“拯救”NA-1株鹅源副黏病毒微型基因组的构建

Construction of the mini-genome of NA-1 GPMV for rescue

根据GenBank上已发表的NA-1株GPMV全序列设计并合成2对特异性引物,克隆得到GPMV的Leader及Tralier序列,与T7启动子、丁肝病毒核酶序列及T7终止子重叠PCR连接后,将连接产物克隆至改造的pVAX-1载体中,并用增强型绿色荧光蛋白基因代替GPMV的整个编码区,只保留与病毒复制、转录和病毒包装相关的调控序列,酶切、测序及荧光鉴定后,与pCI-NP、pCI-P及pCI-L等3个辅助质粒共转染可表达T7RNA聚合酶的VT7细胞系,结果绿色荧光蛋白得到表达,表明成功构建了"拯救"病毒的微型基因组。

According to the gene sequences that had published in GenBank of Newcastle disease virus NA-1 from goose（GPMV） ,a pair of specific primers were designed for amplifying the two fragments of leader and trailer that were ligased with T7 promoter, HdvRz and T7 terminator with overlap PCR, then cloned them to the pVAX-1 which had been reconstituted. Change the coding region of the GPMV into EGFP,only the replication transcription and en casement of GPMV were remained. After the identification of digestion with splI,sequenced and fluorescence,trans-fected them with the help-plasmid of pCI NP, pCI-P and pCI-L to the VT7 cell lines which could express the T7 RNAP and the EGFP expressed. The result showed that the mini-genome of GPMV were constructed successfully.