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

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.