特异性靶向乙型肝炎病毒X基因的人工转录因子的构建

发布时间：2018-04-25 20:45

本文选题：人工转录因子 + 锌指蛋白　；参考：《重庆医科大学》2012年硕士论文

【摘要】：目的：设计出一种新型的人工转录因子（artificialtranscription factor，ATF），使其能够特异性结合于乙型肝炎病毒（hepatitis B virus，HBV） X基因启动子区域的靶序列，通过下调X基因来干扰和抑制HBV DNA的正常的复制和表达，为乙型肝炎的基因治疗提供实验依据。方法：（1）通过生物信息学工具获取人工锌指蛋白（zinc fingerprotein，ZFP）氨基酸序列，经过密码子优化后构建重组质粒pEGFP-N1-ZFP，，将其转染COS-7真核表达细胞，通过观察绿色荧光蛋白的表达、RT-PCR和Western blot检测其在真核细胞中的表达；（2）构建含有ZFP结合结构域和KRAB效应结构域的ATF，将其转染COS-7真核表达细胞，通过RT-PCR和Western blot检测其在真核细胞中的表达；（3）将pcDNA3.1(+)-ATF转染HepG2.2.15细胞，通过酶联免疫法ELISA、细胞免疫组化、荧光定量方法、RT-PCR等方法检测其对HBVDNA复制和表达的抑制作用。结果：（1）成功构建了pEGFP-N1-ZFP重组质粒，并验证其在COS-7细胞中的表达；（2）成功构建ATF，并验证其具有相应的生物学活性；（3）成功验证了重组质粒pcDNA3.1(+)-ATF能够抑制HepG2.2.15细胞内HBV DNA的复制和表达。结论：针对HBV X基因靶序列设计出来的ATF能够抑制HepG2.2.15细胞内HBV DNA的复制和表达。
[Abstract]:Objective: to design a novel artificial transcription factor-ATFN to specifically bind to the target sequence of the X promoter region of hepatitis B virus (HBV) gene, and to interfere and inhibit the normal replication and expression of HBV DNA by down-regulating X gene. To provide experimental basis for gene therapy of hepatitis B. Methods the amino acid sequence of artificial zinc finger protein (ZFP) was obtained by bioinformatics. The recombinant plasmid pEGFP-N1-ZFP1 was constructed by codon optimization and transfected into COS-7 eukaryotic expression cells. The expression of green fluorescent protein (GFP) in eukaryotic cells was detected by RT-PCR and Western blot. ATFs containing ZFP binding domain and KRAB effect domain were constructed and transfected into COS-7 eukaryotic expression cells. RT-PCR and Western blot were used to detect its expression in eukaryotic cells. PcDNA3.1 (-ATF) was transfected into HepG2.2.15 cells. The inhibition of HBVDNA replication and expression was detected by Elisa, immunohistochemistry and RT-PCR. Results the recombinant plasmid of pEGFP-N1-ZFP was successfully constructed, and its expression in COS-7 cells was verified. The recombinant plasmid pcDNA3.1 (pDNA3.1) was proved to be able to inhibit the replication and expression of HBV DNA in HepG2.2.15 cells. Conclusion: ATF designed for the target sequence of HBV X gene can inhibit the replication and expression of HBV DNA in HepG2.2.15 cells.
【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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