稳定表达T7RNA聚合酶的细胞系的构建及鉴定  

Construction and identification of cell-line of stable expressing T7 RNA polymerase

作　　者：母连志[1,2] 孙玉章[1,2] 丛彦龙[1,2] 李少丽[1,2] 张晓东[1,2] 王广美[1,2] 王晓莉[1,2] 丁壮[1,2] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]吉林大学人兽共患病教育部重点实验室,吉林长春130062

出　　处：《中国兽医学报》2011年第1期26-28,54,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30771606);吉林省自然科学基金资助项目(201015114)

摘　　要：设计1对特异性引物扩增痘苗病毒VTF7-3株的T7RNA聚合酶基因并连接真核表达载体,利用Lipo-fectamineTM2000将重组质粒转染BHK-21细胞,G418筛选2周后,转入验证质粒pT7-HN,经Western-blot和间接免疫荧光鉴定后证实获得了1株能稳定表达T7RNA聚合酶的VT7细胞系。构建的VT7细胞系传30代还能够稳定的表达T7RNA聚合酶,为下一步建立有效的鹅源副黏病毒病毒反向遗传操作平台奠定了基础。According to the gene sequences of strain VTF7-3 published in GenBank,a pair of specific primers were designed for amplifying the complete gene of T7 RNA polymerase and ligasing with pCI-neo vector.Recombinant plasmid pCI-T7 pol was transfected into BHK-21 cells by LipofectamineTM 2000.The cells were selected in a medium containing geneticin（G418） for two weeks,a help-plasmid of pT7-HN was transfected and detected by Western-blot and indirect immuno fluorescence assay.The result showed that VT7 cell line expressing T7 RNA polymerase stably at least 30 passages was constructed successfully,which provides an excellent platform for rscue of GPMV strain NA-1.

关 键 词：T7RNA聚合酶 克隆 VT7细胞系 病毒拯救 

分 类 号：S852.65[农业科学—基础兽医学]