论文部分内容阅读
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 Rab9 GTPase short hairpin RNA (shRNA) expression vector and observe its inhibitory effect on the expression of Rab9 GTPase gene and the proliferation of wild strain of measles virus in vitro. Methods Two pairs of Rab9 GTPase gene-specific shRNAs were designed and synthesized according to the sequence of Rab9 GTPase in GenBank. The recombinant vectors were cloned into the expression vector and constructed into recombinant expression vector. The recombinant plasmid was transfected into U937 cells by restriction enzyme digestion and sequence analysis. The expression of Rab9 GTPase mRNA and protein in transfected cells was detected by RT-PCR and Western blotting. The virus titer was determined by standard plaque assay. Flow cytometry The changes of apoptosis rate were detected. The mRNA levels of double-stranded RNA dependent protein kinase (PKR) and 2’-5 ’oligoadenosine synthase (OAS-1) in transfected cells were detected by RT-PCR. Results Restriction analysis and sequence analysis confirmed that shRNA expression vector targeting Rab9 GTPase gene was successfully constructed. Both shRNAs could specifically inhibit the expression of Rab9 GTPase mRNA and protein in U937 cells with the highest inhibition rates of (90.5 ± 0.2)% and (92.1 ± 0.3)%, respectively. The plaque assay showed that shRNAs could effectively inhibit the expression of measles virus The proliferation rate of wild-type strain was up to more than 90% in vitro. The apoptosis rate of transfected cells did not change obviously by flow cytometry. The mRNA level of PKR and OAS-1 did not change significantly before and after transfection. Conclusion Rab9 GTPase specific shRNA expression vector was successfully constructed. shRNAs inhibit the proliferation of wild type measles virus by inhibiting the expression of Rab9 GTPase specifically.