组氨酸标签标记的口蹄疫重组病毒的制备及鉴定  

Generation and Identification of a Histidine Tagged Recombinant Foot and Mouth Disease Virus

作　　者：杨波[1,2] 杨帆[2] 王松豪[2] 张岩[2] 岳城[1] 郑海学[2] 殷宏[2] 

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,国家口蹄疫参考实验室,兰州730016

出　　处：《畜牧兽医学报》2014年第6期960-966,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(31302118);甘肃省科技重大专项计划项目(1302NKDA027);国家863计划项目(2011AA10A211-1);中国农业产业体系(CARS-39)

摘　　要：为了鉴定口蹄疫病毒(FMDV)插入位点并获得标记的重组病毒,本研究利用口蹄疫病毒反向遗传操作技术,在RGD基序后第9位和第10位氨基酸处(+9)引入组氨酸(His)标签,获得了含有His标签的FMDV全长cDNA克隆(命名为prAsia1-9His)。将prAsia1-9His质粒转染BHK-21细胞后,能够出现典型的细胞病变,对收获的病毒分别用RT-PCR、间接免疫荧光进行鉴定,结果证实,成功拯救了含His标签的重组病毒(命名为rAsia1-9His),该重组毒株能够稳定表达His标签。最后,比较测定了其对BHK-21细胞和乳鼠的致病性,结果表明,该重组毒株与亲本毒株(rAsia1)具有相似的生物学特性。含有标签标记的口蹄疫病毒的成功拯救,为深入研究口蹄疫病毒的致病机制以及分子标记疫苗奠定了基础。To identify a feasible insertion site in the foot-and-mouth disease virus(FMDV)and obtain a tagged recombinant virus,in this study,we have used reverse genetics to introduce a histidine(His)tag into the ninth site in the downstream of RGD motif in FMDV and obtain His tagged full-length-cDNA clone,named prAsia1-9 His.We transfected the prAsia1-9 His into BHK-21 cells and typical cytopathic effect(CPE)were found in the transfacted BHK-21 cells.Then,RT-PCR,indirect immunofluorescence assay and sequence analysis were performed,the results showed that the recombinant virus named as rAsia1-9 His was successfully rescued and the His tag was stably maintained.Finally,we compared rAsia1-9 His with the parental virus(named rAsia1) for pathogenicity to BHK-21 cells and suckling mouse,the results showed that they had similarity in the biological characteristics.The successful rescue of tagged FMDV lays a foundation for further study of molecular pathogenesis and marker vaccine of FMDV.

关 键 词：口蹄疫 标签 标记疫苗 

分 类 号：S852.65[农业科学—基础兽医学]