利用光镜-电镜关联方法研究发热伴血小板减少综合征病毒核蛋白的亚细胞定位  

Intracellular location of severe fever with thrombocytopenia syndrome bunyavirus NP protein with correlative light and electron microscopy based on miniSOG

作　　者：牛国宇 张尊 夏百成 刘淑慧 高旭 芜为[2] 邹小辉[2] 鲁茁壮[2] 洪涛[2] Niu Guoyu, Zhang Zun, Xia Baicheng, Liu Shuhui, Gao Xu, Wu Wei, Zou Xiaohui, Lu Zhuozhuang, Hong Tao.(School of Public Health and Management, Weifang Medical University, Weifang 261053, China ; National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China ; Henan Chemical Technician College, Kaifeng 475000, China)

机构地区：[1]潍坊医学院公共卫生与管理学院,261053 [2]中国疾病预防控制中心病毒病预防控制所,北京100052 [3]河南化工技师学院,开封475000

出　　处：《中华实验和临床病毒学杂志》2018年第1期75-79,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：山东省自然科学基金（ZR2016CL03）;国家自然科学基金（31400156）;国家重点研发计划（2017YFC1200503）

摘　　要：目的 通过miniSOG蛋白标记技术观察发热伴血小板减少综合征病毒（SFTSV）核蛋白NP在不同时相下的细胞内定位及特点.方法 miniSOG是新发现的能够被电子显微镜解析的蛋白标签.通过PCR方法克隆SFTSV核蛋白NP与miniSOG融合基因NPSOG,将其构建至真核表达载体,转染细胞;之后对不同转染时相的细胞进行原位固定、光氧化、制备超薄切片,用电镜观察细胞内NPSOG蛋白的表达及分布特点.结果 携带NPSOG基因的质粒转染细胞后,核蛋白NP在胞浆及核周表达,并逐渐聚集,连接内质网、高尔基体等细胞器在胞浆中形成体积较大、形状不规则的包涵体样结构,未见其他亚细胞结构改变.结论 SFTSV核蛋白NP单独表达即可形成包涵体,无需其他病毒蛋白协助;包涵体的形成需要一定数量蛋白的定向移动并聚集,这期间可能涉及蛋白与宿主细胞器的相互作用.Objective To study the intracellular location and characteristic of SFTSV NP protein in different phases using mini singlet oxygen generator （miniSOG） labeling technique.Metbods MiniSOG is a recently-invented genetically-encoded tag for EM.MiniSOG-fused SFTSV NP （NPSOG） gene was cloned by PCR,and inserted into pcDNA3.0 plasmid to form pTPL-NPSOG,which was used to transfect 293 cells.The transfected cells of different phases were fixed in 2.5% glutaraldehyde in situ,stained with DAB through the photooxidation activity of miniSOG,and used to prepare ultrathin sections.Intracellular location and characteristic of SFTSV NP protein in different phases were studied by observing the sections under transmission electron microscope.Results After transfecting the plasmid with NPSOG to 293 cells,NP protein was expressed in cytoplasm and peri nucleus,and gradually aggregated,which connected with endoplasmic reticulum and Golgi apparatus to form larger volume and irregular inclusion bodies in cytoplasm.No obvious subcellular structure changes were found.Conclusions The SFTSV nucleoprotein can be expressed separately to form inclusion bodies without the assistance of other viral proteins.The formation of inclusion bodies requires the directional movement and aggregation of a certain number of NP proteins,which may involve the interaction of NP protein and host organelles during this period.

关 键 词：MiniSOG SFTSV 核蛋白 光镜.电镜关联技术 

分 类 号：R512.8[医药卫生—内科学]