新城疫病毒NP和P基因辅助质粒的构建及鉴定

Construction and identification of NP and P helper plasmids containing genes of the Newcastle disease virus

目的构建新城疫病毒JL02/2000株P和NP基因真核表达质粒,并在BHK-21细胞中表达,为该病毒的反向遗传操作系统的建立奠定了基础。方法根据新城疫病毒(JL02/2000毒株)的全基因组中的NP、P基因分别设计引物,应用RT-PCR方法扩增NP基因和P基因,将扩增的NP基因和P基因片段连接到pMD18-T载体上,限制性内切酶EcoRⅠ和XbaⅠ酶切鉴定确认后回收目的条带,分别插入到真核表达载体pCI上,经测序确认pCI-NP、pCI-P辅助质粒的构建。将pCI-NP、pCI-P转染BHK-21细胞,两次换液,72h后回收细胞,经RT-PCR检测NP和P基因片段。结果构建的辅助质粒pCI-NP和pCI-P经双酶切和测序鉴定到目的片段。pCI-NP、pCI-P转染后,RT-PCR检测到NP和P基因片段。结论新城疫病毒pCI-NP、pCI-P辅助质粒构建成功,pCI-NP、pCI-P可在BHK-21细胞中表达,为该病毒的反向遗传操作系统的建立奠定了基础。

Objectives To construct eukaryotic expression plasmids containing the NP and P genes of the Newcastle disease virus （JL02/2000 strain） and to express them in BHK-21 cells in order to lay the foundation for reverse genetic engi neering of the Newcastle disease virus. Methods Primers for the NP and P genes were designed in accordance with the complete genome sequence of Newcastle disease virus （JL02/2000 strain）. The NP and P genes were amplified using RT- PCR, ligated to the vector pMD-18T, and then inserted into a pCI eukaryotic expression vector. Once the vectors were identified using restriction enzyme digestion with EcoR I and Xba I . The constructed helper plasmids pCI-NP and pCI-P were confirmed using sequencing, pCI-NP and pCI-P were transfected into BHK-21 cells. The cells were then treated and the culture solution was changed twice. Cells were collected after 72 hours. RT PCR was used to detect the NP and P genes. Results The target sequences pCI-NP and pCI-P were constructed and identified using restriction enzyme diges tion and sequencing. The NP and P genes were detected using RT-PCR after the transfection of pCI-NP and pCI-P. Conclusion The helper plasmids pCI-NP and pCI-P were constructed and successfully expressed in BHK-21 cells accord ing to RT-PCR. This study has laid the foundation for the reverse genetic engineering of the Newcastle disease virus.