利用荧光金纳米团簇探针研究猪伪狂犬病毒入侵宿主细胞的分子细节

Fluorescent gold nanoclusters are new types of luminescent imaging probes, and the sizes of them approach the Fermi wavelength of electrons. Fluorescent gold nanoclusters possess many unique optical properties (such as tunable emission-wavelength, good photochemical stability, high quantum yield) and favorable biocompatibility. As an important potential tool, fluorescent gold nanoclusters can also be used to study the molecular process by which virus invades into a host cell. This project focuses on studying the invasion into PK15 cell of porcine pseudorabies virus, a virus having caused great damage to animal husbandry. Different types of virus probes for detection and imaging will be prepared using fluorescent gold nanoclusters. Various technologies, including multi-photon laser scanning confocal microscope, steady-state and lifetime fluorescence spectrometery, and fluorescence resonance energy transfer imaging, will be used for monitoring the virus invasion in in-situ and real time manners. Qualitative and quantitative analysis will be conducted on the viral DNA release and replication, as well as on its protein expression. The trafficking of the virus within the cell, the assembly and disassembly of primary capsule membrane involving UL31 and UL34 proteins will be traced dynamically. These analyses will reveal the molecular details of the pseudorabies virus entering cells. In conclusion, the project will broaden our knowledge of the pathogenesis of pseudorabies virus; provide potential targets for antiviral research; furthermore serve as an important reference for understanding molecular pathogenesis of other viruses.

荧光金纳米团簇是近年来发展起来的新型光学成像探针，其尺寸与电子费米波长接近，具有独特的光学性质（如发射光谱可调、光稳定性良好、荧光量子产率高）和良好的生物相容性，是研究病毒入侵宿主细胞分子细节的重要潜在工具。本项目以危害养殖业的猪伪狂犬病病毒为模型，设计合成不同类型基于荧光金纳米团簇的病毒检测及成像探针，采用多光子激光扫描共聚焦显微镜、稳态/瞬态荧光光谱仪、荧光共振能量转移成像等技术，对猪伪狂犬病毒入侵PK15细胞的过程进行原位实时监测，对感染后病毒的DNA释放及复制、蛋白表达进行定性、定量分析，对病毒在细胞内的运动轨迹、病毒UL31和UL34蛋白初级囊膜化及丢失过程进行动态示踪，从更深层次揭示伪狂犬病毒入侵宿主细胞的分子细节。本研究不仅可以从更开阔的视野认识伪狂犬病毒的致病机理，为抗病毒药物的研发提供潜在靶标，而且还可为其他病毒的分子致病机理研究提供重要借鉴。

荧光金纳米团簇是近年来发展起来的新型光学成像探针，其尺寸与电子费米波长接近，具有独特的光学性质（如发射光谱可调、光稳定性良好、荧光量子产率高）和良好的生物相容性，是研究病毒入侵宿主细胞分子细节的重要潜在工具。本项目以氯金酸、牛血清白蛋白、谷胱甘肽、硝酸银、组氨酸等为原料，合成了牛血清白蛋白修饰的金簇、金银簇、组氨酸及谷胱甘肽混合修饰的金簇、碳点及CdTe量子点，研究了金簇、碳点及量子点对PK-15、Macr-145细胞的影响，金簇、碳点及量子点对伪狂犬病毒复制的影响，采用金簇为探针，研究了伪狂犬病毒吸附、复制等过程，发现病毒对细胞膜的吸附不完全依赖硫酸肝素的作用，病毒对细胞核膜的通透性具有严重破坏作用，通过改变金簇的结构，发现组氨酸修饰的金簇可以进入细胞核并对病毒的复制产生明显的抑制作用，而谷胱甘肽和组氨酸混合修饰的金簇对病毒的复制没有明显的影响。本研究不仅可以从更开阔的视野认识伪狂犬病毒的致病机理，而且还可为开发新型抗病毒药物提供重要借鉴。.在课题执行过程中，撰写《纳米荧光探针》学术著作1部，发表论文8篇，其中SCI收录论文7篇，另有2篇论文已经投稿，1篇论文正整理之中。参加国内学术会议13人次，做学术报告3次，培养博士研究生2名，硕士研究生6名。