| 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，apoE-EGFP）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。 |
| 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 |
| DOI：10.16781/j.0258-879x.2016.12.1470 |
| 投稿时间：2016-04-24修订日期：2016-06-07 |
| 基金项目:国家重点基础研究发展规划项目（2015CB554301）. |
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| Apolipoprotein E-EGFP-expressing cell lines supporting assembly of infectious hepatitis C virus particles |
| YANG Zai-li1\2,ZHAO Fan-fan2,WANG Gang1,LONG Gang2* |
(1. College of Life Science, Shanghai University, Shanghai 201900, China;
2. Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China
* Corresponding author) |
| Abstract: |
| 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. |
| Key words: hepatitis C virus apolipoprotein E green fluorescence proteins cell lines |
