【摘要】 目的构建Rab9 GTPase短发夹RNA(shRNA)表达载体,观察其对Rab9 GTPase基因表达和麻疹病毒野生株体外增殖的抑制作用。方法参照GenBank中Rab9 GTPase基因序列设计合成2对Rab9 GTPase基因特异性shRNA,定向克隆入表达载体,构建重组表达载体,酶切鉴定和序列分析证实后脂质体法转染U937细胞,然后感染麻疹病毒野生株,逆转录聚合酶链反应(RT-PCR)和免疫印迹技术(Western blot)检测转染细胞内Rab9 GTPase mRNA和蛋白质的表达水平;标准蚀斑试验测定病毒滴度;流式细胞仪检测细胞凋亡率的变化;RT-PCR检测转染细胞内双链RNA依赖蛋白激酶(PKR)和2′-5′寡腺甙酸合成酶(OAS-1)的mRNA水平。结果酶切和序列分析证实,成功构建了靶向Rab9 GTPase基因的shRNA表达载体。2个shRNAs均可特异性抑制U937细胞内Rab9 GTPase mRNA和蛋白质的表达,最高抑制率分别为(90.5±0.2)%和(92.1±0.3)%;蚀斑试验结果表明,shRNAs可以有效抑制麻疹病毒野生株体外增殖,其抑制率可达到90%以上;流式细胞仪检测转染后细胞的凋亡率无明显变化;RT-PCR检测PKR和OAS-1的mRNA水平转染前后无明显变化。结论成功构建Rab9 GTPase特异性shRNA表达载体。shRNAs通过特异性抑制Rab9 GTPase基因表达抑制麻疹病毒野生株体外增殖。更多还原

【Abstract】 Objective To construct a Rab9 GTPase-specific short hairpin RNA(shRNA) expression vector and to investigate the inhibitory effect of the shRNA on the expression of Rab9 GTPase and on the proliferation of the wild-type measles virus in vitro. Methods Based on the Rab9 GTPase gene sequence, two Rab9 GTPase gene-specific shRNAs were designed and cloned into the expression vector pSUPER.neo+EGFP to generate the recombinant expression vector pSUPER-R1 and pSUPER-R2. pSUPER-R1 and pSUPER-R2 were transfected into U937 cells via liposomes, and the cells were then infected with wild-type measles virus. The expression of Rab9 GTPase mRNA was assayed by RT-PCR, and the expression of the Rab9 GTPase protein was observed by Western blot assay. The titers of measles virus were determined by standard plaque assay. Flow cytometry was performed to determine the ratio of apoptosis. The expression of double-stranded RNA-dependent protein kinase (PKR) mRNA and 2′-5′ oligoadenylate synthetase (OAS-1) mRNA was assayed by RT-PCR. Results Enzyme digestion analysis and DNA sequencing confirmed that pSUPER-R1 and pSUPER-R2 were successfully constructed. Compared with the control group, Rab9 GTPase gene-specific shRNAs could significantly inhibit the expression of Rab9 GTPase mRNA and protein in U937 cells, with a highest inhibition rate of (90.5±0.2)% and (92.1±0.3)%, respectively. Measles virus proliferation was specifically suppressed by shRNAs targeting Rab9 GTPase gene, with an inhibition rate of over 90%. Flow cytometry showed that the apoptotic rate of the cells was not changed after transfection. PKR mRNA and OAS-1 mRNA remained unchanged. Conclusion Rab9 GTPase-specific shRNA expression vectors that specifically inhibit the expression of Rab9 GTPase and the proliferation of wild type measles virus in vitro have been successfully constructed.更多还原