摘要
近年来红外荧光素的研发及相应的标记技术的进展在生物医学领域的研究中备受关注,通过与尖端仪器的结合在一系列生物样本的检测中得到广泛的应用.然而,红外荧光标记技术在病毒感染生物学研究中的应用却少见报道.本文建立了Alexa Fluor 647(AF647)红外荧光素标记重组2型腺相关病毒(AAV2)的方法,并通过凝胶电泳的银染、红外荧光成像和透射电镜分析等手段,对标记病毒的结构和形态与未标记的病毒进行了细致的比较,同时进一步鉴定了其感染293T细胞后的转导活性.结果发现,AAV2在标记后的理化性质和感染活性均未发生改变.应用狭缝印迹和激光共聚焦显微成像技术,分别对标记病毒的细胞摄取效率和在宿主细胞内的分布进行描述,为红外标记AAV2在研究中的应用提供了范例.AF647标记的AAV2为腺相关病毒的感染动力学研究提供了新的手段,具有敏感度高、抗荧光漂白和低非特异干扰等优点,并且对直接有效地结合形态学分析和生化分子生物学检测,具有重要的应用价值.
Infrared fluorochromes have been increasingly attractive in biomedical researches for their latest developments and associated labeling techniques. Joint with the cutting edge novel equipments, infrared dyes have been applied in the detections of various biological samples. However, their applications to study the biology of viral infections were rarely reported. In this article, we first established a method using Alexa Fluor 647 （AF647） to label adeno-associated virus seral type 2 （AAV2）; then characterized the labeled viruses using SDS-PAGE, silver staining and TEM analyses compared with the non-labeled native viruses; and meanwhile tested their transduction activities in 293T cells. We found that the chemical and biological properties of recombinant AAV2 were not altered after AF647 labeling. Finally, to demonstrate the potential applications of the labeled viruses, we evaluated the viral uptake efficiency and the intracellular distribution of AF647 labeled AAV2 using slot blot assays and confocal microscopy. Our results have shown that AF647 labeling was a valuable approach to investigate the dynamic of AAV infections for its advantages of high sensitivity, less photo bleaching and low non-specific interference. The capability of obtaining both morphological and biochemical data using AF647 labeled viruses is particularly attractive.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2008年第12期1152-1157,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
北京市属市管高等学校人才强教计划(No.BAHED2007-2008)
国家自然科学基金项目(No.30771062)资助~~
