【作者】 林园园；

【导师】 林菊生；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 经绿色荧光蛋白(green fluorescent protein，GFP)标记后的病毒，可以在活细胞中显示并研究它们。但GFP并不适合标记乙型肝炎病毒(hepatitis B virus，HBV)，因为HBV是一种体积较小的病毒体，它的内部空间狭小，无法容纳GFP这样一个含有238个氨基酸的大片段的掺入。最近有报道称：融合了四半胱氨酸标签(tetracysteine tag，TC tag)的目的蛋白可以被一种双砷荧光染料在活细胞中特异地标记。这种双砷标记技术为活细胞中HBV的荧光示踪和研究又提供了新的机遇。【目的】制造重组的经双砷染料标记的荧光HBV，从而在活细胞中示踪并研究HBV。【方法】应用分子克隆和PCR定点突变技术，以含1.3倍HBV基因组的载体为模板，在其编码的核心蛋白的免疫优势c／el表位(the immunodominant c／el site)附近插入一个特殊的TC标签，这个标签可以同双砷荧光染料特异地结合。构建所得的重组的HBV载体被转染入HepG2细胞中用于表达TC嵌合型核心蛋白。应用Westernblotting，免疫荧光以及双砷标记分析这种蛋白的表达和亚细胞定位。此外，表达这种TC嵌合型核心蛋白的细胞用双砷染料印迹后，继续被培养，用于制造荧光HBV病毒体。应用投射电子显微镜(transmission electron microscopy，TEM)和激光共聚焦显微镜鉴定荧光HBV病毒体的产生，并进一步调查这些荧光病毒对DMSO处理过的HepG2细胞的感染性。最终，应用激光共聚焦显微镜在活细胞中直接观察荧光HBV的转运。【结果】编码TC嵌合型核心蛋白的HBV载体被成功构建，转染了这些HBV载体的细胞可以表达TC嵌合型核心蛋白并制造成熟的HBV病毒体。而且，细胞中结合了双砷染料的TC嵌合型核心蛋白可以特异地发出荧光，并组装入HBV核心颗粒中进而形成荧光HBV病毒体，这些病毒体保留了同野生型HBV病毒体类似的感染性。这些荧光HBV在活细胞中的转运可以通过激光共聚焦显微镜，直观而便捷地观察到。【结论】本研究应用双砷标记技术，首次制造出了荧光HBV病毒体，它让我们可以在活细胞中示踪并研究HBV的转运。这种荧光HBV可以作为一种非常有价值的工具，用于在活细胞中深入地研究HBV生命周期中的动态过程以及病毒与宿主之间的相互作用。更多还原

【Abstract】 Production of virus tagged with green fluorescent protein (GFP) contributes tovisualizing and studying virus in living cells. But GFP tag, as a 238-amino acide largeinserted fragment, is not suitable for labeling Hepatitis B virus (HBV) that is a compactvirion with limited internal space. It was recently reported that protein of interestgenetically inserted with a smaller size tetracysteine (TC) tag could be specially labeled bya biarsenical fluorescent dye in living cells. This biarensical labeling approach offers us anew opportunity to fluorescently label HBV virion for visualizing and studying HBV inliving cells.【Objective】To generated one recombinant HBV fluorescently labeled with biarsenicaldye, which could be visualized and studied in living cells.【Methods】By using a molecular clone and PCR based site directed mutagenensis, a TCtag that could be specially bound with a biarsenical fluorescent dye was genetically insertednear the immunodominant c/el site of HBV core protein encoded by 1.3 time HBV vector.The recombinant HBV vectors were transfected into HepG2 cells to express TC-taggedcore proteins. The expression and subcellular localization of TC-tagged core proteins wereanalyzed by western blotting, immunofluorescence and biarsenical dye labeling. Furthermore, these transfected cells expressing TC-tagged core proteins were stained withbiarsenical dye, and then maintained to produce fluorescent HBV virions. Confocalmicroscopy and transmission electron microscopy (TEM) were used to analyze theproduction of fluorescent HBV virions. And the infectivity of fluorescent HBV virions toDMSO-treated HepG2 cells was investigated. Finally, the translocation of fluorescent HBVin living cells was observed by time-lapse confocal microscopy.【Results】The HBV vector encoding TC-tagged core protein were successfullyconstruted. The cells transfected with the HBV vector could express TC-tagged coreproteins and produce mature HBV virions. Moreover, TC-tagged core proteins bound withbiarsenical dye could specifically fluoresce in cells and be incorporated into nucleocapsidto form fluorescent HBV virions. The recombinant fluorescent HBV virions retained theirinfectivity as wild-type ones. The translocation of fluorescent HBV in living cells could bedirectly and conveniently observed by time-lapse confocal microscopy.【Conclusion】To the best of our knowledge, this is the first time to generate fluorescentHBV virions with biarsenical labeling and to visualize their trafficking in living cells. Thefluorescent HBV may become one highly valuable tool for further studying detaileddynamic processes of HBV life cycle and interaction of HBV with host in live-imagingapproach.更多还原