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目的设计与构建特异性结合乙型肝炎病毒增强子1(HBV Enh1)的锌指蛋白人工转录因子(ZFP-ATF),检测在Hep G2.2.15细胞内的表达、对细胞生长影响及观察其抑制HBV DNA复制与表达的作用。方法构建含结合结构域ZFP与抑制性效应结构域Krüppel相关盒(KRAB)的人工转录因子(ATF),进行相关修饰、优化后,将人工合成ATF核酸序列插入真核表达质粒载体pc DNA3.1(~+),测序鉴定其正确性,X-treme GENE HP转染Hep G2.2.15细胞,共聚焦显微镜下观察ATF蛋白表达情况,CCK-8法检测ATF对细胞生长的影响,ATF转染24、48、72 h后,采用实时定量PCR、ELISA、Western blot法检测对HBV DNA复制与表达的抑制作用。结果成功构建pc DNA3.1-ATF(nls-ZFP-KRAB-FLAG)真核表达载体,ATF在Hep G2.2.15细胞中能正常表达,且对细胞生长无明显的影响,ATF具有抑制HBV DNA复制与表达的作用,在转染72 h后抑制作用达最高,抑制率达68.0%。结论构建的ATF在Hep G2.2.15细胞中能够正常表达且无毒性作用,可特异性结合HBV Enh1靶序列并具有抑制HBV DNA复制与表达的作用。
Objective To design and construct Zinc finger protein artificial transcription factor (ZFP-ATF) that specifically binds to HBV Enh1 and detect its expression in Hep G2.2.15 cells, and to observe its effect on cell growth HBV DNA replication and expression. Methods The artificial transcription factor (ATF) containing the binding domain ZFP and KRAB of the inhibitory effector domain was constructed and modified. After the optimization, the synthetic ATF nucleic acid sequence was inserted into the eukaryotic expression plasmid pcDNA3.1 (~ +). Sequencing was used to identify the correctness of ATF protein expression. X-treme GENE HP was transfected into Hep G2.2.15 cells. The ATF protein expression was observed by confocal microscopy. The effect of ATF on cell growth was detected by CCK-8 assay. After 48 and 72 h, the inhibition of HBV DNA replication and expression was detected by real-time PCR, ELISA and Western blot. Results The eukaryotic expression vector pcDNA3.1-ATF (nls-ZFP-KRAB-FLAG) was successfully constructed. ATF could be expressed normally in Hep G2.2.15 cells without any significant effect on cell growth. ATF inhibited HBV DNA replication With the effect of expression, the inhibition reached the maximum at 72 h after transfection, with the inhibition rate of 68.0%. CONCLUSION: ATF can be expressed normally and non-toxic in Hep G2.2.15 cells. It can specifically bind HBV Enh1 target sequence and inhibit HBV DNA replication and expression.