NDV-F基因稳定表达P815细胞株的建立

在成功地构建NDV-F基因真核表达载体pCDNA3-NDV-F的基础上，应用脂质体LipofectamineTM2000介导使其转染小鼠肥大细胞瘤细胞株（P815），以G418加压筛选（600μg/ ml）得到稳定的克隆并扩大培养成系。应用Trizol法提取细胞RNA，经RT-PCR鉴定，转染细胞在1600bp处出现目的条带。收集转染细胞，经Western blot和免疫荧光法检测到目的蛋白的表达。进一步将获得的细胞株在液氮中冻存6个月以检测其稳定性，实验证明该细胞株仍能很好的表达F基因。本实验采用脂质体法已成功建立了能够稳定表达NDV-F基因细胞株，为进一步研究ND核酸疫苗的效果奠定良好的实验基础。

Establishment of P815 Cell Line Stably Expressing NDV-F gene

After the eukaryotic expression vector pcDNA3-NDV-F was constructed successfully, the recombinant was transfected into P815 cell by LipofectamineTM 2000, and the cells were screened with G41 8 to obtain stable clone. The total RNA was prepared from transfected cells according to the instructions of the Trizol reagent and the 1600bp fragment was obtained by RT-PCR. Westem blotting and immunofluorescence were used to detect the expression of NDV-F protein. In order to detect the stability of the transfected cell line, the cell line was freezed in the liquild nitrogen for six months. The experiments proved NDV–F gene were expressed well. These results suggested that the stable transfected P815 cell line was successfully established by Lipofectin, which provides solid foundation for further experimental studies on the function NDV nucleotide vaccine.