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RNAi技术在抗HIV-1治疗研究中已显示出巨大潜力,获得可高效特异抑制HIV-1的RNAi元件是进行相关研究的重要基础。miRNA在抑制和表达方式上相比siRNA具有更多的优势。本研究即探讨构建可高效特异靶向HIV-1的人工miRNA元件。选择以保守性较好的HIV-1pol基因为靶区筛选高效保守的RNAi序列,设计了16个可靶向pol区高保守区段的RNAi靶点,构建表达载体与HIV-1感染性克隆进行共转染抑制实验,筛选显示pol1026序列兼具高保守性及高抑制效率特点。以天然miR-30a为基础骨架构建了靶向pol1026靶点的人工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(miR-1026E)。通过与携带靶序列的报告质粒的共转染实验证明miR-1026E具有良好的靶点特异性。本研究进一步构建了携带miR-1026E表达元件的重组慢病毒,转导MT-4细胞并对转导后细胞进行克隆化筛选,获得稳定整合miR-1026E表达元件的MT-4-miR1026E细胞克隆,该细胞在体外攻毒实验中可高效抑制HIV-1的复制,具有显著的抑制HIV-1的能力。同时应用实时RT-PCR方法检测显示,miR-1026E在细胞中不会影响内源性代表miRNA(miR-181与miR-16)的表达水平和干扰素效应相关基因stat1的表达水平,具有良好的特异性。所获得的可特异高效抑制HIV-1复制的人工miRNA元件可为抗HIV-1研究提供重要参考。
RNAi technology has shown great potential in the research of anti-HIV-1 treatment. Obtaining RNAi element that can efficiently and specifically inhibit HIV-1 is an important basis for related research. miRNAs have more advantages in terms of inhibition and expression than siRNAs. This study explored the construction of artificial miRNA elements that efficiently and specifically target HIV-1. Sixteen RNAi targets that can target the highly conserved region of pol region were designed by selecting highly conserved RNAi sequences from the well-conserved HIV-1pol gene as the target region, and constructing the expression vector and HIV-1 infectious clone Co-transfection inhibition experiments, screening showed pol1026 sequence with both high conservation and high inhibitory efficiency. An artificial miRNA element targeting pol1026 target was constructed based on the natural miR-30a framework. The artificial miRNA element that can effectively inhibit the expression of HIV-1 was successfully obtained by co-transfection with HIV-1 infectious clone plasmid miR-1026E). Co-transfection experiments with reporter plasmids carrying target sequences demonstrated that miR-1026E has good target specificity. In the present study, a recombinant lentivirus carrying the miR-1026E expression element was further constructed and transduced into MT-4 cells. The transduced cells were cloned and screened to obtain MT-4-miR1026E cell clones stably integrated with miR-1026E expression elements. The cells can inhibit the replication of HIV-1 efficiently in vitro and have a significant ability of inhibiting HIV-1. Meanwhile, real-time RT-PCR assay showed that miR-1026E did not affect the expression of endogenous miRNAs (miR-181 and miR-16) and the expression of interferon-related gene stat1 in the cells with good Specificity. The obtained artificial miRNA elements that can specifically and highly inhibit HIV-1 replication can provide important reference for anti-HIV-1 research.