建立基于miniSOG标签的光镜-电镜关联方法观察腺病毒Ⅸ蛋白的亚细胞定位

Study of intracellular location of adenovirus protein Ⅸ with correlative light and electron microscopy based on miniSOG

目的 建立光镜-电镜关联方法观察腺病毒蛋白Ⅸ的细胞内定位.方法 miniSOG是新发现的能够被荧光显微镜和电子显微镜解析的蛋白标签.通过PCR方法克隆腺病毒结构蛋白Ⅸ与miniSOG的融合基因ⅨSOG,将其构建至真核表达载体,转染细胞;通过荧光显微镜确定ⅨSOG蛋白表达的细胞,之后对转染细胞进行原位固定、光氧化、制备超薄切片,用电镜观察细胞内ⅨSOG融合蛋白的表达及分布情况.结果 在蓝光激发下,miniSOG能够发射绿色荧光.定位选择光氧化形成嗜锇颗粒的ⅨSOG表达细胞进行电镜观察,结果显示Ⅸ蛋白在细胞核内多个区域形成点状聚集或包涵体.结论 建立了依赖miniSOG标签的的光镜-电镜关联方法,该方法可用于病毒蛋白细胞内定位研究.

Objective To establish a method of correlative light and electron microscopy （CLEM） to study the intracellular location of adenovirus protein Ⅸ.Methods MiniSOG （mini singlet oxygen generator） is a recently-invented genetically-encoded tag for CLEM.MiniSOG-fused adenovirus Ⅸ gene （ⅨSOG） was cloned by PCR,and inserted into pcDNA3 plasmid to form pTPL-ⅨSOG,which was used to transfect 293 cells.ⅨSOG expressing cells could be distinguished under fluorescence microscope due to the emission of green fluorescence of miniSOG.The transfected cells were fixed in 2.5% glutaraldehyde in situ,stained with diaminobenzidine（DAB） through the photooxidation activity of miniSOG,and used to prepare uhrathin sections.Intracellular location of ⅨSOG was studied by observing the sections under transmission electron microscope.Results Eukaryotic expression plasmid carrying ⅨSOG fusion gene was constructed.ⅨSOG expressing cells were selected for DAB photooxidation and preparation of ultrathin sections.ⅨSOG fusion mainly formed punctate aggregations or inclusions in the nucleus.Conclusions The correlative light and electron microscopy method based on miniSOG was successfully established,and it could be used to study the intracellular localization of viral proteins.