对进行交联,进一步组装得到微米级的具有半螺旋结构的纤维。此外,采用荧光标记的蛋白质及DNA重复实验,进一步证实了组装的纤维是由使用的蛋白质与DNA组装而成。另外还使用了原子力显微镜表征了纤维表面的微观结构,初步推测纤维由片层结构平行组装而成。 第四章:本章研究的内容为采用两性交联剂Sulfo-SMCC,将脱铁蛋白与DNA交联,然后通过DNA的碱基互补配对将脱铁蛋白连接起来,随着DNA进一步的杂化,脱铁蛋白进一步组装成微米级纤维。首先,交联剂Sulfo-SMCC结构中的羟基琥珀酰亚胺(NHS酯)在pH=7.4的溶液中与脱铁蛋白表面游离的氨基结合,形成稳定的酰胺键,从而得到交联剂与Apoferrtin的复合物Apoferritin-SMCC。上述溶液除去多余的交联剂后分为两等份,分别加入等量碱基序列互补配对的巯基修饰的DNA溶液,巯基DNA上的巯基与Apoferritin-SMCC复合物上的马来酰亚胺基团形成稳定的硫醚键,从而实现了将蛋白质与DNA交联,得到蛋白质与DNA的复合物Apoferritin-DNA。上述两份溶液均除去多余的巯基DNA后,室温下混合,孵化一定时间,利用DNA的碱基互补配对功能将蛋白质与蛋白质连接起来。最后,随着DNA的进一步杂化,更多的蛋白质通过DNA碱基互补配对交联,进一步组装得到微米级具有半螺旋结构的纤维。采用扫描电子显微镜、琼脂糖凝胶电泳、光学显微镜、共聚焦荧光显微镜及原子力显微镜表征了组装的纤维。通过原子力显微镜对纤维表面微观结构的观察,初步推测DNA与脱铁蛋白首先组装得到片层,然后这些片层以平行的方式组装得到具有半螺旋结构的纤维。 第五章:本论文研究的主要内容是三种蛋白质的自组装,以及采用的多种检测手段对组装体结构的初步表征。主要做了三项工作,第一是采用脱铁蛋白、DNA及金纳米粒子这三个物质通过一定的方式组织得到花一样的组装体。第二是采用交联剂将牛血清白蛋白与巯基DNA交联,利用DNA碱基互补配的功能使牛血清白蛋白组装形成具有一定周期半螺旋结构的微米级纤维。第三是采用交联剂将脱铁蛋白与巯基DNA进行交联,利用DNA的碱基互补配对功能使脱铁蛋白交联,随后通过DNA的进一步杂化,使脱铁蛋白组装成为具有一定周期半螺旋结构的微米级纤维。
英文题目 Study on the self-assembling of supermolecular aggregation using DNA with the mediation of protein
英文主题词 Protein,DNA,self-assembly,supramolecular, apoferritin
英文摘要 Chapter one: The related contents of molecular self-assembly and supramolecular chemistry are introduced in this section. The research progress and application of DNA and protein self-assembly are reviewed. The ideas and significance of this paper were put forwarded. Chapter two: The content of this research considers the use of apoferritin-assisted DNA to guide gold nanoparticles to assemble into flower-like structures. This chapter selects protein apoferritin, which can respond to pH value. When the apoferritin solution pH value is adjusted to 2, apoferritin will dissociate and 24 subunits are obtained. The prepared gold nanoparticles (particle size of 5 nm) are then mixed with the above subunit solution. The next process involves the pH value of the mixed solution being slowly adjusted to neutral, the subunits become recombinant and the protein will recover the original globular structure. In the process of reorganisation of apoferritin, the gold nanoparticles are caged in the cage of apoferritin. After the excess gold nanoparticles in the solution are removed and concentrated, the solution is divided into two parts, with each part added to a thioled DNA solution. Moreover, the base sequence of the added thioled DNA is complementary to each other. Thioled DNA combines with gold nanoparticles caged in the apoferritin through the hydrophilic channel of the protein. The excess thioled DNA of the two solutions is removed and concentrated respectively, following which the two solutions are mixed. The annealing of the mixture is performed following a programmed cooling using a single cycle of Polymerase Chain Reaction (PCR) procedure. This approach can lead to further self-assembly of gold nanoparticles. The assembly flowers are then characterised by transmission electron microscopy, scanning electron microscopy, polyacrylamide gel electrophoresis and atomic force microscopy. The components of the assembled flowers are confirmed by various means of testing, initially confirming that the protein, DNA and gold nanoparticles self-assemble into flower-like structures. It is preliminarily confirmed that protein, DNA and gold nanoparticles assemble into the obtained flower-structure through screw dislocations. Chapter three: This chapter focuses on using an amphoteric crosslinking agent, Sulfo-SMCC, to connect Bovine Serum Albumin (BSA) and DNA. Bovine Serum Albumin is then connected through the complementary base pairing of DNA. After that, BSA is further assembled into micron fibre through further DNA hybridisation. Firstly, crosslinker Sulfo-SMCC reacts with the amino-group of protein BSA to produce a maleimide-activated protein BSA-SMCC. After removing the excess of crosslinker and by-products, the maleimide-activated BSA-SMCC solution is divided into two parts, and thioled DNA solution is added to the above solutions respectively. Moreover, the base sequence of the added thioled DNA is complementary base paring. The maleimide-activated protein (BSA-SMCC) reacts with thiolated DNAs in the appropriate molar ratio and produces BSA-SMCC-DNAs through an addition reaction. The H-S bond of thiolated DNA is added to the double bond in the SMCC of BSA-SMCC. Subsequently, the above two BSA-DNA complex solutions are concentrated and mixed after removing the excess of thioled DNA. BSA molecules are connected with each other through the strict complementary base pairing of DNA function. Finally, the polymer BSA-SMCC-DNAs are self-assembled into fibres with half helical structure via further complementary base paring of the DNAs. In addition, fluorescently labelled protein (FITC-BSA) and DNA (Cy5-DNA) are used to repeat the experiment under the same conditions. The results further confirm that the assembly is composed of protein BSA and DNA. The microstructure of the fibres is observed using atomic force microscopy (AFM). The assembled mechanism of the fibre is that BSA and DNA firstly assemble into lamellar structure and these lamellae are then assembled into fibres in parallel. Chapter four: The content of this chapter describes how a protein (apoferritin) is connected with thioled DNA using an amphoteric crosslinking agent Sulfo-SMCC and the protein apoferritin is further assembled into micron fibre through further DNA hybridisation. Firstly, crosslinker Sulfo-SMCC reacts with the amino-group of apoferritin to produce a maleimide-activated protein, Apoferritin-SMCC. After removing the excess of crosslinker and by-products, the maleimide-activated Apoferritin-SMCC solution is divided into two parts, and thioled DNA solution is added to the above solutions respectively. Moreover, the base sequence of the added thioled DNA is complementary base paring. The maleimide-activated protein (Apoferritin-SMCC) reacts with thiolated DNAs in the appropriate molar ratio and produces Apoferritin-DNAs through addition reaction. The H-S bond of thiolated DNA is added to the double bond in the SMCC of Apoferritin-SMCC. Subsequently, the above two Apoferritin-DNA complex solutions are concentrated and mixed after removing the excess of thioled DNA. Apoferritin molecules are connected to each other through strict complementary base pairing of DNA function. Finally, the polymer Apoferritin-DNAs self-assemble into fibres with half helical structure via further complementary base paring of the DNAs. The fibres are presented by Scanning Electron Microscopy (SEM), agarose gel electrophoresis, Optical Microscopy (OM), confocal fluorescence microscopy and atomic force microscope (AFM) respectively. The microstructure of the assembled fibre is observed by using atomic force microscopy. The results suggest that DNA and apoferritin first self-assemble lamellar structure and then these lamellar structures assemble into fibres in a parallel way. Chapter five: The main content of this paper is about the self-assembling of three proteins. The assembled structure is preliminarily characterised through various methods. Three parts of the work are considered in this paper. Firstly, a flower-like structure is obtained by using apoferritin, DNA and gold nanoparticles to assemble through several steps. Secondly, a cross-linking agent is used to connect BSA with thioled DNA. The micro fibre with half periodic helical structure is obtained through the complementary base pairing of DNA. Thirdly, DNAs-conjugated apoferritin assembled into fibres via DNA hybridisation is demonstrated. The morphology of the two fibres are observed by optical microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM), and the assembly mechanism is then analysed and discussed. Protein molecules are first linked by DNA molecule and form a lamellae structure, then these lamellae further assemble into fibres in a parallel manner.
学术讨论
主办单位时间地点报告人报告主题
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学术会议
会议名称时间地点本人报告本人报告题目
2016年第三届国际化学与生物科学会议组委会 2016.3.23-2016.3.25 荷兰阿姆斯特丹 Fold DNA and Gold Nanoparticles into Nanoflower with Mediation of Apoferritin
2017年生物医学工程大学生菁英创新创业论坛主办方 2016.06.01 江苏省科技工作者活动中心 脱铁蛋白辅助DNA指导金纳米粒子组装成纳米花
第十一届中美华人纳米论坛 2016.6.18-2016.6.20 南京大学仙林校区
     
代表作
论文名称
Assembling gold nanoparticles into flower-like structures by complementary base pairing of DNA molec
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
周建成 正高 教授 博导 东南大学
叶树集 正高 教授 博导 中国科学技术大学
黄军 正高 教授 博导 南京工业大学
张天柱 正高 教授 博导 东南大学
王志飞 正高 教授 博导 东南大学
      
答辩秘书信息
姓名职称工作单位备注
程林 副高 副教授 东南大学