HBV人工转录因子的制备及其靶向抑制HBV增强子活性研究

Preparation of artificial transcription factors to HBV and its targeted inhibitory effect on activity of HBV enhancer

目的构建可靶向性识别HBV增强子的锌指蛋白人工转录因子真核表达载体,检测其在真核细胞内的表达、对细胞增殖影响及生物学活性分析。方法应用锌指蛋白(zinc finger protein,ZFP)设计工具Zinc finger tools 3.0获取人工转录因子(artificial transcription factor,ATF)结合结构域并融合抑制性效应结构域KRAB,全基因合成ATF克隆进真核表达载pcDNA3.1(+),酶切、测序来鉴定构建载体的正确性,X-tremeGENE HP转染pcDNA3.1-ATF于HepG2细胞,采用RT-PCR、免疫荧光及Western blot检测ATF mRNA与蛋白的表达情况;CCK-8检测不同浓度的ATF表达载体转染后对细胞增殖的影响;双荧光素酶报告基因检测ATF对HBV增强子序列的靶向性抑制作用。结果成功构建pcDNA3.1-ATF真核表达载体;在HepG2细胞中能够正常表达;CCK-8检测ATF对细胞增殖生长无明显的毒性作用;双荧光素酶报告基因分析ATF能靶向性结合增强子并抑制报告基因的表达。结论通过基因工程的方法构建pcDNA3.1-ATF的真核表达载体可正常表达且无明显细胞毒性作用,可靶向性识别HBV增强子并具有相应的生物学活性。

Objective To prepare a zinc finger protein eukaryotie expression vector of artificial transcription factor targeting HBV enhancer and determine its expression in eukaryotic cells and the effect on cell proliferation and other biological activities. Methods Using the zinc finger protein （ZFP） design tool Zinc finger tool 3.0 to obtain DNA-binding domain of the artificial transcription factors （ATF）, combing with an inhibitory effector structure domain KRAB, cloning total synthesis ATF gene into eukaryotic expression vector pcDNA3.1 （ ＋ ）, confirmed correctness by using enzyme cleavage and sequencing, pcDNA3. 1-ATF were transformed into HepG2 cells by X-tremeGENE HP. The mRNA and protein expression of ATF were detected by RT-PCR, immunofluorescence staining and Western blotting. The cell proliferation was tested by CCK-8 kit by using transfection of ATF expression vector at different concentrations. Dual luciferase report gene was used to detect whether ATF recognizing HBV sequence and exerted eukaryotic expression vector was successfully constructed and targeted inhibition. Results pcDNA3.1-ATF expressed in HepG2 cells. CCK-8 assay indicated that ATF had no obvious toxic effect on cell proliferation. Dual luciferase report gene analysis specifically combined with enhancer and then inhibited report gene expression. Conclusion eukaryotic expression vector can normally express and have no cytotoxic effect in HepG2 engineering, which can target HBV enhancer and have corresponding biological activities. showed that ATF pcDNA3.1-ATF cells by genetic