| 稳定表达T7 RNA聚合酶IBRS-2细胞系的建立以及利用该细胞系对SVDV的体内拯救 |
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| 引用本文： | 郑海学,田 宏,靳 野,吴锦艳,尚佑军,刘湘涛,谢庆阁. 稳定表达T7 RNA聚合酶IBRS-2细胞系的建立以及利用该细胞系对SVDV的体内拯救[J]. 生物化学与生物物理进展, 2008, 35(4): 449-456 |
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| 作者姓名： | 郑海学 田 宏 靳 野 吴锦艳 尚佑军 刘湘涛 谢庆阁 |
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| 作者单位： | 1. 中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,农业部畜禽病毒学重点开放实验室,兰州,730046;广东省农业科学院兽医研究所,广州,510640
2. 中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,农业部畜禽病毒学重点开放实验室,兰州,730046 |
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| 基金项目： | 国家重点基础研究发展计划(973计划) , 国家科技支撑计划 |
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| 摘 要： | 利用PCR从λ溶原性细菌DE3中扩增出T7 RNA聚合酶基因,定向克隆进逆转录病毒载体pBABEpuro,得到阳性重组质粒pTTBABEpuro.把该重组质粒与pVSV-G共感染GP2-293包装细胞系,形成假型病毒.通过polybrene介导假型病毒感染IBRS-2细胞,利用嘌呤霉素进行传代筛选,形成细胞系IBRST7.对不同代次的IBRST7细胞基因组进行PCR和RT-PCR鉴定,结果表明,T7 RNA聚合酶基因在细胞传代过程中能稳定存在,并能表达目的蛋白的mRNA.为了鉴定T7 RNA聚合酶在IBRST7细胞内是否具有转录活性,扩增口蹄疫病毒(FMDV)的内部核糖体进入位点(IRES)片段和EGFP基因,定向克隆于原核表达载体pET-43.Ia-c( )中,构建了T7启动子控制下转录的具有非帽依赖性表达的重组质粒pIERS-EGFP-ET,把该质粒转染IBRST7细胞,能够在紫外显微镜下观察到绿色荧光,说明EGFP得到了表达,表明IBRST7细胞系内的T7 RNA聚合酶具有转录活性.然后,利用该细胞系成功拯救出具有感染性的猪水泡病病毒(SVDV),并对其生物学功能进行了鉴定.该细胞系的建立为利用T7 RNA聚合酶转录系统体内高效拯救病毒提供了基础.该拯救病毒的策略使RNA拯救简化为一步快速的拯救方法,为进一步探索SVDV病毒致病的分子机制及研制新型SVD疫苗奠定了良好的基础.
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| 关 键 词： | T7RNA聚合酶 IBRST7细胞系 假型病毒 SVDV 病毒拯救 逆转录病毒载体 感染性克隆 稳定表达 聚合酶 细胞系 利用 SVDV Cells Recovery RNA Polymerase Cell Line 疫苗 分子机制 方法 快速 简化 策略 转录系统 生物学功能 猪水泡病 感染性 绿色荧光 |
| 收稿时间： | 2007-08-29 |
| 修稿时间： | 2007-08-29 |
Establishment of IBRS-2 Cell Line Stably Expressing T7 RNA Polymerase and Recovery of SVDV From IBRST7 Cells |
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| ZHENG Hai-Xue,TIAN Hong,JIN Ye,WU Jin-Yan,SHANG You-Jun,Liu Xiang-Tao and XIE Qing-Ge. Establishment of IBRS-2 Cell Line Stably Expressing T7 RNA Polymerase and Recovery of SVDV From IBRST7 Cells[J]. Progress In Biochemistry and Biophysics, 2008, 35(4): 449-456 |
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| Authors: | ZHENG Hai-Xue TIAN Hong JIN Ye WU Jin-Yan SHANG You-Jun Liu Xiang-Tao XIE Qing-Ge |
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| Affiliation: | Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China; Veterinary Research Institute of Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China |
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| Abstract: | |
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| Keywords: | T7 RNA polymerase(T7 RNAP) IBRS-2 cell pseudotype virus retroviral vector infectious clone |
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