表达绿色荧光蛋白的重组鸡传染性支气管炎病毒的构建  

Expression of Green Fluorescent Protein Using an Infectious cDNA Clone of Infectious Bronchitis Virus

作　　者：周生[1] 唐梦君[1,2] 戴亚斌[1] 刘梅[1] 赵宝华[1] 程旭[1] 吕晓娟[1] 

机构地区：[1]中国农业科学院家禽研究所,扬州225003 [2]农业部家禽品质监督检验测试中心,扬州225003

出　　处：《病毒学报》2011年第1期11-17,共7页Chinese Journal of Virology

基　　金：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

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。An infectious cDNA clone of H120 vaccine strain of infectious bronchitis virus （IBV） was con- structed 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 ORFSa open reading frame in the pMDTM9 plasmid was replaced by an enhanced green fluorescent protein （EGFP） gene. Recombinant plasmids were digested by the restriction enzyme Bsa I, and all the cDNA fragments were recovered. By using appropriate ligation strategy, the genomic cDNA of H120 strain were reconstituted. Then genome RNA was synthesized in vitro by T7 RNA polymerase and transfected into BHK-21 cells. Recombinant virus expressing the green fluorescent protein was rescued and identified by RT-PCR and sequencing. The characteristics of recombinant virus were evaluated by passage in embryonated chicken eggs. This study showed that the 5a ORF is a good candidate for an insertion site of recombinant genes for the development of IBV infectious cDNA clone as a gene transfer vector.

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 

分 类 号：S852.659.6[农业科学—基础兽医学]