| M蛋白基因shRNA抑制PRRSV在Marc145细胞中复制的研究 |
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| 引用本文： | 黄娟,姜平,李玉峰,蒋文明.M蛋白基因shRNA抑制PRRSV在Marc145细胞中复制的研究[J].中国农业科学,2008,41(1):259-264. |
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| 作者姓名： | 黄娟 姜平 李玉峰 蒋文明 |
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| 作者单位： | 1. 青岛农业大学
2. 南京农业大学动物医学院/农业部动物疫病诊断与免疫重点开放实验室,南京,210095 |
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| 基金项目： | 国家自然科学基金
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江苏省自然科学基金
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高等学校博士学科点专项科研项目 |
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| 摘 要： | 【目的】通过靶向PRRSV基因组中的M基因的siRNA来抑制PRRSV在Marc145细胞中的复制。【方法】构建4个能转录小发夹RNA（shRNA）的质粒，将其与靶蛋白表达质粒共转染HEK293A细胞，观察荧光或进行半定量PCR；或将其转染Marc145细胞，感染PRRSV后进行IFA、TCID50和实时PCR检测。【结果】shRNA表达质粒对M融合蛋白表达的抑制率约为50％，使M真核质粒表达蛋白的mRNA水平降低54%～64%，表达的shRNA在PRRSV感染后48 h使病毒的TCID50和mRNA水平均降低到1/10～1/100倍，间接免疫荧光结果表明shRNA表达质粒转染孔的荧光细胞数显著减少。【结论】shRNA表达质粒特异性的抑制了靶蛋白M和PRRSV的复制，靶向PRRSV基因组M基因不同区域的siRNA可以作为控制该病毒传播的候选策略。
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| 关 键 词： | PRRSV RNA干扰 M蛋白基因 |
| 收稿时间： | 2006-07-17 |
| 修稿时间： | 2007-03-06 |
Inhibition of Porcine Reproductive and Respiratory Syndrome Virus Replication by Short Hairpin RNA Targeting to M Protein Gene |
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| HUANG Juan,JIANG Ping,LI Yu-feng,JIANG Wen-ming.Inhibition of Porcine Reproductive and Respiratory Syndrome Virus Replication by Short Hairpin RNA Targeting to M Protein Gene[J].Scientia Agricultura Sinica,2008,41(1):259-264. |
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| Authors: | HUANG Juan JIANG Ping LI Yu-feng JIANG Wen-ming |
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| Abstract: | Abstrct:【Objective】To evaluate the inhibition ability of shRNAs targeting to M protein gene of Porcine Reproductive and Respiratory Syndrome virus;【Method】shRNA-expressing plasmids were constructed and delivered to HEK293A or Marc145 cells,efficiency of RNA interfering was assayed by semiquantitative RT-PCR, indirect immunofluorescence assay(IFA), virus titre of TCID50 and real time PCR;【Results】After cotransfection with fusion-protein expressing plasmids, the fluorescence in HEK293A cells treated by shRNA expressing plasmids became obviously weaker comparing to those cotransfected with target gene expressing plasmid and pSUPER plasmid. Different shRNA expressing vectors were also cotransfected into HEK293A cells with vectors expressing M proteins of PRRSV, and the mRNA level of M protein was inhibited to 54%-64% assayed by semi-quantitative PCR. These plasmids expressing shRNA were also delivered into Marc145 cells. After infection, these cells showed a significant decrease in virus yield when compared to control cells, by detection using virus titers (TCID50), IFA and real-time RT-PCR; 【Conclusion】shRNA targeting to M protein gene of PRRSV could inhibit M protein expression and PRRSV replication in Marc145 cells. |
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| Keywords: | PRRSV RNA interference (RNAi M protein gene |
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