| 禽呼肠孤病毒σNS和σC基因shRNA载体构建及其干扰效果测定 |
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| 引用本文： | 熊文婕,谢芝勋,刘加波,庞耀姗,谢志勤,邓显文,谢丽基.禽呼肠孤病毒σNS和σC基因shRNA载体构建及其干扰效果测定[J].西南农业学报,2012,25(2):697-702. |
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| 作者姓名： | 熊文婕 谢芝勋 刘加波 庞耀姗 谢志勤 邓显文 谢丽基 |
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| 作者单位： | 1. 防城港市动物疫病预防控制中心,广西防城港,538001
2. 广西壮族自治区兽医研究所,广西南宁,530001 |
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| 基金项目： | 国家百千万人才工程入选专项资金项目，广西特聘专家专项经费，广西自然科学基金项目，广西留学回国人员基金项目 |
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| 摘 要： | 禽呼肠孤病毒σC基因是病毒粘附蛋白,σNS基因具有结合单链RNA活性。根据siRNA靶序列设计原则,设计并合成siRNA模板并克隆到shRNA表达载体pSilencer-CMV 4.1 neo。分别构建了针对σC基因的shRNA载体C1、C2、C3和针对σNS基因的shRNA载体NS1、NS2、NS3。将构建的shRNA载体和阴性对照分别与表达σC和σNS基因的融合蛋白的真核表达载体pEGFP-σC及pEGFP-σNS共转染DF-1细胞。荧光显微镜观察结果表明,6个shRNA片段不同程度地抑制各自融合蛋白的表达。Real-time PCR检测结果表明,C3和NS1体外干扰病毒复制的效果最佳。
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| 关 键 词： | 禽呼肠孤病毒 shRNA 干扰 |
Construction of Short Hairpin RNA Vector of Avian Reovirus Targeting σNS & σC Genes and Determination of Its Interference Effect |
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| XIONG Wen-jie
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XIE Zhi-xun
,
LIU Jia-bo
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PANG Yao-shan
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XIE Zhi-qin
,
DENG Xian-wen
,
XIE Li-ji.Construction of Short Hairpin RNA Vector of Avian Reovirus Targeting σNS & σC Genes and Determination of Its Interference Effect[J].Southwest China Journal of Agricultural Sciences,2012,25(2):697-702. |
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| Authors: | XIONG Wen-jie XIE Zhi-xun LIU Jia-bo PANG Yao-shan XIE Zhi-qin DENG Xian-wen XIE Li-ji |
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| Institution: | 1.Fangchenggang City Animal Disease Prevention and Control Center,Guangxi Fangchenggang 538001,China;2.Guangxi Veterinary Research Institute,Guangxi Nanning 530001,China) |
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| Abstract: | Avian reovirus σC gene was virus adhesion proteins,and σNS gene had the activity of binding single strand RNA.According to the siRNA target sequences,siRNA templates were designed,synthesized and then cloned into the shRNA expression vector pSilencer-CMV 4.1 neo.ShRNA vector C1,C2,C3 for σC gene and shRNA vectors for the σNS gene NS1,NS2,NS3 were constructed separately.ShRNA vectors of σC and σNS gene and negative control shRNA were co-transfected into DF-1 cells with the eukaryotic expression vector pEGFP-σC and pEGFP-σNS,respectively.Fluorescence microscopy results indicated that the constructed 6 shRNA fragments could inhibit the expression of fusion protein in some degrees.Real-time PCR tests demonstrated that C3 and NS1 had the best interference effect to the viral replication in vitro. |
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| Keywords: | Avian reovirus shRNA(short hairpin RNA) Interference |
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