表达载脂蛋白E-增强型绿色荧光蛋白的细胞系支持感染性丙型肝炎病毒的组装  

Apolipoprotein E-EGFP-expressing cell lines supporting assembly of infectious hepatitis C virus particles

作　　者：杨再立[1,2] 赵凡凡[2] 王刚[1] 龙钢[2] YANG Zai-li ZHAO Fan-fan WANG Gang LONG Gang(College of Life Science, Shanghai University, Shanghai 201900,China Institute Pasteur of Shanghai,Chinese Academy of Sciences,Shanghai 200031,China)

机构地区：[1]上海大学生命科学学院,上海201900 [2]中国科学院上海巴斯德研究所,上海200031

出　　处：《第二军医大学学报》2016年第12期1470-1474,共5页Academic Journal of Second Military Medical University

基　　金：国家重点基础研究发展规划项目(2015CB554301)~~

摘　　要：目的探讨表达载脂蛋白E-增强型绿色荧光蛋白(apolipoprotein E-enhanced green fluorescence protein,apoEEGFP)的细胞系是否支持感染性丙型肝炎病毒(HCV)颗粒的组装。方法利用shRNA基因沉默技术建立apoE稳定下调的Huh7.5.1细胞系,然后通过基因工程技术在该细胞系中建立稳定表达apoE-EGFP融合蛋白的细胞系。将HCV RNA转染进入野生型细胞(Huh7.5.1细胞)、对照组sh-NT细胞(转导非靶向野生型细胞基因的shRNA质粒的细胞)、apoE下调的Huh7.5.1细胞(sh-apoE细胞)以及表达apoE-EGFP融合蛋白的sh-apoE细胞(apoE-EGFP细胞)中,收集病毒液;通过半数组织培养感染剂量(TCID50)方法检测释放到Huh7.5.1、sh-NT、sh-apoE和apoE-EGFP细胞培养上清液中的HCV的滴度。利用免疫荧光技术检测apoE与HCV的结构蛋白E2的相互作用,利用蛋白质印迹法检测用具有特异亲和性FLAG-gel纯化的HCV颗粒表面的apoE-EGFP的表达。结果 apoE-EGFP融合蛋白在apoE-EGFP细胞系中高效表达;apoE-EGFP细胞来源的HCV的感染性与Huh7.5.1、sh-NT细胞相比差异无统计意义;在apoE-EGFP细胞系中,apoE-EGFP融合蛋白与HCV结构蛋白E2存在共定位,并且可以在HCV颗粒表面上检测到apoE-EGFP融合蛋白。结论 apoE-EGFP融合蛋白是HCV颗粒的组分,apoE-EGFP细胞系支持感染性HCV颗粒的组装。Objective To investigate whether cell lines expressing apolipoprotein E-enhanced green fluorescence protein （apoE-EGFP） can support assembly of infectious hepatitis C virus particles. Methods apoE stably down-regulated Huh7. 5. 1 cell lines （sh-apoE cell lines） were established by shRNA gene silencing technique, and cell lines expressing apoE-EGFP fusion protein （apoE-EGFP cell lines） were established. The culture supernatants of wild-type Huh7. 5. 1 cells, control cells （sh-NT cells）,sh-spoE cells and apoE-EGFP cells transfected with HCV RNA were collected and the HCV titer of supernatants was determined by TCID50 . The interaction of apoE with HCV structure protein E2 was examined by immunofluorescence and confocal microscopy, and the expression of apoE-EGFP on the surface of HCV particles purified by FLAG-specific affinity gel was analyzed by Western blotting analysis. Results The apoE-EGFP fusion protein was highly expressed in sh-apoE cell lines. The infectivity of HCV from apoE-EGFP cell culture supernatant was not significantly different with those of HCV from Huh7. 5. 1 and sh-NT cells. apoE-EGFP infused protein had fluorescent co-location with HCV structural protein E2, and was detected on the surface of HCV particles purified by FLAG-specific affinity gel. Conclusion The apoE-EGFP fusion protein is an important component of HCV particles and apoE-EGFP cell lines can support the assembly of HCV particles.

关 键 词：丙型肝炎病毒 载脂蛋白E 绿色荧光蛋白质类 细胞系 

分 类 号：R373.21[医药卫生—病原生物学]