表达基因Ⅶ型新城疫病毒F蛋白的重组鸡痘病毒的构建和筛选  

Construction and selection of a recombinant fowlpox virus expressing F protein of genotype Ⅶ of Newcastle disease virus

作　　者：辛舒[1,2] 田明尧[2] 曹佳媛 薛斐[2] 杜寿文[2] 谭鹏[1,2] 肖朋朋[2] 陈兴[1,2] 郭海宁[1,2] 金宁一[1,2] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]广西大学动物科学技术学院,广西南宁530004

出　　处：《中国兽医科学》2015年第3期235-240,共6页Chinese Veterinary Science

摘　　要：为了构建并筛选表达新城疫病毒(NDV)F蛋白的重组鸡痘病毒(rFPV-F),通过PCR扩增F基因,克隆至pMD18-T Simple载体,进行DNA序列分析和遗传进化关系分析。将测序正确的F基因克隆至笔者所在实验室自行构建的新型鸡痘病毒穿梭载体pTS2GFPV150中,获得重组质粒pTS2GFPV150-F。然后将其与鸡痘病毒FPV282E4株共转染鸡胚成纤维细胞进行同源重组,挑取表达增强型绿色荧光蛋白的噬斑,经多轮筛选获得重组病毒rFPV-F,应用PCR、RT-PCR、Western-blot等方法对重组鸡痘病毒进行鉴定。结果显示,成功获得NDV F基因及重组F基因的鸡痘病毒rFPV-F,并证实F基因已被整合到鸡痘病毒基因组中,且在感染细胞内被成功表达。本研究成功获得了1株表达NDV F基因的重组鸡痘病毒,为下一步开展动物体内免疫研究奠定了基础。To construct and select a recombinant fowlpox virus(rFPV-F)containing Newcastle disease virus(NDV)fusion gene,the gene was amplified by RT-PCR,cloned into pMD18-T Simple Vector,and then analyzed through DNA sequencing and genetic evolutionary analyses.The F gene was then inserted into a fowlpox virus shuttle vector pTS2GFPV150,resulting in the recombinant plasmid pTS2GFPV150-F.The recombinant plasmid was transfected into chicken embryo fibroblast(CEF)cells by infecting with fowlpox virus 282E4 strain,and the recombinant fowlpox virus rFPV-F was screened by enhanced green fluorescent protein plaques.PCR,RT-PCR and Western-blot were used to detect the expression of NDV-F in the infected CEF cells.In result,NDV F gene and rFPV-F were obtained,and the fusion gene was successfully expressed in CEF cells.In addition,the recombinant fowlpox virus rFPV-F has been obtained.In conclusion,this study obtains a recombinant fowlpox virus which provides the good foundation for future study of NDV F immunity experiment in animals.

关 键 词：新城疫病毒 基因Ⅶ型 F蛋白 鸡痘病毒穿梭载体 同源重组 

分 类 号：S852.65[农业科学—基础兽医学]