shRNA干扰Rab9 GTPase表达对麻疹病毒野生株体外增殖的抑制作用  

shRNA-mediated silencing of the Rab9 GTPase gene inhibits the proliferation of wild-type measles virus in vitro

作　　者：史俊岩[1] 王舰[1] 王斯[1] 王美莲[1] 罗恩杰[1] 

机构地区：[1]中国医科大学基础医学院病原生物学教研室,辽宁沈阳110001

出　　处：《中国病原生物学杂志》2009年第6期416-419,422,共5页Journal of Pathogen Biology

基　　金：辽宁省教育厅基金项目(No.2008758)

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。Objective To construct a Rab9 GTPase-specific short hairpin RNA（shRNA） expression vector and to inves-tigate 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 transfeeted 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 deter-mined 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 pSU- PER-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.

关 键 词：Rab9 GTPASE SHRNA 麻疹病毒野生株 增殖 

分 类 号：R373.11[医药卫生—病原生物学]