靶向UL27shRNA抑制Ⅱ型单纯疱疹病毒在HEK293细胞中的复制

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中国生物化学与分子生物学报 ›› 2013, Vol. 29 ›› Issue (8) : 759-766.

研究论文

靶向UL27shRNA抑制Ⅱ型单纯疱疹病毒在HEK293细胞中的复制

吕延成*，潘晓瑜，周丹丹，袁俊杰，黄畅

作者信息 +

Targeting UL27 shRNA Inhibits Herpes Simplex Virus Type ⅡReplication in HEK293 Cells

Lv Yan-Cheng*, PAN Xiao-Yu, ZHOU Dan-Dan, YUAN Jun-Jie, HUANG Chang

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文章历史 +

摘要

按照shRNA(small hairpin RNA)设计原则，针对Ⅱ型单纯疱疹病毒(herpes simplex virus type 2, HSV-2)的UL27基因序列保守区域筛选设计、合成4条干扰靶序列并构建表达UL27序列特异性siRNA(short interfering RNA)的质粒载体pGPU6/GFP/Neo．通过脂质体介导重组表达载体转染HEK293细胞 (human embryonic kidney 293 cell)再接种HSV-2．采用实时荧光定量PCR(real-time fluorescent quantitative PCR)技术检测UL27各组的mRNA转录水平，终点滴定法检测细胞上清液中的病毒滴度，四甲基偶氮唑盐(four methyl thiazolyl tetrazolium, MTT)法测定细胞存活率,Western印迹法检测蛋白表达效果．结果显示，UL27shRNA75组对UL27基因mRNA表达抑制效果最佳，同时能显著抑制感染细胞的CPE(cytopathic effect, CPE)，降低上清液中的病毒感染滴度，提高细胞的生存率，抑制UL27基因的蛋白表达．提示本研究构建的pGPU6/GFP/Neo-UL27表达载体能在细胞水平上不同程度地干扰HSV-2 UL27基因表达，抑制HSV-2在HEK293细胞中复制．

Abstract

Based on the rule of shRNA design, four fragment targeting conserved sequences of UL27 gene was synthesized and cloned into a plasmid vector pGPU6/GFP/Neo which expressed siRNA specifically targeting UL27 gene. The expression vectors were transfected into human embryonic kidney(HEK) 293 cells with liposome, and then inoculated with herpes simplex virus type 2（HSV-2）. The expression of HSV-2 UL27 mRNA were detected with realtime fluorescent quantitative PCR. Cytopathic effect (CPE) of HSV-2 infected cells was observed, and the TCID50 in supernatants collected from HSV-2 infected cells was determined by endpoint assay. Survival rate of cells were determined by MTT (four methyl thiazolyl tetrazolium). The expression of HSV-2 UL27 protein were detected by Western blotting. The results showed that the UL27shRNA75 caused a 75.17% inhibition of UL27 mRNA expression, which significantly blocked the CPE, and reduced viral titer by 10^-3, and improved the survival rate of cells, as well as inhibition of UL27 protein expression. The results demonstrated that the constructed pGPU6/GFP/ Neo-UL27 could differently interfere with HSV-2 UL27 gene expression at cell level and inhibited the replication of HSV-2 in HEK293 cells.

导出引用

吕延成，潘晓瑜，周丹丹，袁俊杰，黄畅. 靶向UL27shRNA抑制Ⅱ型单纯疱疹病毒在HEK293细胞中的复制[J]. 中国生物化学与分子生物学报, 2013, 29(8): 759-766

Lv Yan-Cheng, PAN Xiao-Yu, ZHOU Dan-Dan, YUAN Jun-Jie, HUANG Chang. Targeting UL27 shRNA Inhibits Herpes Simplex Virus Type ⅡReplication in HEK293 Cells[J]. Chinese Journal of Biochemistry and Molecular Biology, 2013, 29(8): 759-766

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基金

贵州省科学技术基金（黔科合J字No.［2008］2313号）和贵州省优秀科技教育人才省长专项资金项目（黔省专合字No.(2009）122号）资助

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收稿日期	修回日期	出版日期
2013-01-28	2013-04-23	2013-08-20
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2013-08-20

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