[设计用于乙肝病毒核心启动子的锌指蛋白抑制HepG2.2.15细胞中HBV的转录]

[Zinc finger protein designed to hepatitis B virus core promoter inhibit the transcription of HBV in HepG2.2.15 cells].

作者信息

Chen Cong, Li Xin, Guo Ning, Zhang Hua, Wu Zhong-jun

机构信息

Department of Hepatobiliary Surgery, Chongqing Medical University, Chongqing, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2012 Feb;28(2):160-2.

PMID:22304776

Abstract

AIM

To observe the inhibition of hepatitis b virus(HBV) transcription in vitro by zinc fingrer protein(ZFP) which was designed specifically to bind DNA sequence in the HBV core promoter(Cp).

METHODS

The sequence of HBV Cp was submitted to the Zinc Finger Tools and selected a sequence from the Cp as the target site of ZFP. The nucleic acid sequence of ZFP was synthesised and cloned into pEGFP-N1 or pcDNA3.1(+) to construct the recombinant plasmid pEGFP-N1/ZFP-Flag or pcDNA3.1(+)/ZFP. COS-7 cells were transfected with pEGFP-N1 or pEGFP-N1/ZFP-Flag, the expression of ZFP was observed by green fluorescent microscope, RT-PCR and Western blot. HepG2.2.15 cells were transfected with pcDNA3.1(+) or pcDNA3.1(+)/ZFP, HBeAg and HBV DNA levels in cell supernatant were detected by ELISA and FQ-PCR, HBV mRNA was tested by RT-PCR.

RESULTS

ZFP can express in COS-7 cells.In th presence of ZFP, HBeAg expression and HBV DNA level was significantly reduce(P<0.05), and HBV mRNA was dramatically decreased.

CONCLUSION

ZFP can express in eukaryotic cells and inhibit the transcription of HBV in vitro.

摘要

目的

观察特异性设计用于结合乙肝病毒（HBV）核心启动子（Cp）中DNA序列的锌指蛋白（ZFP）在体外对HBV转录的抑制作用。

方法

将HBV Cp序列提交至锌指工具，从Cp中选择一个序列作为ZFP的靶位点。合成ZFP的核酸序列并克隆到pEGFP-N1或pcDNA3.1(+)中，构建重组质粒pEGFP-N1/ZFP-Flag或pcDNA3.1(+)/ZFP。用pEGFP-N1或pEGFP-N1/ZFP-Flag转染COS-7细胞，通过绿色荧光显微镜、RT-PCR和蛋白质免疫印迹法观察ZFP的表达。用pcDNA3.1(+)或pcDNA3.1(+)/ZFP转染HepG2.2.15细胞，通过ELISA和荧光定量PCR检测细胞上清液中的HBeAg和HBV DNA水平，用RT-PCR检测HBV mRNA。