HBV人工转录因子的制备及其抑制HBV复制的实验研究

发布时间：2018-03-18 19:47

本文选题：锌指蛋白　切入点：人工转录因子　出处：《重庆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：构建可靶向性识别HBV增强子的锌指蛋白（zinc fingerprotein，ZFP）人工转录因子（Artificial Transcription Factor，ATF）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，通过靶向性识别HBV增强子来抑制HBV DNA复制与表达作用，为探索防治HBV感染的方法提供实验依据。方法：(1) ATF真核表达质粒载体pcDNA3.1-ATF的设计与构建。（2）将pcDNA3.1-ATF转染HepG2细胞，采用RT-PCR、免疫荧光及Western-blot检测ATF mRNA与蛋白的表达；采用CCK-8检测不同浓度的ATF对细胞增殖的影响；应用荧光素酶报告基因系统检测ATF对HBV增强子靶序列的抑制作用。（3）通过pcDNA3.1-ATF转染HepG2.2.15细胞，采用免疫荧光检测ATF在细胞内的表达，同时评价ATF对细胞生长的影响，分别利用荧光定量、Western-blot、RT-PCR方法检测对HBV DNA复制与表达的抑制作用。结果：（1）成功构建pcDNA3.1-ATF真核表达质粒载体。（2）ATF在HepG2细胞中能够正常表达且对细胞增殖生长无明显的毒性作用；双荧光素酶报告基因系统分析ATF能靶向性结合HBV增强子序列，具有抑制报告基因的表达的作用。（3）转染pcDNA3.1-ATF表达质粒于HepG2.2.15细胞，成功验证ATF具有抑制HepG2.2.15细胞内HBVDNA复制与表达的作用，在转染72h后抑制作用达最高。实验结果提示ATF可特异性结合HBV EnhI靶核苷酸序列并表现出相应的生物学活性。结论：通过基因工程的方法设计与构建靶向HBV EnhI核酸序列的真核表达质粒载体pcDNA3.1-ATF，其在细胞内能够正常表达，且对细胞生长无明显细胞毒性作用，，并具有结合HBV EnhI靶序列，抑制HBVDNA复制与表达的生物学作用。
[Abstract]:Objective: to construct the eukaryotic expression vector of zinc finger protein (ZFP), an artificial transcription factor of zinc finger protein (ZFP), which can recognize HBV enhancer, and to detect its expression in eukaryotic cells, and to analyze its effect on cell proliferation and biological activity. The target recognition of HBV enhancer was used to inhibit the replication and expression of HBV DNA, and to provide experimental evidence for the prevention and treatment of HBV infection. Methods: the design and construction of ATF eukaryotic expression plasmid pcDNA3.1-ATF were used to transfect pcDNA3.1-ATF into HepG2 cells. The expression of ATF mRNA and protein was detected by RT-PCR, immunofluorescence and Western-blot, and the effect of ATF at different concentrations on cell proliferation was detected by CCK-8. Luciferase reporter gene system was used to detect the inhibitory effect of ATF on the target sequence of HBV enhancer. HepG2.2.15 cells were transfected with pcDNA3.1-ATF. The expression of ATF in the cells was detected by immunofluorescence, and the effect of ATF on cell growth was evaluated. The inhibition of HBV DNA replication and expression was detected by RT-PCR. Results the eukaryotic expression plasmid vector of pcDNA3.1-ATF was successfully constructed, which could express normally in HepG2 cells and had no obvious toxic effect on cell proliferation and growth. Double luciferase reporter gene system was used to analyze the targeting binding of ATF to HBV enhancer sequence. PcDNA3.1-ATF expression plasmid was transfected into HepG2.2.15 cells. It was proved that ATF could inhibit the replication and expression of HBVDNA in HepG2.2.15 cells. The inhibitory effect was the highest at 72 h after transfection. The results showed that ATF could specifically bind to the target nucleotide sequence of HBV EnhI and exhibit corresponding biological activity. Conclusion: the eukaryotic expression plasmid pcDNA3.1-ATF targeting the nucleic acid sequence of HBV EnhI was designed and constructed by genetic engineering. The plasmid pcDNA3.1-ATF can be expressed normally in cells, and it has no cytotoxic effect on cell growth and has binding to HBV EnhI target sequence. Biological effects of inhibiting HBVDNA replication and expression.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.62

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