鸡痘病毒复制非必需位点的筛选及鉴定  

Identification of fowlpox virus nonessential gene locus for the foreigner integration

作　　者：张雪梅[1] 赵妍[1,2] 张晓彩[1] 王云峰[1,3] 

机构地区：[1]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/禽呼吸道病创新团队,黑龙江哈尔滨150001 [2]东北农业大学动物科学与技术学院,黑龙江哈尔滨150030 [3]哈尔滨动物生物制品国家工程研究中心有限公司,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2015年第6期422-425,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家科技支撑计划课题(2015BAD12B05);中国博士后科学基金面上资助(2014M560245)

摘　　要：为筛选新的高效的鸡痘病毒(FPV)中外源基因的插入位点,本研究选择FPV ORF161与ORF162之间的基因间隔区作为重组位点,构建表达增强型绿色荧光蛋白(EGFP)的转移载体,以表达鸡传染性喉气管炎病毒g B基因的重组FPV(r FPV-g B)为亲本病毒,通过在鸡胚成纤维细胞(CEF)中同源重组,并经过蚀斑纯化筛选获得能够稳定表达gfp报告基因的重组病毒,命名为r FPV-g B161gfp。将r FPV-g B161gfp与亲本病毒连续传代20代,每5代进行荧光显微镜观察、PCR检测及复制动力学比较,结果表明重组病毒遗传稳定性良好,而且重组病毒与亲本病毒生长特性一致。本研究鉴定的新外源基因插入位点为构建多价重组FPV疫苗奠定了基础。To identify the non-essential gene for fowlpox virus （FPV） replication, two DNA fi＇agments between FPV ORF161 and ORF162 were amplified by PCR and cloned into pUC18 flanking the gfp gene as the homologous arms to construct transfer recombinant plasmid, which was transfected into the pre-rFPV-gB （the recombinant FPV expressing gB gene of infectious bronchitis of chicken） infected chicken embryo fibroblast （CEF） to rescue the recombinant FPV （rFPV）of rFPV-gB 161gfp. The rFPV were proved to be genetically stable when passed in CEF for 20 passages according to the GFP expression and PCR detection of the g09 gene. Moreover, the replicaton dynamics were compared for the rFPV and the parental virus which showed they had the similar replication curves. Our study provided a basis for the development multible valent rFPV vaccine.

关 键 词：重组鸡痘病毒 插入位点 增强型绿色荧光蛋白 

分 类 号：S852.65[农业科学—基础兽医学]