为构建携带Strep标签的禽流感病毒(Avian influenza virus,AIV)A/Goose/Hunan/109/2014(H5N6,HN109)反向遗传操作系统,RT-PCR扩增AIV HN109株8个基因片段,分别连接至pBD双向转录表达载体.通过点突变技术将NS1基因的77~84位的氨基酸替换成Strep-tag.将8个重组质粒共转染293T细胞,48 h后收取细胞上清接种至9~11日龄SPF鸡胚,并检测血凝效价.结果显示,本研究成功拯救病毒株rHN109-NS1-Strep.rHN109-NS1-Strep的8个基因片段序列与亲本毒素 HN109株一致;rHN109-NS1-Strep与亲本毒HN109株在MDCK细胞上复制水平相似,在小鼠体内病毒复制能力相似.结果表明,本研究已成功构建携带Strep-tag的H5N6亚型禽流感病毒反向遗传操作系统,为研究该病毒的分子致病机理、传播机制及新型疫苗的研制奠定了基础.%To establish a reverse genetics system of H5N6 Avian influenza virus with Strep-tag, the strain A/Goose/Hunan/109/2014 (H5N6,HN109 strain)was rescued carrying a Strep-tag in the NS1 protein. The whole genome including 8 segments of H5N6 AIV HN109 strain was amplified by RT-PCR and then cloned into pBD vector. The corresponding sequence at position 77-84 in NS1 was replaced by Strep-tag (WSHPQFEK) through site-directed mutagenesis. The resulting 8 recombinant plasmids were used for co-transfection into 293T cells. The supernatant of 293T cells was collected at 48 h post-transfection and inoculated into 9-day-old specific pathogen free (SPF) chicken embryonated eggs. Then Strep-bearing virus (rHN109-NS1-Strep) was rescued and identified by hemagglutination assay. Sequence analysis confirmed that the 8 gene fragments were consistent with the planned design. The rHN109-NS1-Strep showed indistinguishable replication level from the parental strain HN109 both in MDCK cells and mice. All of these results confirmed the success of the reverse genetics system of Avian influenza virus H5N6 with Strep-tag, which provided a technological platform for further study on the molecular pathogenicity, transmission and development of novel vaccines.
展开▼
