新城疫病毒Clone 30株辅助表达系统的构建及真核表达

Construction and Eukaryotic Expression of Recombinant Plasmids of Newcastle Disease Virus Clone 30 Strain

为研究新城疫病毒NP、P和L蛋白作用机理和新城疫病毒反向遗传操作系统,构建新城疫病毒Clone 30株NP、P和L基因的真核表达载体。根据Gen Bank中新城疫病毒Clone 30株的全长序列(Y18898.1),设计3对特异性引物,用RT-PCR法扩增出新城疫病毒Clone 30株的NP、P和L基因,将其克隆到真核载体pCI-neo上。通过酶切和测序验证克隆正确,得到重组质粒pCI-NP、pCI-P和pCI-L,瞬时转染到BHK-21细胞中。经RT-PCR、Western blot、间接免疫荧光试验分别检测NP、P和L蛋白在BHK-21细胞中的表达。结果表明,重组质粒pCI-NP、pCI-P和pCI-L构建成功,为后期新城疫病毒反向遗传操作系统的构建奠定了基础。

In order to study the mechanism of NP,P and L protein and the reverse genetic operating system of Newcastle disease virus, the eukaryotic expression vector of NP, P and L gene of Newcastle disease virus Clone 30 strain was constructed. According to the full-length sequence of Clone 30 strain published in GenBank （Y18898.1）, 3 pairs of specific primers were designed,and NP,P and L genes of Clone 30 strain were amplified by RT-PCR,and the genes were cloned into the eukaryotic expression vector pCI-neo. The cloning was veryfied by restriction enzyme digestion and sequencing. The recombinant plasmid pCI-NP, pCI-P and pCI-L were obtained, and the recombinant plasmid was transiently transfected into BHK-21 cells. The expression of NP,P and L protein in BHK-21 cells were detected by RT-PCR,Westeru blot and indirect immunofluorescence assay. The results showed that the recombinant plasmid pCI-NP,pCI-P and pCI-L were successfully constructed,which laid a foundation for the construction of the reverse genetic operating system of Newcastle disease virus.