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Position: Home > Articles > Selection for Effective miRNA Targeting VP2 Gene of Infectious Bursal Disease Virus Jiangsu Journal of Agricultural Sciences 2010,26 (6) 1270-1276

靶向传染性法氏囊病病毒(IBDV)VP2有效miRNA的筛选

作者：

王永娟;崔平福;孙怀昌

单位：

昆山出入境检验检疫局;扬州大学兽医学院;江苏畜牧兽医职业技术学院江苏省兽用生物制药高技术研究重点实验室

关键词：

传染性法氏囊病病毒(IBDV);VP2;miRNA;荧光共聚焦;流式细胞术

摘要：

为筛选出能有效抑制传染性法氏囊病病毒(IBDV)VP2基因复制的miRNA,采用RT-PCR方法从病毒基因组中进行结构蛋白VP2的基因扩增与序列测定,构建融合表达载体pVP2-EGFP。根据VP2测序结果,借助genscript软件设计针对VP2的5条miRNA,分别命名为miVP2A、miVP2B、miVP2C、miVP2D和miVP2E,将合成的5条miRNAs分别插入到pRFPRNAiC中形成miVP2s表达载体,这些载体分别与pVP2-EGFP共转染DF-1细胞系,通过NortheringBlotting方法检测miVP2s的表达,利用荧光共聚焦显微镜定性、流式细胞术定量的方法进行有效miVP2s的筛选。结果显示:miVP2s能成功表达;转染miVP2s表达载体组的绿色荧光蛋白(GFP)强度和数量都明显弱于或少于阴性对照组;miVP2s对VP2的抑制效率为59.7%到78.5%。表明所设计的miRNAs对VP2均有抑制作用,其中miVP2A和miVP2E效果最为显著。

译名：

Selection for Effective miRNA Targeting VP2 Gene of Infectious Bursal Disease Virus

作者：

WANG Yong-juan1,CUI Ping-fu2,SUN Huai-chang3(1.Jiangsu Animal Husbandry and Veterinary College,Jiangsu Provincial Key Laboratory of Veterinary Bio-pharmaceutical High-tech Research,Taizhou 225300,China;2.Kunshan Entry-exit Inspection and Quarantine Bureau,Kunshan 215300,China;3.College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China)

关键词：

infectious bursal disease virus(IBDV);VP2;miRNA;fluorescent microscopy;flow cytometry

摘要：

To get effective miRNAs targeting the VP2 gene of infectious bursal disease virus(IBDV),VP2 gene from IBDV genomic RNA was amplified by RT-PCR,then was cloned into pEGFP and a reporter vector pVP2-EGFP was structured.After that,five miRNAs(named miVP2A,miVP2B,miVP2C,miVP2D and miVP2E,respectively) targeting VP2 gene of IBDV were predicted using a web-based tool softwear(genscript),and they were inserted into miRNA expression vector(pRFPRNAiC) and co-transfected into DF-1 cells with pVP2-EGFP.Expression of miVP2s were demonstrated by Northern dot blotting,and their silencing effect were tested by fluorescent microscopy and flow cytometry.The results showed that miVP2s can successfully express.After expression vectors were transfected with miVP2s,the number of GFP-positive cells and total fluorescence were obviously decreased than thore of control,and the inhibition rate of miVP2s against VP2 gene expression were in the range from 59.7% to 78.5%.These miVP2s can efficiently inhibit VP2 replication,among which,miVP2A and miVP2E are more effective.

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