Construction of the tetracycline regulated expression vector of HBV-S gene and its expression in eukaryotic cells

LIANG Li1; LEI Yu1; NI Yan1; WANG Shu-qiang1; PENG Feng-ying2; ZHOU Zhi1; CHEN Ya-xi1; REN Hong1

doi:10.13406/j.cnki.cyxb.2011.07.013

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Construction of the tetracycline regulated expression vector of HBV-S gene and its expression in eukaryotic cells

更新时间：2024-01-17

- Construction of the tetracycline regulated expression vector of HBV-S gene and its expression in eukaryotic cells
- Journal of Chongqing Medical University Vol. 36, Issue 7, Pages: 833-836(2011)
- 作者机构：
  
  1. 重庆医科大学附属第二医院感染病科
  2. 重庆医科大学病毒性肝炎研究所
  3. 重庆医科大学附属第一医院传染科
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.2011.07.013
  CLC： R512.62
- Published：2011，
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[1]梁丽,雷宇,倪艳,王蜀强,彭凤英,周智,陈压西,任红.HBV-S基因四环素调控双表达载体的构建及其调控表达研究[J].重庆医科大学学报,2011,36(07):833-836.

LIANG Li1, LEI Yu1, NI Yan1, et al. Construction of the tetracycline regulated expression vector of HBV-S gene and its expression in eukaryotic cells. [J]. Journal of Chongqing Medical University 36(7):833-836(2011)
[1]梁丽,雷宇,倪艳,王蜀强,彭凤英,周智,陈压西,任红.HBV-S基因四环素调控双表达载体的构建及其调控表达研究[J].重庆医科大学学报,2011,36(07):833-836. DOI： 10.13406/j.cnki.cyxb.2011.07.013.

LIANG Li1, LEI Yu1, NI Yan1, et al. Construction of the tetracycline regulated expression vector of HBV-S gene and its expression in eukaryotic cells. [J]. Journal of Chongqing Medical University 36(7):833-836(2011) DOI： 10.13406/j.cnki.cyxb.2011.07.013.

摘要

目的:构建在同一载体中含乙肝病毒表面抗原基因（Hepatitis B virus surface gene

HBV-S）和四环素阻抑蛋白基因的双表达载体

研究在真核细胞中四环素对S基因表达的调控。方法:在双表达载体pVITRO3一个启动子后的多克隆位点插入四环素阻抑蛋白基因

用含四环素操纵子的启动子取代另一个启动子

并在其多克隆位点插入S基因

在同一载体中构建成S基因的四环素调控表达系统。用脂质体将重组质粒转染HepG2细胞

潮霉素400μg/ml进行抗性筛选

20 d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（0.5、1.0、2.0、3.0μg/ml）进行诱导

用逆转录PCR和微粒子酶免疫分析法检测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

表达HBsAg水平极低

72 h时S/N值平均为1.57

在不同浓度的四环素诱导下

HBsAg表达量明显增加

72 h时S/N平均值分别为4.24、4.23、4.18、4.20

未发现与四环素的浓度之间有剂量关系。结论:成功构建了在同一质粒中表达HBV-S基因和四环素阻抑蛋白基因的双表达调控载体

在不同浓度四环素诱导下均有HBsAg表达

并随时间延长而表达量增高

但在同一时间点无明显差异。

Abstract

Objective: To construct a co-expression vector which expresses both HBsAg and Tet Repressor protein and in which the expression of HBsAg can be regulated by tetracycline

and to study the regulating role of tetracycline in the HBV-S expression in HepG 2 cell.Methods:Tetracycline repressor(TetR)gene was insert into the first multiple cloning site

which was just after the first promotor in pVITRO3

a co-expression vector

then the second promotor was partly replaced by another one which contained tetracycline operator.Then

HBs gene was inserted into the second multiple cloning site.The constructed vector was transfected into HepG2 by means of lipofectamine.And 400 μg/ml Hygromycin B was used to kill the untransfected host cell line.After 20 days selection

the cell line which expressed HBsAg stably after being induced by tetracycline was generated.Then the HBsAg expression was induced by various concentrations of tetracycline(0.5

1.0

2.0

3.0 μg/ml).And HBsAg was detected by means of RT-PCR and MEIA(Microparticle Enzyme Immunoassay)to determine the appropriate concentration of tetracycline.Results:HBsAg was expressed little without tetracycline in transfected HepG2.The average S/N in 72 hour was 1.57.And the S/N of HBsAg was increased remarkably but had no difference after being induced by various concentrations of tetracycline.The average S/N in 72 hour was 4.24

4.23

4.18 and 4.20 respectively.Conclusion:The basal expression was very low in the tetracycline regulated expression vector of HBV-S gene without inducer.After being induced by various concentrations of tetracycline

HBsAg expression was sharply increased with the incubation time prolongation

but the expression quantity showed no difference at the same time point.

关键词

乙肝病毒四环素调控表达

Keywords

hepatitis B virustetracyclineregulated expression

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