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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。
OBJECTIVE: To design a zinc finger protein (ZFP) that specifically binds to hepatitis B virus core promoter (HBVCp), and to observe the inhibitory effect of ZFP on HBV transcription in vitro. METHODS: Zinc finger specifically binding to HBV Cp was designed using Zinc finger tools. The ZFP nucleotide sequence was synthesized and then cloned into pEGFP-N1 and pcDNA3.1 (+) to construct eukaryotic expression plasmids pEGFP-N1 / ZFP-Flag and pcDNA3.1 (+) / ZFP. The expression of pEGFP-N1 / ZFP-Flag in COS-7 was identified by inverted fluorescence microscope, RT-PCR and Western blot. The expression of pEGFP-N1 / The contents of HBeAg and HBV DNA in the supernatant were detected by FQ-PCR and the levels of HBV mRNA in the cells were detected by RT-PCR. Results: ZFP was normally expressed in COS-7 cells. Compared with the empty plasmid group, the amount of HBV DNA and the content of HBeAg in the cell culture supernatant of ZFP group were significantly decreased (P <0.05), and the intracellular HBV mRNA was also significantly decreased. Conclusion: Artificial ZFP can be expressed normally in eukaryotic cells and can effectively inhibit the transcription of HBV.