| 저자(한글) |
Li, Zhongtang,Luo, Wei,Fu, Yu,Jiang, Qinghu,Liu, Jiming,Wu, Zhongjun |
| 초록 |
Objective To construct a zinc finger protein-artificial transcription factor (ZFP-ATF) specifically bound to hepatitis B virus (HBV) enhancer 1 (Enh1), then detect its expression in HepG2.2.15 cells, observe its effect on cell growth and its role in inhibiting the replication and expression of HBV DNA. Methods The zinc finger protein (ZFP) specifically bound to HBV Enh1 was fused with a Krvppel-associated box (KRAB) transcriptional repression domain to produce an artificial transcription factor (ATF). After relative modification and optimization, the synthetic ATF nucleic acid sequence was inserted into the eukaryotic expression plasmid vector pcDNA3.1(+) and its sequence was identified. Then pcDNA3.1-ATF was transfected into HepG2.2.15 cells through X-tremeGENE HP. The expression of ATF was detected by confocal microscope. The impact of ATF on cell growth was measured by CCK-8 assay. The inhibitory effect on HBV DNA by ATF was assured by real-time quantitative PCR (qRT-PCR), ELISA and Western blotting at 24, 48 and 72 hours after transfection. Results The pcDNA3.1-ATF (nls-ZFP-KRAB-FLAG) eukaryotic expression plasmid vector was successfully constructed. ATF was expressed in HepG2.2.15 cells as expected without obvious influence on cell growth. ATF repressed the replication and expression of HBV DNA, especially at 72 hours post-transfection when the inhibition rate was 68%. Conclusion The synthetic ATF can be expressed in HepG2.2.15 cells normally without toxic effect on the cells and exert an inhibitory effect on the replication and expression of HBV DNA by specifically bound to HBV Enh1. |
