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
发布时间：2018-10-05 11:08
【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。
[Abstract]:Objective to investigate whether the cell lines expressing apolipoprotein E-enhanced green fluorescent protein (apolipoprotein E-enhanced green fluorescence protein,apoEEGFP) support the assembly of infectious hepatitis C virus (HCV) particles. Methods shRNA gene silencing technique was used to establish a stable down-regulated Huh7.5.1 cell line of apoE, and then a cell line stably expressing apoE-EGFP fusion protein was established by genetic engineering technique. HCV RNA was transfected into wild-type cells (Huh7.5.1 cells), control sh-NT cells (shRNA plasmids transducted non-target wild-type cells) Huh7.5.1 cells (sh-apoE cells) and sh-apoE cells expressing apoE-EGFP fusion protein (apoE-EGFP cells). The viral solution was collected and the titer of HCV released into the supernatant of Huh7.5.1,sh-NT,sh-apoE and apoE-EGFP cells was detected by half of the tissue culture infection dose (TCID50) method. The interaction between apoE and HCV structural protein E2 was detected by immunofluorescence technique, and the expression of apoE-EGFP on the surface of HCV particles purified by specific affinity FLAG-gel was detected by Western blot. Results there was no statistical significance between the infection of HCV derived from apoE-EGFP fusion protein and that of Huh7.5.1,sh-NT cells in apoE-EGFP cell line, and the co-localization of HCV fusion protein and HCV structural protein E2 in apoE-EGFP cell line. ApoE-EGFP fusion protein can be detected on the surface of HCV particles. Conclusion apoE-EGFP fusion protein is a component of HCV particles, which supports the assembly of infective HCV particles in apoE-EGFP cell line.
【作者单位】： 上海大学生命科学学院;中国科学院上海巴斯德研究所;
【基金】：国家重点基础研究发展规划项目(2015CB554301)~~
【分类号】：R512.63
本文编号：2253151
[Abstract]:Objective to investigate whether the cell lines expressing apolipoprotein E-enhanced green fluorescent protein (apolipoprotein E-enhanced green fluorescence protein,apoEEGFP) support the assembly of infectious hepatitis C virus (HCV) particles. Methods shRNA gene silencing technique was used to establish a stable down-regulated Huh7.5.1 cell line of apoE, and then a cell line stably expressing apoE-EGFP fusion protein was established by genetic engineering technique. HCV RNA was transfected into wild-type cells (Huh7.5.1 cells), control sh-NT cells (shRNA plasmids transducted non-target wild-type cells) Huh7.5.1 cells (sh-apoE cells) and sh-apoE cells expressing apoE-EGFP fusion protein (apoE-EGFP cells). The viral solution was collected and the titer of HCV released into the supernatant of Huh7.5.1,sh-NT,sh-apoE and apoE-EGFP cells was detected by half of the tissue culture infection dose (TCID50) method. The interaction between apoE and HCV structural protein E2 was detected by immunofluorescence technique, and the expression of apoE-EGFP on the surface of HCV particles purified by specific affinity FLAG-gel was detected by Western blot. Results there was no statistical significance between the infection of HCV derived from apoE-EGFP fusion protein and that of Huh7.5.1,sh-NT cells in apoE-EGFP cell line, and the co-localization of HCV fusion protein and HCV structural protein E2 in apoE-EGFP cell line. ApoE-EGFP fusion protein can be detected on the surface of HCV particles. Conclusion apoE-EGFP fusion protein is a component of HCV particles, which supports the assembly of infective HCV particles in apoE-EGFP cell line.
【作者单位】： 上海大学生命科学学院;中国科学院上海巴斯德研究所;
【基金】：国家重点基础研究发展规划项目(2015CB554301)~~
【分类号】：R512.63
【相似文献】
相关期刊论文 前6条
1 曹尚,邵一鸣,蒋岩,范秀娟,潘品良,姚均,冯毅,邢辉;M嗜性HIV-1 毒株一过性感染T细胞系的研究[J];中华实验和临床病毒学杂志;1999年02期
2 洪世雯;;V_8蛋白酶诱发HBV感染HepG2细胞系[J];传染病信息;1997年02期
3 陈智;HTLV-1感染的细胞系MT-2对HCV的易感性[J];国外医学(流行病学传染病学分册);1995年03期
4 吴衍恒;谢颖;刘洪波;师舞阳;;C6/36细胞系分离登革1型病毒的方法学探讨[J];中国热带医学;2013年05期
5 吴还梅;孙俊;孟哲峰;张晓燕;徐建青;;HIV-1感染上调细胞系中泛素样蛋白ISG15表达[J];病毒学报;2013年05期
6 ;[J];;年期
，本文编号：2253151
本文链接：https://www.wllwen.com/yixuelunwen/xiaohjib/2253151.html
最近更新
教材专著
