ccr5、vif和pol基因的人工miRNA的构建与筛选

发布时间：2018-01-10 18:31

本文关键词：ccr5、vif和pol基因的人工miRNA的构建与筛选　出处：《南华大学》2012年硕士论文　论文类型：学位论文

更多相关文章： HIV RNAi miRNA vif pol ccr5

【摘要】：背景及目的：RNAi在HIV-1的治疗研究中得到了广泛的应用,构建高效并且安全的RNAi抗病毒元件是开展相关研究的基础。siRNAs或miRNAs可以特异地结合靶mRNA，引起靶mRNA降解或翻译抑制，最终导致基因特异性沉默。辅助受体（CCR5或CXCR4）是HIV进入被感染细胞所必需的。研究发现，基因CCR5delta32序列缺失可以减缓或抵抗HIV-1感染，但机体未出现免疫异常。基因vif是感染性因子，可能影响游离HIV感染性、病毒体的产生和体内传播。基因pol编码多种酶，如逆转录酶，蛋白酶，对HIV的复制有重要作用。本实验分别以ccr5，vif，pol为靶基因，构建miRNA,从中筛选出来基因效率较高的miRNAs。方法：第一步，选择保守型较好的ccr5，vif，pol基因为靶区，，合成基因序列，克隆入载体，转染293T细胞，最后通过qPCR筛选高效表达ccr5、vif和pol基因的293T细胞。第二步，根据基因序列设计引物并分别合成针对ccr5、vif和pol基因的人工miRNA各4对。第三步，以天然miR-155为基础骨架构建了分别靶向ccr5，vif，pol的人工miRNA元件，通过与HIV-1感染性克隆质粒的共转染抑制实验验证获得了可有效抑制HIV-1表达的人工miRNA元件。通过与携带靶序列的报告质粒的共转染实验证明ccr5-miRNA，vif-miRNA和pol-miRNA具有良好的沉默效果。结果：通过干扰效果评价，得到了干扰效果较强的ccr5-miRNA、vif-miRNA和pol-miRNA，其对靶基因的干扰效果分别达到了72%、68%和76%。结论：得到了高效表达ccr5，vif，pol基因的293T细胞；构建和筛选到了各种基因沉默效率最好的miRNA，其干扰效率分别为72%、68%和76%。
[Abstract]:Background and objective: RNAi has been widely used in the treatment of HIV-1. The construction of efficient and safe RNAi antiviral elements is the basis of related research. SiRNAs or miRNAs can specifically bind to target mRNAs and cause target mRNA degradation or translation inhibition. The coreceptor CCR5 or CXCR4 is necessary for HIV to enter infected cells. Gene CCR5delta32 deletion can slow down or resist HIV-1 infection, but there is no immune abnormality. Gene vif is an infectious factor, which may affect the infection of free HIV. The gene pol encodes a variety of enzymes, such as reverse transcriptase and protease, which play an important role in the replication of HIV. MiRNAs with high gene efficiency were selected from miRNAs. Methods: in the first step, the conserved ccr5 vifpol gene was selected as the target region, the gene sequence was synthesized and cloned into the vector and transfected into 293T cells. Finally, 293T cells expressing ccr5vif and pol genes were screened by qPCR. In the second step, primers were designed according to gene sequence and synthesized respectively for ccr5. In the third step, the artificial miRNA elements targeting ccr5 vifopol were constructed based on the skeleton of natural miR-155. An artificial miRNA element was obtained by co-transfection inhibition experiment with HIV-1 infectious clone plasmids. The co-transfection experiment with the reporter plasmid carrying target sequence showed that the artificial miRNA element could effectively inhibit the expression of HIV-1. Prove ccr5-miRNA. Vif-miRNA and pol-miRNA have good silencing effect. Results: through the evaluation of interference effect, we got the good interference effect of ccr5-miRNA-vif-miRNA and pol-miRNA. Its interference effect on target gene reached 72% and 76% respectively. Conclusion: 293T cells with high expression of ccr5 vifopol gene were obtained. The miRNAs with the best gene silencing efficiency were constructed and screened, and the interference efficiency was 722% and 76%, respectively.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346

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