本文關(guān)鍵詞： 乙肝病毒 X基因啟動(dòng)子 鋅指蛋白 HepG2.2.15細(xì)胞　出處：《重慶醫(yī)科大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：構(gòu)建人工鋅指蛋白(zinc finger protein, ZFP)特異性結(jié)合于乙肝病毒X蛋白(hepatitis B virus X protein, HBX)基因啟動(dòng)子，觀察人工鋅指蛋白在體外對(duì)乙型肝炎病毒(hepatitis B virus, HBV)復(fù)制和轉(zhuǎn)錄的抑制作用。 方法：用脂質(zhì)體LipofectamineTM2000將重組質(zhì)粒pcDNA3.1-ZFP或pcDNA3.1-NC分別轉(zhuǎn)染HepG2.2.15細(xì)胞分別作為實(shí)驗(yàn)組（ZFP組）和對(duì)照組（NC組）。 1.用免疫印跡法（Western blot）檢測(cè)實(shí)驗(yàn)組和對(duì)照組細(xì)胞中HBX蛋白的含量。 2.用酶聯(lián)免疫吸附測(cè)定法（ELISA）檢測(cè)兩組上清液中乙型肝炎核心相關(guān)抗原(hepatitis B e antigen, HBeAg)水平。 3.用實(shí)時(shí)熒光定量PCR（qPCR）檢測(cè)兩組上清液中HBV DNA含量。 4.用RT-PCR檢測(cè)實(shí)驗(yàn)組和對(duì)照組細(xì)胞內(nèi)HBV mRNA水平。 結(jié)果：重組質(zhì)粒轉(zhuǎn)染入HepG2.2.15細(xì)胞后； 1.Western blot檢測(cè)所得的HepG2.2.15細(xì)胞中HBX蛋白表達(dá)水平，相比對(duì)照組，實(shí)驗(yàn)組中HBX蛋白表達(dá)水平明顯下降，，差異具有統(tǒng)計(jì)學(xué)意義。 2.相比對(duì)照組，ZFP組細(xì)胞培養(yǎng)上清液中HBeAg含量下降了33.8%（t=3.887,P=0.003）。 3.ZFP組細(xì)胞培養(yǎng)上清液中HBV DNA拷貝量相對(duì)NC組下降了51.7%（t=23.079,P=0.000）。 4.測(cè)得實(shí)驗(yàn)組細(xì)胞內(nèi)HBV mRNA水平，與對(duì)照組相比，明顯減少。 結(jié)論：經(jīng)過(guò)上述研究證明我團(tuán)隊(duì)前期人工設(shè)計(jì)的可特異性結(jié)合于HBX的ZFP可于HepG2.2.15細(xì)胞中有效抑制HBV轉(zhuǎn)錄及HepG2.2.15細(xì)胞中HBX的表達(dá)。
[Abstract]:Objective: to construct an artificial zinc finger protein (ZINC) finger protein. ZFP specifically binds to hepatitis B virus X protein (HBX) gene promoter of hepatitis B virus X protein. To observe the inhibitory effect of artificial zinc finger protein on replication and transcription of hepatitis B virus (HBV) in vitro. Methods:. Transfection of Recombinant plasmid pcDNA3.1-ZFP or pcDNA3.1-NC into HepG2.2.15 cells by Liposome LipofectamineTM2000. As an experimental group (. ZFP group) and control group (NC group). 1. Western blotting was used to detect the content of HBX protein in experimental group and control group. 2.Enzyme-linked immunosorbent assay (Elisa) was used to detect hepatitis B e antigen in supernatant of two groups. HBeAg level. 3. The content of HBV DNA in supernatant of two groups was determined by real time fluorescence quantitative analysis. 4. RT-PCR was used to detect the level of HBV mRNA in the cells of the experimental group and the control group. Results: the recombinant plasmid was transfected into HepG2.2.15 cells. 1. The expression level of HBX protein in HepG2.2.15 cells was detected by Western blot. Compared with the control group, the expression level of HBX protein in the experimental group was significantly lower than that in the control group. The difference is statistically significant. 2.Compared with the control group, the content of HBeAg in the supernatant of cell culture decreased by 33.8%. 3. The copy amount of HBV DNA in the supernatant of ZFP group decreased by 51.7% compared with NC group. 4. The level of HBV mRNA in the experimental group was significantly lower than that in the control group. Conclusion:. Through the above research, it is proved that the ZFP specifically designed by our team to bind to HBX can effectively inhibit HBV transcription in HepG2.2.15 cells and HepG2.2.15 cells in HepG2.2.15 cells. Expression of HBX.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R512.62

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