高效分离1型单纯疱疹病毒感染细胞外泌体的研究  

Optimized exosome isolation protocol for herpes simplex virus type 1 infected cells

作　　者：张莉 王娇[1] 王湛[1] 李颖[1] 王慧[1] 宋敬东[2] 刘宏图 Zhang Li;Wang Jiao;Wang Zhan;Li Ying;Wang Hui;Song Jingdong;Liu Hongtu(National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;State Key Laboratory for Infectious Disease Prevention and Control,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Center for Biosafety Mega-Science,Chinese Academy of Science,Wuhan 430071,China;Chinese Center for Disease Control and Prevention-Wuhan Institute of Virology,Chinese Academy of Sciences Joint Research Center for Emerging Infectious Diseases and Biosafety,Wuhan 430071,China;Key Laboratory of Medical and Viral Diseases of the Ministry of Health,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京102206 [2]中国疾病预防控制中心病毒病预防控制所,传染病预防控制国家重点实验室,北京102206 [3]中国科学院生物安全大科学研究中心,武汉430071 [4]中国疾病预防控制中心-中国科学院武汉研究所新发传染病与生物安全联合研究中心,武汉430071 [5]中国疾病预防控制中心病毒病预防控制所,卫生部医学病毒和病毒病重点实验室,北京102206

出　　处：《中华实验和临床病毒学杂志》2021年第6期619-625,共7页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的建立并优化1型单纯疱疹病毒(herpes simplex virus type 1,HSV-1)感染HEp-2细胞外泌体的分离方案。方法收集HSV-1感染HEp-2的细胞培养上清,经差速离心和滤膜过滤对上清液进行预处理,使用外泌体提取试剂(total exosome isolation,TEI)结合尺寸排除色谱法(size-exclusion chromatography,SEC)的组合方案对粗提的外泌体进行分离和纯化,最后经超滤浓缩,获得高纯度外泌体。使用3种检测方法对纯化的外泌体进行表征鉴定。结果Western blot结果显示,HSV-1感染或未感染HEp-2细胞外泌体均含有外泌体标志蛋白TSG101和CD9,另外,HSV-1感染细胞外泌体含有gB、VP16和ICP5等3种病毒蛋白。透射电镜观察显示,纯化的外泌体近似圆形,具有膜结构,直径50~150 nm,未发现典型HSV-1病毒颗粒。NTA结果显示,纯化的外泌体峰值粒径均为112.2 nm;单纯HEp-2细胞来源的粒子峰值浓度为3.4×10^(9)个粒子/ml,HSV-1感染细胞来源的粒子峰值浓度为4.1×10^(9)个粒子/ml。结论本研究建立了高效优化的HSV-1感染细胞外泌体分离纯化方案。Objective To establish and optimize the isolation scheme of exosomes from herpes simplex virus type 1(HSV-1)infected HEp-2 cells.Methods The supernatant of HSV-1 infected HEp-2 cells were collected and pretreated with differential centrifugation and filtration,then purified by total exosome isolation(TEI)in combination with size-exclusion chromatography(SEC);and enriched the exosome with ultrafiltration.Western blotting,transmission electron microscopy(TEM)and nanoparticle tracking analysis(NTA)were performed to characterize the purified exosomes.Results The purified exosomes from HEp-2 cells with or without HSV-1 infection showed positive signals of exosome-specific markers(TSG101 and CD9),and the expression of virus protein gB,VP16 and ICP5 were detected in exosomes derived from HSV-1 infected cells.The purified exosomes from HEp-2 cells with or without HSV-1 infection showed a typical round-shaped morphology under TEM,with a mean diameter of 50~150 nm;meanwhile,no typical HSV-1 virus particles were detectable.The peak diameter of purified exosomes was 112.2 nm,furthermore,the peak concentration of exosomes from HEp-2 control cells was 3.4×10^(9) particles/ml,while the peak concentration of exosomes from HSV-1 infected cells was 4.1×10^(9) particles/ml.Conclusions We accomplished an efficient and optimized approach for the isolation and purification of exosomes from HSV-1 infected cells.

关 键 词：1型单纯疱疹病毒 HEP-2细胞 外泌体 分离 纯化 

分 类 号：R373.11[医药卫生—病原生物学]