本文選題：鋅指蛋白　切入點：人工轉(zhuǎn)錄因子　出處：《重慶醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：構(gòu)建可靶向性識別HBV增強子的鋅指蛋白（zinc fingerprotein，ZFP）人工轉(zhuǎn)錄因子（Artificial Transcription Factor，ATF）真核表達載體，檢測其在真核細胞內(nèi)的表達、對細胞增殖影響及生物學(xué)活性分析，通過靶向性識別HBV增強子來抑制HBV DNA復(fù)制與表達作用，為探索防治HBV感染的方法提供實驗依據(jù)。 方法：(1) ATF真核表達質(zhì)粒載體pcDNA3.1-ATF的設(shè)計與構(gòu)建。（2）將pcDNA3.1-ATF轉(zhuǎn)染HepG2細胞，采用RT-PCR、免疫熒光及Western-blot檢測ATF mRNA與蛋白的表達；采用CCK-8檢測不同濃度的ATF對細胞增殖的影響；應(yīng)用熒光素酶報告基因系統(tǒng)檢測ATF對HBV增強子靶序列的抑制作用。（3）通過pcDNA3.1-ATF轉(zhuǎn)染HepG2.2.15細胞，采用免疫熒光檢測ATF在細胞內(nèi)的表達，同時評價ATF對細胞生長的影響，分別利用熒光定量、Western-blot、RT-PCR方法檢測對HBV DNA復(fù)制與表達的抑制作用。 結(jié)果：（1）成功構(gòu)建pcDNA3.1-ATF真核表達質(zhì)粒載體。（2）ATF在HepG2細胞中能夠正常表達且對細胞增殖生長無明顯的毒性作用；雙熒光素酶報告基因系統(tǒng)分析ATF能靶向性結(jié)合HBV增強子序列，具有抑制報告基因的表達的作用。（3）轉(zhuǎn)染pcDNA3.1-ATF表達質(zhì)粒于HepG2.2.15細胞，成功驗證ATF具有抑制HepG2.2.15細胞內(nèi)HBVDNA復(fù)制與表達的作用，在轉(zhuǎn)染72h后抑制作用達最高。實驗結(jié)果提示ATF可特異性結(jié)合HBV EnhI靶核苷酸序列并表現(xiàn)出相應(yīng)的生物學(xué)活性。 結(jié)論：通過基因工程的方法設(shè)計與構(gòu)建靶向HBV EnhI核酸序列的真核表達質(zhì)粒載體pcDNA3.1-ATF，其在細胞內(nèi)能夠正常表達，且對細胞生長無明顯細胞毒性作用，，并具有結(jié)合HBV EnhI靶序列，抑制HBVDNA復(fù)制與表達的生物學(xué)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R512.62

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