摘要
目的应用质粒介导的RNA干扰技术研究siRNA抑制截短HCMV IE2基因的表达的作用。方法以pLL3．7质粒为模板,采用半套式PCR的方法扩增出含U6启动子的siRNA表达片段,连接pMD-T simple vector构建效应质粒表达重组体pMD-T-shRNAR1和pMD-T-shRNAR2。同时采用RT- PCR方法从HCMV AD169株基因组中调取目的截短基因IE2,定向克隆到带有绿色荧光蛋白(GFP)作为报告基因的真核表达载体pEGFP-C1上,构建HCMV AD169株截短IE2基因重组表达质粒pEGFP- C1-IE2;用脂质体共转染效应质粒和靶基因表达重组体至Hep-2细胞中,荧光显微镜下观察效应质粒的RNA干涉作用,并观察RNA干扰的时效和量效改变。结果三组质粒重组体构建成功,克隆到的2个shRNA表达载体对截短IE2基因的表达都有干涉作用。在与靶基因质粒量比为1:1和1:2时,其中pMD-T-shRNAR1 48～72 h间干涉效应为100％,可完全封闭IE2的表达;pMD-T-shRNAR2使IE2处于极低水平表达,48-72 h间干涉效应达到98．4％～99％。结论通过质粒介导的RNAi技术能够有效地抑制HCMV IE2基因的表达,为治疗HCMV感染的药物开发提供了思路。
Objective To study the inhibited effect of siRNA on the expression of truncated region of HCMV IE2 gene by vector-based RNAi technology. Methods After designing two siRNA sequences R1 and R2 based truncated region of HCMV IE2 gene, siRNA expression fragments containing U6 promoter were amplified from pLL3.7 by semi-nested PCR, siRNA expression vectors as effector plasmid pMD-T-shRNAR1 and pMD-T-shRNAR2 were constructed by cloning siRNA expression fragments into pMD-T simple vector. Meanwhile, the truncated region of HCMV IE2 gene was obtained from HCMV mRNA by RT-PCR. Eukaryotic expression vector pEGFP-C1-IE2 as target plasmid was constructed by cloning truncated region of HCMV IE2 into pEGFP-C1 on special direction. After effector and target plasmids were transfected into Hep-2 cell by different ratio by lipofectin mediation, effect of RNAi could be observed at different time points by fluorescence microscope. Results Three recombinant vectors were constructed by test-based PCR, restriction digestion and gene sequencing methods. Two obtained shRNA expression vectors were available. One which interference effect was about 100% could stop expression of truncated region of HCMV IE2 gene from 48 h to 72 h. When effector and target plasmids ration was 1 ： 1 and 1：2, the other which interference effect was 98.4%-99% could obviously inhibit expression of region of HCMV IE2 gene in the same time. Conclusion Expression of HCMV IE2 gene can be effectively inhibited by vector-based RNAi technology. This will offer a kind of study path in following： what is molecular action of HCMV IE2 gene in HCMV infection and proliferation？ Can HCMV IE2 gene be accepted as an object if medicine study？
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2006年第7期593-597,共5页
Chinese Journal of Microbiology and Immunology
基金
安徽省科技厅"十五"生物医药重大科技专项(编号:01303003)
教育部科学技术重点项目(编号:01052)
关键词
载体介导RNA干涉
人巨细胞病毒
截短IE2基因
绿色荧光蛋白
转染
Vector-based RNAi
Human cytomegalovirus（HCMV）
Truncated region of IE2 gene
Green fluorescence protain（GFP）
Trausfection
