| 鸡传染性法氏囊病病毒Gt株感染性分子克隆的构建 |
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| 引用本文： | 祁小乐,高宏雷,高玉龙,邓小芸,步志高,王晓燕,王笑梅.鸡传染性法氏囊病病毒Gt株感染性分子克隆的构建[J].中国农业科学,2007,40(10):2343-2349. |
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| 作者姓名： | 祁小乐 高宏雷 高玉龙 邓小芸 步志高 王晓燕 王笑梅 |
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| 作者单位： | 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/禽传染病研究室,哈尔滨,150001 |
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| 基金项目： | 国家重点基础研究发展计划(973计划) |
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| 摘 要： | 【目的】建立高效鸡传染性法氏囊病病毒（IBDV）拯救平台，为深入研究该病毒基因组的结构与功能奠定基础。【方法】在IBDV Gt株全基因组中引入分子标签（A节段：EcoR V酶切位点；B节段：PstI酶切位点）。在基因组两端分别引入锤头状核酶结构（HamRz）和丁肝病毒核酶结构（HdvRz)。将带有分子标签和核酶结构的IBDV基因组插入载体pCAGGS的β肌动蛋白启动子下游，构建IBDV感染性克隆pCAGGmGtAHRT和pCAGGmGtBHRT，LipofectamineTM2000介导共转染DFI细胞，进行病毒拯救研究。 【结果】 构建的IBDV感染性克隆可在DFI、Vero/E6、Vero/P12等3种细胞上高效拯救出病毒。RT-PCR、间接免疫荧光、电镜等均检测到了拯救的IBDV，且存在分子标签。拯救毒在CEF上能产生特有的砂砾状CPE，且TCID50与亲本毒差异不显著，具有相似的生物学特征。【结论】构建的RNA聚合酶ⅢBDV拯救系统高效、稳定、简便、经济，且具有良好的细胞普适性。
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| 关 键 词： | 传染性法氏囊病病毒 感染性分子克隆 病毒拯救 分子标签 核酶 |
| 收稿时间： | 2006-6-15 |
| 修稿时间： | 2006-06-15 |
Development of Infectious Molecular Clones in Infectious Bursal Disease Virus Gt Strain |
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| QI Xiao-le,GAO Hong-lei,GAO Yu-long,DENG Xiao-yun,BU Zhi-gao,WANG Xiao-yan,WANG Xiao-mei.Development of Infectious Molecular Clones in Infectious Bursal Disease Virus Gt Strain[J].Scientia Agricultura Sinica,2007,40(10):2343-2349. |
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| Authors: | QI Xiao-le GAO Hong-lei GAO Yu-long DENG Xiao-yun BU Zhi-gao WANG Xiao-yan WANG Xiao-mei |
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| Institution: | Division of Avian lnfectiouss Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute Chinese Academy of Agricultural Sciences, Harbin 150001 |
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| Abstract: | Abstract: Infectious bursal disease (IBD) ,mediatied by infectious bursal disease virus (IBDV),is one of the causive agents which cause significant losses to the poultry industry. In this research, EcoRⅤ site or PstⅠsite ,as the genetic tags,was introduced separately into the A segment or B segment of the IBDV Gt strain.The full-length genome was flanked by hammerhead ribozyme(HamRz) and hepatitis delta ribozyme(HdvRz) sequence. The full-length genome containing genetic tags flanked by HamRz and HdvRz were arranged downstream of the Beta chincken actin promoter of the vector pCAGG.The recombinant plasmid ，which were infectious clones,were named pCAGGmGtAHRT and pCAGGmGtBHRT.These clones can infection DFⅠcell effectively.The results of RT-PCR,indirect immunofluorescence,electron microscope showed IBDV was rescued successfully. The genome of rescued IBDV have genetic tags.The rescued IBDV could cause CPE on CEF,and the TCID50 of rescued IBDV was similar to Gt.The method of rescuing verus was the basis for further researching IBDV. |
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| Keywords: | Infectious bursal disease virus Infectious molecular clones Virus rescue Genetics tags Ribozyme |
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