Construction of the tetracycline regulating expression system of HBV-S gene and its expression in eukaryotic cells

LEI Yu; et al

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Construction of the tetracycline regulating expression system of HBV-S gene and its expression in eukaryotic cells

更新时间：2024-01-17

- Construction of the tetracycline regulating expression system of HBV-S gene and its expression in eukaryotic cells
- Journal of Chongqing Medical University Issue 10, Pages: 1020-1022(2007)
- 作者机构：
  
  1. 重庆医科大学附属第二医院肝病治疗研究中心
  2. 重庆医科大学附属第二医院肝病治疗研究中心,重庆,400010
- 作者简介：
- 基金信息：
- DOI：
  CLC： R373
- Published：2007，
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[1]雷宇,黎燕,王蜀强,陈压西,周智,任红.乙型肝炎病毒S基因四环素调控体系的构建及其在真核细胞中的表达[J].重庆医科大学学报,2007(10):1020-1022.

LEI Yu, et al. Construction of the tetracycline regulating expression system of HBV-S gene and its expression in eukaryotic cells. [J]. Journal of Chongqing Medical University (10):1020-1022(2007)
[1]雷宇,黎燕,王蜀强,陈压西,周智,任红.乙型肝炎病毒S基因四环素调控体系的构建及其在真核细胞中的表达[J].重庆医科大学学报,2007(10):1020-1022. DOI：

LEI Yu, et al. Construction of the tetracycline regulating expression system of HBV-S gene and its expression in eukaryotic cells. [J]. Journal of Chongqing Medical University (10):1020-1022(2007) DOI：

摘要

目的:构建乙型肝炎病毒S基因的四环素调控表达载体

并研究在真核细胞中四环素对S基因的调控表达

以期用于基因治疗实验。方法:将乙型肝炎病毒S基因插入启动子内含有2个四环素超纵子（TO）的质粒pcDNA4

并用脂质体将重组质粒pcDNA4-S和含有四环素抑制基因的质粒pcDNA6共转染入真核细胞HepG2中

用Zeocin（400μg/ml）及Blasticidin（10μg/ml）进行抗性筛选

约20天形成抗性克隆

建立在四环素诱导下能稳定表达S基因的细胞系。以不同浓度的四环素（0.5μg/ml

1.0μg/ml

2.0μg/ml

3.0μg/ml）进行诱导

用微粒子酶免疫分析法（MEIA）检测S抗原的表达

以确定四环素的最佳浓度。结果:转染了重组质粒的HepG2细胞

在无四环素环境下HBsAg表达量很低

当加入不同浓度的四环素诱导后

HBsAg表达量明显增加

但所测HBsAg的S/N值无明显差异。结论:成功构建了乙型肝炎病毒S基因四环素调控表达载体

并在真核细胞中进行了四环素的调控表达。

Abstract

Objective:To construct tetracycline regulating expression system of HBV-S gene

and study its regulating expression of HBVAg by tetracycline in HepG 2 cells. Methods:Insert HBs gene into the multiple cloning site of pcDNA4

which contain tetracycline operator in the promoter

and the constructed vector with pcDNA6 was co-transferred into HepG2 by means of lipofectamine

and use 400μg/ml Zeocin and 10μg/ml Blasticidin to kill the untransfected host cell line. After 20 days’select

the cell line which express HBsAg stablely after induced by tetracycline was generated. Then the HBsAg expressing was induced by varying concentration of tetracycline(0.5μg/ml

1.0μg/ml

2.0μg/ml

3.0μg/ml).Amount of HBsAg was detected by means of MEIA (Microparticle Enzyme Immunoassay) to determine the appropriate concentration of tetracycline. Result:HBsAg had been expressed at low quantity without tetracycline in transfected HepG2. However the S/N of HBsAg were high but not different after induced by varying concentration of tetracycline. Conclusion:Tetracycline regulating expression system of HBV-S gene was constructed successfully and HBsAg was highly expressed in the vector induced by tetracycline.

关键词

乙肝病毒四环素调控表达

Keywords

hepatitis B virustetracyclineRegulating expression

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Related Institution

Department of infectiaus,the Second Affiliated Hospital,Chongqing Medical University

Institute of Hepatiecs Viral,Chongqing Medical University

Department of Infectious,the Second Affiliated Hospital,Chongqing Medical University

Department of Diseases Therapy Center,the Second Affiliated Hospital,Chongqing Medical Univesity

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