人工转录因子的设计以抑制乙肝病毒的转录

发布时间：2018-12-20 12:36

【摘要】：目的设计特异性结合乙肝病毒核心启动子的人工转录因子并观察其对乙肝病毒转录的抑制作用。方法 1、利用Zine finger tools设计特异性靶向HBV核心启动子的锌指蛋白，获得锌指蛋白的靶序列及氨基酸序列，并对该蛋白的理化性质和结构特征进行分析。 2、将ZFP氨基酸序列逆向翻译成核苷酸序列并进行优化后人工合成。用带有核定位信号(NLS)的引物扩增ZFP片段并将其克隆至真核表达质粒pcDNA3.1(+)，构建pcDNA3.1(+)-ZFP，然后以此为基础插入带有Flag tag的KRAB序列，构建人工转录因子的真核表达质粒pcDNA3.1(+)-ATF。 3、脂质体转染重组质粒pcDNA3.1(+)-ATF到COS-7细胞，应用RT-PCR和Western Blot分别从mRNA水平和蛋白质水平检测ATF在真核细胞中的表达情况。 4、用脂质体将重组质粒pcDNA3.1(+)-ATF或pcDNA3.1(+)分别转染HepG2.2.15细胞，ELISA和FQ-PCR检测上清液中HBeAg和HBVDNA含量，RT-PCR检测细胞内HBV mRNA表达，免疫细胞化学检测细胞内核心抗原的表达。结果 1、锌指蛋白在HBV核心启动子上的靶序列为：5'-AATGTCAACGACCGACCT-3' 2、锌指蛋白的氨基酸序列为：LEPGEKPYKCPECGKSFSTKNSLTEHQRTHTGEKPYKCPECGKSFSQSGHLTEHQRTHTGEKPYKCPECGKSFSDPGNLVRHQRTHTGEKPYKCPECGKSFSDSGNLRVHQRTHTGEKPYKCPECGKSFSDPGALVRHQRTHTGEKPYKCPECGKSFSTTGNLTVHQRTHTGKKTS 3、ZFP结构特征分析为：等电点9.20，分子量19.69，稳定指数为31.41，显示为稳定结构。二级结构预测提示该蛋白含有6段Ｃ2Ｈ2型锌指单元序列，分别位于其氨基酸残基序列的第8~30、36~58、64~86、92~114、120~142及148~170位。同源建模也提示其含有6个连续的锌指结构。StructureAssessment对建模结果进行评估，结果显示空间结构稳定。 4、pcDNA3.1(+)-ATF转染COS-7细胞后，经RT-PCR及Western Blot检测，，ATF mRNA和融合有Flag tag的ATF蛋白表达明显增加，而转染pcDNA3.1(+)的细胞内则没有相应的表达，说明ATF在真核细胞内是能够正常表达的。 5、ATF重组质粒转染入HepG2.2.15细胞后，细胞上清液中的HBVDNA和HBeAg含量明显减少，细胞内的HBV mRNA及HBcAg也显著降低，说明ATF能够有效抑制HBV的转录。结论利用生物信息学方法设计了能特异性结合HBV核心启动子的锌指蛋白，并以此为基础构建了完整的人工转录因子。实验证明设计的人工转录因子能够在真核细胞内正常表达，并对细胞模型中的HBV转录起抑制作用。
[Abstract]:Objective to design the artificial transcription factors specifically binding to HBV core promoter and to observe its inhibitory effect on HBV transcription. Methods 1. Zinc finger protein targeting the core promoter of HBV was designed by Zine finger tools. The target sequence and amino acid sequence of zinc finger protein were obtained, and the physical and chemical properties and structural characteristics of the protein were analyzed. 2. The amino acid sequence of ZFP was converse translated into nucleotide sequence and optimized for artificial synthesis. The ZFP fragment was amplified with a primer with nuclear localization signal (NLS) and cloned into eukaryotic expression plasmid pcDNA3.1 (), to construct pcDNA3.1 ()-ZFP, and then inserted into the KRAB sequence with Flag tag. Construction of eukaryotic expression plasmid pcDNA3.1 ()-ATF. of artificial transcription factors 3. The recombinant plasmid pcDNA3.1 ()-ATF was transfected into COS-7 cells by liposome. The expression of ATF in eukaryotic cells was detected by RT-PCR and Western Blot from mRNA level and protein level, respectively. 4Recombinant plasmids pcDNA3.1 ()-ATF or pcDNA3.1 () were transfected into HepG2.2.15 cells by liposome, HBeAg and HBVDNA contents in supernatant were detected by ELISA and FQ-PCR, HBV mRNA expression was detected by RT-PCR. The expression of core antigen was detected by immunocytochemistry. Results 1. The target sequence of zinc finger protein on the core promoter of HBV was 5- AATGTCAACGACCGACCT-3'2, and the amino acid sequence of zinc finger protein was as follows: isoelectric point 9.20, molecular weight 19.69, and amino acid sequence of zinc finger protein as follows: isoelectric point 9.20, molecular weight 19.69, amino acid sequence of zinc finger protein as follows. The stability index is 31.41, showing a stable structure. The prediction of the secondary structure suggested that the protein contained six C2H2 zinc finger units, located at position 830 / 36 / 586 / 864 / 866 / 92C / 114120 / 142 and at position 148170of the amino acid residues, respectively. The homologous modeling also indicates that it contains six consecutive zinc finger structures. StructureAssessment evaluates the modeling results and the results show that the spatial structure is stable. 4After transfection of COS-7 cells with pcDNA3.1 ()-ATF, the expression of, ATF mRNA and ATF fused with Flag tag was detected by RT-PCR and Western Blot, but there was no corresponding expression in pcDNA3.1 () transfected cells. These results suggest that ATF can be expressed normally in eukaryotic cells. After transfection into HepG2.2.15 cells, the contents of HBVDNA and HBeAg in the supernatant were significantly decreased, and the HBV mRNA and HBcAg in the cells were also significantly decreased, which indicated that ATF could effectively inhibit the transcription of HBV. Conclusion the zinc finger protein which can specifically bind to the core promoter of HBV was designed by bioinformatics, and a complete artificial transcription factor was constructed based on it. The results show that the designed artificial transcription factors can express normally in eukaryotic cells and inhibit the transcription of HBV in the cell model.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

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