构建特异性结合HBVX基因启动子的人工锌指蛋白以抑制HepG2.2.15细胞中HBV的转录

发布时间：2018-02-04 18:47

本文关键词： 乙肝病毒 X基因启动子锌指蛋白 HepG2.2.15细胞　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：构建人工锌指蛋白(zinc finger protein, ZFP)特异性结合于乙肝病毒X蛋白(hepatitis B virus X protein, HBX)基因启动子，观察人工锌指蛋白在体外对乙型肝炎病毒(hepatitis B virus, HBV)复制和转录的抑制作用。方法：用脂质体LipofectamineTM2000将重组质粒pcDNA3.1-ZFP或pcDNA3.1-NC分别转染HepG2.2.15细胞分别作为实验组（ZFP组）和对照组（NC组）。 1.用免疫印迹法（Western blot）检测实验组和对照组细胞中HBX蛋白的含量。 2.用酶联免疫吸附测定法（ELISA）检测两组上清液中乙型肝炎核心相关抗原(hepatitis B e antigen, HBeAg)水平。 3.用实时荧光定量PCR（qPCR）检测两组上清液中HBV DNA含量。 4.用RT-PCR检测实验组和对照组细胞内HBV mRNA水平。结果：重组质粒转染入HepG2.2.15细胞后； 1.Western blot检测所得的HepG2.2.15细胞中HBX蛋白表达水平，相比对照组，实验组中HBX蛋白表达水平明显下降，，差异具有统计学意义。 2.相比对照组，ZFP组细胞培养上清液中HBeAg含量下降了33.8%（t=3.887,P=0.003）。 3.ZFP组细胞培养上清液中HBV DNA拷贝量相对NC组下降了51.7%（t=23.079,P=0.000）。 4.测得实验组细胞内HBV mRNA水平，与对照组相比，明显减少。结论：经过上述研究证明我团队前期人工设计的可特异性结合于HBX的ZFP可于HepG2.2.15细胞中有效抑制HBV转录及HepG2.2.15细胞中HBX的表达。
[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.
【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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