表达鸡新城疫病毒F、HN基因和传染性喉气管炎病毒gB基因的重组鸡痘病毒的构建及其特性研究  

Construction and Characterization of a Recombinant Fowlpox Virus Co-expressing F,HN Genes of Newcastle Disease Virus and gB Gene of Infectious Larygnotracheitis Virus

作　　者：孙惠玲[1,2,3] 王云峰[4] 苗得园[5] 张培君[5] 智海东[4] 徐灵龙[4] 王玫[4] 童光志[1,2] 汪明[1] 

机构地区：[1]中国农业大学动物医学院 [2]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,哈尔滨150001 [3]北京市农业科学院畜牧兽医研究所,北京100094 [4]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 [5]北京市农业科学院畜牧兽医研究所

出　　处：《生物工程学报》2006年第6期931-939,共9页Chinese Journal of Biotechnology

基　　金：国家高技术研究与发展计划项目(No.2001AA213041);北京市科技计划项目(No.H030730011030)资助。~~

摘　　要：利用同源重组将新城疫病毒(NDV)的F和HN基因、传染性喉气管炎病毒(ILTV)的gB基因以及报告基因LacZ插入鸡痘病毒(FPV)的017株的复制非必需区,其中NDV的F、HN基因、ILTV的gB基因以及报告基因LacZ是在早晚期启动子LP2EP2的控制下,大肠杆菌报告基因LacZ在晚期启动子P11的控制下。经过10轮蓝斑纯化获得了包含了NDV的F和HN基因、ILTV的gB基因以及报告基因LacZ的重组鸡痘病毒,称为rFPV-F/HN/gB/LacZ。经PCR方法证明rFPV-F/HN/gB/LacZ基因组中含有NDV的F基因、HN基因和ILTVgB基因;间接免疫荧光试验和Western-blot试验表明NDV的F、HN蛋白和ILTVgB蛋白在rFPV-F/HN/gB/LacZ感染的CEF细胞中获得表达。与亲本毒相比,重组病毒在病毒的复制和致鸡胚成纤维细胞的病变方面无显著不同。这证明了在鸡痘病毒载体的一个复制非必需区可以同时插入多个禽类病原的多个外源基因,为制备多价基因工程疫苗奠定了基础。The Fusion （F） and Haemagglutinin-Neuraminidase （HN） genes of Newcastle disease virus （NDV） and the glycoprotein B （gB） gene of infectious laryngothracheitis virus （ILTV） as well as a LacZ reporter gene were all inserted into a nonessential gene of fowlpox virus （FPV） 017 strain by homologous recombination. The NDV and ILTV genes were each under the control of a fowlpox virus immediate early/late promoter （LP2EP2） while the LacZ reporter gene expression cassette was regulated by a P11 late promoter. A recombinant FPV harboring the F, HN and gB genes as well as the LacZ gene, designated as rFPV-F/HN/gB/LacZ, was obtained after ten cycles of blue plaque purification. The presence of the NDV and ILTV genes was confirmed by PCR. The expression of the recombinant proteins in rFPV-F/HN/gB/LacZ were characterized by Western blot （ F and gB proteins） and indirect immunofluorescence test （ F, HN and gB proteins）. The results demonstrated that all four foreign proteins, which were encoded within a 10kb gene fragment, could be expressed authentically and efficiently. Compared to the parental virus, rFPV-F/HN/gB/LacZ showed no obvious difference with respect to virus replication and cytopathogenic effects in chicken embryo fibroblasts （CEF） cell culture. Overall, our work suggests that FPV can be a useful live virus vector for the expression of multi- foreign genes against multiple avian pathogens.

关 键 词：重组鸡痘病毒 新城疫病毒 传染性喉气管炎病毒 F基因 HN基因 GB基因 

分 类 号：S852.5[农业科学—基础兽医学] Q789[农业科学—兽医学]