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RNAi技术在艾滋病治疗研究中已展现出巨大的潜力,兼具高效抑制特性和保守性的siRNA靶位是其获得成功应用的重要基础。本研究选择以HIV-1 vif基因为靶区筛选高效保守的RNAi序列,共选择设计了30个识别不同位点的siRNA序列,以pSUPER为载体构建了相应的shRNA表达质粒。通过与pNL4-3质粒在293FT细胞中进行共转染抑制实验,以及对初筛获得的高效序列进行保守性分析显示siRNA-vif37序列具有高效抑制效率和较好的保守性特征。通过与pGL3-vif报告质粒的共转染实验证明siRNA-vif37具有vif基因抑制特异性。带有shRNA-vif37表达元件的重组慢病毒转导后的MT-4细胞在HIV-1NL4-3体外攻毒实验中可显示出较有效的抑制病毒复制的能力,本研究进一步对转导后细胞进行克隆化筛选,获得稳定整合shRNA-vif37表达元件的MT-4-vif37细胞克隆,该细胞具有显著的抑制病毒复制的能力,在高攻毒剂量下仍可获得良好的抑制效果。本研究为进一步应用RNAi技术进行新型艾滋病治疗方法研究提供了重要基础。
RNAi technology has shown great potential in the research of AIDS treatment. The target of siRNA with high efficiency of inhibition and conservation is an important basis for its successful application. In this study, highly efficient and conservative RNAi sequences were selected by targeting HIV-1 vif gene. A total of 30 siRNA sequences were designed and selected, and the corresponding shRNA expression plasmids were constructed by using pSUPER as a vector. Conservative analysis by cotransfection with pNL4-3 plasmid in 293FT cells and conservative analysis of the highly efficient sequences obtained from the preliminary screening showed that the siRNA-vif37 sequence has high efficiency of inhibition and better conservative characteristics. Co-transfection experiments with pGL3-vif reporter plasmid demonstrated that siRNA-vif37 has the vif gene inhibitory specificity. The recombinant lentivirus transfected MT-4 cells with shRNA-vif37 expression element showed a more potent inhibitory effect on viral replication in HIV-1 NL4-3 in vitro challenge study. The further study of transduced cells The clones were screened and cloned to obtain MT-4-vif37 cell clone stably integrated with shRNA-vif37 expression element. The cell has significant ability of inhibiting virus replication and good inhibitory effect can be obtained even at high challenge dose. This study provides an important basis for further research on new AIDS treatment using RNAi technology.