ATF抑制HBV的表达与拉米夫定的作用研究比较

发布时间：2018-03-02 10:33

本文关键词： 人工锌指蛋白真核表达乙型肝炎病毒 X蛋白拉米夫定　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:构建特异性结合乙型肝炎病毒（Hepatitis B virus, HBV）X基因启动子（X gene promoter，Xp）的锌指蛋白（zinc finger protein，ZFP），并与拉米夫定体外抗乙肝病毒效果进行比较。方法:1.设计特异性结合HBV Xp的ZFP并载入真核表达载体中，构建重组质粒nls-ZFP-kRAB-Flag （pcDNA3.1+，ATF）、nls-ZFP-Flag（pcDNA3.1+，ZFP1）、nls-kRAB-Flag（pcDNA3.1+，，ZFP2）。2.通过酶切、测序鉴定重组质粒的正确性。3.Western blot和细胞免疫荧光技术鉴定重组质粒在HepG2.2.15细胞中的表达情况。4.将重组质粒转染HepG2.2.15细胞，设空白组（Blank组）、ATF组、ZFP1组、ZFP2组、空质粒组（Empty vctor组）和拉米夫定组（3TC组），分别培养2d、6d、10d。用Western blot检测各组HBx蛋白的表达情况；FQ-PCR检测各组HBV DNA复制的拷贝数；ELASA技术检测各组HepG2.2.15细胞上清中HBsAg和HBeAg的浓度。结果:1:特异性的ATF能在HepG2.2.15细胞中表达。2: ATF和3TC对HepG2.2.15细胞中X蛋白和HBV DNA的表达在三个时间点具有明显抑制作用（P0.05)，且ATF的抑制作用强于3TC（P0.05），6d和10d作用强于2d（P0.05）。3: ATF组和3TC组上清中HBsAg、HBeAg的浓度显著降低，即ATF组和3TC组对HBsAg、HBeAg的抑制率高与其余三组（P0.01），且ATF的抑制率高于3TC（P0.01）。4: ZFP1和ZFP2对X蛋白和HBV DNA无抑制作用。结论:人工设计的特异性ATF在真核细胞中能正常表达，并和拉米夫定一样都有明显抗HBV作用，他们的作用机制不同，且作用强于拉米夫定。
[Abstract]:Aim: to construct a zinc finger protein (ZINC finger protein HBV)X) specifically bound to hepatitis B virus (HBV)X) promoter X gene promoter, and to compare the antiviral effect of lamivudine with lamivudine in vitro. Methods 1. To design a ZFP specifically bound to HBV XP and load it into eukaryotic expression vector, construct the recombinant plasmid nls-ZFP-kRAB-Flag pcDNA3.1 + ATFP-Flagagnls-ZFP-Flagus pcDNA3.1 nls-kRAB-FlagDNA pc3.1nls-kRAB-FlagDNA pc3.1 + ZFP2N2.The recombinant plasmid pcDNA3.1 was digested by enzyme digestion, and the recombinant plasmid pcDNA3.1 was inserted into the eukaryotic expression vector. The correctness of the recombinant plasmid was confirmed by sequencing. 3. Western blot and immunofluorescence technique were used to identify the expression of the recombinant plasmid in HepG2.2.15 cells. The recombinant plasmid was transfected into HepG2.2.15 cells. Empty plasmid group (empty vctor group) and lamivudine group (3TC group) were cultured for 2 days, 6 days and 10 days, respectively. The expression of HBx protein in each group was detected by Western blot. The copy number of HBV DNA replication was detected by FQ-PCR and the concentration of HBsAg and HBeAg in the supernatant of HepG2.2.15 cells in each group was detected by ELASA technique. Results the specific ATF in HepG2.2.15 cells showed significant inhibitory effects on the expression of X protein and HBV DNA in HepG2.2.15 cells at three time points, and the inhibitory effect of ATF on the expression of X protein and HBV DNA in HepG2.2.15 cells was stronger than that in 3TCU P0.05D and 10d groups. 3The concentration of HBsAg HBeAg in the supernatant of TC group was significantly lower than that in the control group. In other words, the inhibition rate of ATF and 3TC on HBsAg HBeAg was higher than that of the other three groups, and the inhibitory rate of ATF was higher than that of 3TCX P0.01HBeAg. 4: ZFP1 and ZFP2 had no inhibitory effect on X protein and HBV DNA. Conclusion: the artificially designed specific ATF can express normally in eukaryotic cells and has the same anti-#en1# effect as lamivudine. The mechanism of their action is different and stronger than that of lamivudine.
【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;Q78

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