登革Ⅱ型病毒前膜蛋白基因反向重复序列具有干扰病毒复制的作用  

An inverted-repeat RNA construct for silencing dengue virus type 2 pre-membrane gene suppresses viral replication in BHK-21 cells

作　　者：朱娉婷[1] 潘京[1] 郑学礼[1] 

机构地区：[1]南方医科大学公共卫生与热带医学学院病原生物学系,广东广州510515

出　　处：《南方医科大学学报》2013年第5期642-648,共7页Journal of Southern Medical University

基　　金：国家自然科学基金(30972566;81271880)~~

摘　　要：目的构建含有登革Ⅱ型病毒(DENV-2)前膜蛋白(prM)基因反向重复序列(irRNA)的重组质粒,评价其抑制病毒复制的效果。方法 DENV-2接种乳鼠,提取总RNA,在正义和反义引物两端分别加上XhoⅠ和EcoRⅠ的识别位点,将经RT-PCR扩增的prM全长基因片段插入pcDNA3.1(+)中,形成含有prM反向序列的重组质粒pcDNA-asprM;于另一对正义和反义引物两端引入酶切位点NheⅠ和BglⅡ,经RT-PCR扩增prM全长基因片段插入pMD18-T-vector中,形成含有正向序列的重组质粒pMD18-T-prM。限制性内切酶NheⅠ和KpnⅠ分别酶切重组质粒pMD18-T-prM和重组质粒pcDNA-asprM,得到片段prM和线性化载体pcDNA-asprM,连接构建成含有prM反向重复序列的重组质粒pcDNA-irRNA。筛选的阳性重组质粒pcDNA-irRNA用脂质体法转染至BHK-21细胞中,半定量PCR和实时荧光定量PCR分析评估该质粒抑制登革病毒复制的效果。结果成功构建具有prM反向重复序列的重组质粒pcDNA-irRNA。经转染pcDNA-irRNA的实验组与未转染质粒阳性对照组相比,半定量PCR结果显示DENV-2攻击96 h后NS3的mRNA表达水平下降28%;实时定量PCR分析显示,DENV-2攻击72 h后实验组病毒拷贝数比阳性对照组少1.44倍,攻击96 h后实验组的NS1表达量明显低于阳性对照组(P<0.05)。结论 DENV-2 prM反向重复序列具有干扰病毒复制的效果,为登革病毒基因疫苗的研究提供理论依据。Objective To evaluate the anti-viral effects of a plasmid expressing an inverted-repeat RNA targeting dengue virus type-2 （DENV-2） pre-membrane （prM） gene. Method Suckling mice were inoculated with live DENV-2 in the brain. The total RNA was extracted from the brain tissue of the infected mice, and the prM gene fragments were amplified by RT-PCR and then subcloned into Xho I/EcoR I of the pcDNA3.1（＋） plasmid in antisense orientation to construct the plasmid pcDNA-asprM. DENV-2 prM sequences were also subcloned into pMD18-T-vector in sense orientation to construct the plasmid pMD18-T- prM. pcDNA-irRNA was constructed by inserting in sense orientation the prM fragment isolated from pMD18-T-prM into the Nhe I/Kpn I of pcDNA-asprM. The plasmid pcDNA-irRNA was transfected into BHK-21 cells and the anti-viral effects were analyzed by semi-quantitative PCR and real-time PCR. Results Transfection with the plasmid pcDNA-irRNA caused a reduction of NS3 mRNA expression level by 28% in BHK-21 cells following a 96-h challenge with DENV-2 as compared to the cells without plasmid transfection （positive control）. The viral copies in pcDNA-irRNA-transfected cells was 1.44-fold lower than those in the positive control cells following a 72-h virus challenge, and the mRNA expression levels of NS1 were also significantly lower in the transfected cells at 96 h after viral challenge （P〈0.05） as shown by real-time quantitative PCR. Conclusion The inverted-repeat RNA for DENV-2 prM gene silencing can suppress DENV-2 replication in BHK-21 cells, which provides a basis for developing dengue virus gene vaccine.

关 键 词：登革Ⅱ型病毒 前膜蛋白 反向重复序列 RNA干扰 BHK-21细胞 

分 类 号：R373[医药卫生—病原生物学]