鹅源新城疫病毒拯救体系的建立

Establishment of Reverse Genetics System for NDV Isolated from Goose

参照GenBank上公布的鹅源新城疫病毒ZJI株全序列,设计8对引物,经RT-PCR法从尿囊液中扩增目的片段后,分别克隆进pCR2.1载体,将Ⅰ～Ⅶ7对引物的扩增片段依次亚克隆到TVT7R转录载体中,构建了含NDV全基因组cDNA的转录载体(pNDVZJI),将Ⅴ、Ⅵ和Ⅷ3对引物的扩增片段分别克隆进pCR2.1载体,并亚克隆到表达质粒pCI-neo上,构建了L基因的真核表达载体(pCI-L),pNDVZJI、pCI-L与另外两个辅助表达质粒(pCI-NP和pCI-P)共转染BSR-T7/5细胞,成功拯救出了具有血凝性的鹅源新城疫病毒,ZJI株鹅源新城疫病毒的成功拯救为后续相关研究工作的开展打下了基础。

Eight fragments were amplified and cloned into pCR2.1 vector with the designed primers. The fragments, amplified with primer Ⅰ to Ⅶ, were subcloned into transcription vector to construct the plasmid pNDVZJI which contained the full-length cDNA of NDV ZJI strain. The eukaryotic expression vector pCI-L was constructed by subcloning the fragments, amplified with the primer Ⅴ, Ⅵ and Ⅶ, into the expression vector pCI-neo. The full-length cDNA clone, pNDVZJI, with three helper plasmids, pCI-NP,pCI-P and pCI-L, were cotranfected into BSR-T7/5 cell expressing T7 RNA polymerase. After inoculation of transfected cell culture into embryonated chicken eggs from specific pathogen free（SPF） flock, The NDV of ZJI strain was rescued successfully, which laid a good foundation for the further related research.