【摘要】 为筛选鸡马立克氏病病毒(MDV)gI、gE基因特异性siRNA,本研究以鸡胚成纤维细胞(CEF)的DNA为模板,扩增禽源cU6-3启动子序列,并合成针对gI和gE基因各5个靶位点的10条shRNA序列。通过重叠PCR将cU6-3启动子与shRNA融合扩增制备shRNA表达盒,将制备的shRNA表达盒分别与重组质粒pEGFP-gI或pEGFP-gE共转染CEF细胞,并根据荧光显微镜观察和流式细胞仪检测结果评价siRNA抑制融合荧光蛋白表达的效率。结果表明,gI和gE基因的抑制效率为10%～80%。分别将其中抑制效率最高的一条shRNA插入pGEM-T载体中构建重组表达质粒pcU6-shgI735和pcU6-shgE936,通过不同细胞系稳定干扰试验和抑制病毒复制试验评价其干扰活性。结果显示,pcU6-shgI735和pcU6-shgE936能够明显抑制融合蛋白的表达及MDV在CEF细胞中的增殖,并且禽源U6启动子构建的表达盒在禽源细胞(DF1)中的活性比在哺乳动物细胞(Vero、MDCK)的活性高1.21～1.45倍。本研究鉴定获得了gI和gE基因的特异性siRNA,筛选出能够抑制MDV复制的靶位点,为MDV基因的功能和抗MDV病毒研究奠定了基础。更多还原

【Abstract】 To select and evaluate the siRNA targeting the gI and gE gene of Marek’s disease virus (MDV), ten shRNAs were synthesized and fused with chicken U6-3 (cU6) promoter to construct shRNA expression cassettes by overlapping PCR, respectively. The PCR product of the shRNA expression cassette, together with recombinant plasmid pEGFP-gI or pEGFP-gE,were co-transfected into CEF, respectively, and the fluorescence intensity in the transfected cells were monitored by fluorescence microscopy and flow cytometry. The result showed that the inhibition rate of the shRNA in transfected cells were 8.5% to 79.1%. Furthermore, the shRNA expression cassettes of cU6-shgI735 and cU6-shgE936 with the highest inhibition rate for gI or gE expression were inserted into pGEM-T vector to construct the recombinant plasmid of pcU6-shgI735 and pcU6-shgE936. The recombinant plasmid with the shRNA under the control of cU6 promoter was 1.21 to 1.45 times more efficient in CEF than that in mammalian cells (i.e. Vero or MDCK cells) for reducing the target gene expression. In addition, MDV replication in CEF was reduce to 77.42% and 80.06% by pcU6-shgI735 and pcU6-shgE936, respectively. The results of the study will lay a foundation to investigate the gene function of MDV and anti-MDV research.更多还原