生物型纳米金—一种可提高病毒检测和疫苗制备效力的新兴组合

发布时间：2018-03-01 10:46

本文关键词： 乙型肝炎病毒丙型肝炎病毒检测 DNA疫苗免疫反应抗体金纳米粒子量子点　出处：《浙江大学》2013年博士论文　论文类型：学位论文

【摘要】：病毒是一种导致全球发病率和死亡率持续升高的病原体。快速灵敏的检测系统加高效的疫苗能防止病毒感染和传播。然而目前已有的相关技术存在许多缺陷。随着全球总人口数量和移民数量的不断增长,对于病毒的检测手段和疫苗提出了更高的要求。纳米材料包括了具有非常独特和重要特性的一系列纳米尺度的物质,称为纳米结构。其中金纳米结构是研究得最多的具有独特表面等离子体特性的一类纳米结构。由于它们出色的生物相容性和稳定性,一些金纳米结构在生物医药中的应用潜能正在被开发,它们是病毒检测和疫苗研发中大有希望的候选材料。本论文主要通过下列两方面的研究来评估其在病毒检测和疫苗研发中的作用： 1.联合纳米组件和等离子体的共振传感器技术用于检测乙型肝炎病毒。这一技术主要是建立了一种新型的基于联合纳米组件和等离子体的共振传感器,共振传感器主要由一个核心元件和多个帽状生物纳米配体组成。每个帽状配体是由金纳米粒子和乙肝病毒表面抗原preS2的一段短肽偶联而成；而核心元件是由绿色(3nm)量子点上结合多个能特异性识别preS2短肽的Fab片段而组成的。由于核心元件上的Fab片段可以特异性结合帽状配体上的preS2短肽,两者形成了核心元件-帽状配体纳米组件复合体。当反应体系中有乙肝病毒存在时,核心元件上的Fab片段会竞争性与乙肝病毒preS2短肽结合,从而破坏核心元件-帽状配体纳米组件复合体的形成。用多种技术(如紫外可见吸收光谱术、透射电子显微镜术、动态光散射技术、Zeta电位分析法、傅里叶变换红外光谱术、能量色散x射线分析术、荧光光谱术、琼脂糖凝胶电泳)均证实了核心元件、帽状配体及其上偶联的传感分子以正确的形式(如单独存在或形成复合体)和结构存在。通过比较反应体系中加入的乙肝病毒表面抗原和完整的乙肝病毒颗粒,评估了核心元件-帽状配体纳米组件复合体甩于检测乙肝病毒的灵敏度和特异性,也进一步分析了纳米组件复合体的动力学特征。结果表明,我们首创的联合纳米组件和等离子体的共振传感器技术是一种快速和高灵敏度的病毒检测技术,可以检测到低至1-50个病毒粒子/微升(即10-8摩尔/升)的水平。 2.纳米等离子增强DNA疫苗递送效率用于预防丙型肝炎病毒。该方法研制了一种新型的基于纳米技术,提高肌注DNA疫苗的递送效率,用于预防丙型肝炎病毒。这种新型的递送系统通过在递送DNA疫苗的邻近部位局部注射金纳米结构,并施加几个时间控制电脉冲而达成。我们制备了两种特异性针对丙型肝炎病毒的DNA疫苗质粒,分别称为pVAX1核心与pVAX1-NS3,另又制备了不同大小(5、50与100nm)的金纳米结构。上述制备物的理化特性用紫外可见吸收光谱术、透射电子显微镜术、动态光散射技术、Zeta电位分析法、傅里叶变换红外光谱术和琼脂糖凝胶电泳等技术进行了分析验证。对所开发的单一丙型肝炎病毒DNA质粒及其组合分别进行了测试。另一方面,在不同的小鼠组测试了金纳米结构与pVAX1核心联合给药的效果。结果清楚地表明金纳米粒子即使在很低浓度时也具有提高试验动物免疫反应、增强特异性抗体形成的能力。纳米颗粒的大小对特异性抗体形成的水平影响甚微,而对细胞介导的免疫反应却有显著的影响。表面负电荷能显著影响金纳米粒子增强pVAX1核心质粒DNA疫苗递送和响应的效率。同时,运用透射电子显微镜和HE染色技术对肌肉组织中注射的金纳米粒子进行了检测,证实金纳米粒子的确进入了肌细胞,并导致了持续性的组织溶解和炎症。上述结果提示,我们的联合给药方案中金纳米粒子之所以能提高机体的免疫反应,一方面可能由于增加了对DNA疫苗的摄入,另一方面可能因为招募了更多免疫细胞到注射部位。
[Abstract]:The virus is a cause of morbidity and mortality worldwide continues to rise. The pathogen detection system of rapid and sensitive and efficient vaccine can prevent the virus infection and spread. However, there are many defects in the current existing technology. With the global population and the growing number of immigrants, for the virus detection and vaccine forward high requirements. Nano materials include a series of nanometer scale has a very unique and important characteristics of the material, called nano structure. The gold nano structure is a kind of nano structure has unique surface plasmon characteristics most. Due to their excellent biocompatibility and biological stability, some potential applications of gold nanostructures in biological medicine is being developed, they are the candidate materials for virus detection and vaccine development in great hope. This thesis mainly through the following two aspects Research to assess its role in virus detection and vaccine development:
1. sensor resonance technology and nano assembly and plasma for the detection of hepatitis B virus. This technology is mainly a kind of resonance sensor and nano assembly and plasma based on the model established, resonance sensor is mainly composed of a core element and a plurality of cap shaped nano bio ligand. Each cap is composed of a ligand short peptide conjugated gold nanoparticles and hepatitis B virus surface antigen preS2 and core element is made of green; (3nm) quantum dots with a number of specific recognition of preS2 peptide fragments of Fab and composition. Because the core components on the Fab fragment can specifically bind to preS2 peptide ligands on the cap. They formed the core components - cap ligand complex. When the nano assembly of hepatitis B virus exists in the reaction system, the core component of the Fab fragment will be competitive with hepatitis B virus preS2 binding peptides, To break the core component form a cap ligand complex. Nano components with a variety of techniques (such as UV Vis absorption spectroscopy, transmission electron microscopy, dynamic light scattering, Zeta potential analysis, Fourier transform infrared spectroscopy, energy dispersive X ray fluorescence spectroscopy analysis technique, agarose gel electrophoresis) were confirmed the core element of sensor molecule cap ligand and its coupling to the correct form (such as a separate existence and form complex) structure. Through the comparison with the hepatitis B virus surface antigen in the reaction system and complete the hepatitis B virus particles and evaluate the sensitivity and specificity of core components - cap ligand complex nano assembly rejection in the detection of hepatitis B virus, further analysis the dynamic characteristics of nano composite components. The results show that the resonance sensor with nanometer components and plasma we pioneered the Device technology is a rapid and highly sensitive virus detection technique that can detect as low as 1-50 virus particles / micro liters (10-8 mole / liter).
2. nano plasma enhanced DNA vaccine delivery efficiency for the prevention of hepatitis C virus. The method developed a model based on nanotechnology, improve the delivery efficiency of intramuscular injection of DNA vaccine, for the prevention of hepatitis C virus. This new delivery system in the adjacent area by local injection of gold nanostructures to deliver DNA vaccine, and applied several time control an electrical pulse. We have prepared two kinds of plasmid DNA vaccine specific for hepatitis C virus, called pVAX1 core and pVAX1-NS3, and the other for different size were prepared (5,50 and 100nm) of gold. The physicochemical properties of the nano structure preparation by UV Vis absorption spectroscopy, transmission electron microscopy, dynamic light scattering, Zeta potential analysis, Fourier transform infrared spectroscopy and agarose gel electrophoresis were analyzed. The single type of liver development C Virus plasmid DNA and its combinations were tested. On the other hand, the gold nanostructures and pVAX1 core test in mice of different drug combination effect. The results clearly show that the gold nanoparticles can also improve the experimental animal immune response even at very low concentration, the ability to gain high specificity of antibody formation. The level of success the effect of nano particle size on specific antibody formation, and had significant influence on the cell-mediated immune response. The surface negative charge can significantly affect the efficiency of delivery of core pVAX1 plasmid DNA vaccine and the response enhancement of gold nanoparticles. At the same time, the use of transmission electron microscopy and HE staining technique of gold nanoparticles for injection in muscle tissue the test confirmed that gold nanoparticles do go into muscle cells, and the persistent tissue dissolution and inflammation. These results suggest that our combined drug In the scheme, gold nanoparticles can enhance the immune response of the body. On the one hand, it may increase the intake of DNA vaccine, on the other hand, it may recruit more immune cells to the injection site.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08

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