| 表达绿色荧光蛋白的重组鸡传染性支气管炎病毒的构建 |
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| 引用本文： | 周生,唐梦君,戴亚斌,刘梅,赵宝华,程旭,吕晓娟.表达绿色荧光蛋白的重组鸡传染性支气管炎病毒的构建[J].病毒学报,2011,27(1):11-17. |
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| 作者姓名： | 周生 唐梦君 戴亚斌 刘梅 赵宝华 程旭 吕晓娟 |
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| 作者单位： | 中国农业科学院,家禽研究所,扬州,225003;中国农业科学院,家禽研究所,扬州,225003;农业部家禽品质监督检验测试中心,扬州,225003 |
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| 基金项目： | 江苏省自然科学基金BK2008203; 中国农业科学院家禽研究所科研基金S015 |
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| 摘 要： | 为探讨鸡传染性支气管炎病毒(IBV)作为载体表达外源基因的可行性,本研究根据IBV H120疫苗株的全基因组序列设计引物,采用RT-PCR方法分10个片段对其基因组进行扩增,并克隆至pMD19-T载体中;同时构建IBV基因组5a基因编码区被增强型绿色荧光蛋白(EGFP)基因替换的重组质粒。采用体外拼接策略,将BsaI酶切处理的10个基因片段顺序连接,构建5a基因编码区被EGFP基因替换的基因组全长cDNA,其5’端具有完整的T7 RNA聚合酶启动子核心序列,3’端具有polyA尾巴结构。然后通过T7 RNA聚合酶体外转录系统合成病毒基因组RNA,脂质体转染BHK-21细胞进行病毒拯救。结果表明成功的从基因组全长cDNA拯救出重组病毒H120-5a/EGFP株,其在鸡胚中能有效的复制和传代,并表达绿色荧光蛋白;5a基因的缺失并不影响病毒对鸡胚的致病性。本研究为进一步开展IBV的分子致病机理、载体疫苗等研究奠定了基础。
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| 关 键 词： | 传染性支气管炎病毒 绿色荧光蛋白 重组病毒 病毒拯救 |
Expression of Green Fluorescent Protein Using an Infectious cDNA Clone of Infectious Bronchitis Virus |
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| ZHOU Sheng,TANG Meng-jun,DAI Ya-bin,LIU Mei,ZHAO Bao-hua,CHENG Xu,LU Xiao-juan.Expression of Green Fluorescent Protein Using an Infectious cDNA Clone of Infectious Bronchitis Virus[J].Chinese Journal of Virology,2011,27(1):11-17. |
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| Authors: | ZHOU Sheng TANG Meng-jun DAI Ya-bin LIU Mei ZHAO Bao-hua CHENG Xu LU Xiao-juan |
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| Institution: | ZHOU Sheng1,TANG Meng-jun1,2,DAI Ya-bin1,LIU Mei1,ZHAO Bao-hua1,CHENG Xu1,LU Xiao-juan1(1.Poultry Institute,Chinese Academy of Agricultural Sciences,Yangzhou 225003,China,2.Quality Inspection Center for Poultry of Ministry of Agriculture,China) |
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| Abstract: | An infectious cDNA clone of H120 vaccine strain of infectious bronchitis virus(IBV) was constructed to demonstrate its potential as a gene transfer vector.Primers were designed according to the published genome sequence of H120 strain,and ten cDNA fragments covering the entire genome of H120 strain was amplified by RT-PCR.All the PCR products were ligated into pMD19-T vector and sequenced,and the ORF5a open reading frame in the pMDTM9 plasmid was replaced by an enhanced green fluorescent protein(EGFP) gene.... |
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| Keywords: | infectious bronchitis virus EGFP recombinant virus virus rescue |
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