传染性法氏囊病病毒凋亡调节蛋白表达细胞株的建立

Establishment of stable cell strains expressing apoptotic regular protein of infectious bursal disease virus

构建传染性法氏囊病病毒非结构蛋白基因（NS）的重组表达质粒pEGFP-C2-NS,然后在LipofectamineTM 2000介导下转染Vero细胞,活细胞状态下直接观察EGFP-NS蛋白在细胞中的表达与定位。结果显示,转染后4~5 h出现荧光蛋白表达,24 h达到高峰,呈小点样环核膜与细胞膜附近沉积,经G418筛选2~3周后稳定表达的荧光蛋白主要分布于细胞膜上。G418抗性细胞,经RT-PCR和Western-blot验证NS mRNA及其蛋白表达,提示已建立能稳定表达EGFP-NS蛋白的细胞株,所表达蛋白具有NS和EGFP双重活性,为进一步研究NS蛋白凋亡调节机制打下了基础。

The recombinant plasmid pEGFP-C2-NS was transfected into Vero cells by the LipofectamineTM 2000,ring-like patches of EGFP-NS protein were observed in close proximity to the perinuclear and cellular membrane of Vero cells 4-5 h after transfection,and reached the peak after 24 h,furthermore were distinctively disposed in cellular membrane of the cells which obtained by G418 screening after 2-3 weeks.Transcription and translation of NS gene of IBDV were confirmed by RT-PCR and Western-blot analyses in G418-resistant cell line.The results may be of great value for further investigation of the regular mechanism of NS protein on apoptosis.