设计特异性识别HBV核心启动子的锌指蛋白以抑制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

目的:设计特异性结合乙肝病毒核心启动子(HBVCp)的锌指蛋白(ZFP),观察ZFP在体外对HBV转录的抑制情况。方法:利用Zinc finger tools设计特异性结合HBV Cp的ZFP。将ZFP核苷酸序列人工合成后克隆至pEGFP-N1和pcDNA3.1(+),构建真核表达质粒pEGFP-N1/ZFP-Flag和pcDNA3.1(+)/ZFP。通过倒置荧光显微镜、RT-PCR和Western blot鉴定pEGFP-N1/ZFP-Flag在COS-7中的表达情况;将pcDNA3.1(+)/ZFP转入HepG2.2.15细胞后24 h,采用ELISA和FQ-PCR检测上清液中HBeAg和HBV DNA含量,RT-PCR检测细胞内HBV mRNA水平。结果:ZFP在COS-7细胞中能正常表达。与空质粒组相比,ZFP组细胞培养上清液中HBV DNA拷贝量和HBeAg含量明显降低(P<0.05),细胞内HBV mRNA也显著减少。结论:人工设计的ZFP可以在真核细胞内正常表达,并能有效抑制HBV的转录。

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 expresstion 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（P0.05）,and HBV mRNA was dramatically decreased.CONCLUSION： ZFP can express in eukaryotic cells and inhibit the transcription of HBV in vitro.