新城疫病毒DF-1细胞蚀斑纯化方法的建立

Establishment of Plaque Purification for Newcastle Disease Virus using DF-1 Cell

本研究对DF-1细胞的培养条件及新城疫病毒(NDV)DF-1细胞的蚀斑纯化方法进行了优化摸索,结果显示相较于DMEM培养基,DF-1细胞更适宜在含15%胎牛血清的DMEM/F12培养基中生长,DF-1细胞以3×105/孔的剂量覆盖6孔板能获得最佳的铺板效果,吸附病毒后1.5h覆盖第1层琼脂,48h后覆盖第2层琼脂能获得最佳的蚀斑形成效果。通过对蚀斑纯化株F和HN基因测序分析,结果表明蚀斑纯化后无任何变异。通过此方法可在1个星期之内获得稳定的纯化毒株,方法简单、可重复性强、纯化效率高。

In order to establish a new cell culture system for the purification of Newcastle disease virus （NDV）, NDV was plaque purified by using the chicken embryo fibroblast cell line （DF-1）. The results showed that, compared to the common basal media （DMEM） used in cell culture, DMEM/F12 with 15% fetal bovine serum was better. The optimized purification conditions were as follows, 3 × 10^5 DF-1 cells per well were seeded into 6 well plates for 24 h, then infected with NDV for 1.5 h. After removing all virus from each plate, the first agarose overlay was added and incubated for 48 h before the operating of the second agarose overlay. After purification, the homologies of F and HN genes between the purified virus and the original virus were 100 %, respectively, which indicated no mutation was found during the purification and screening. By using this method, it would take only one week to obtain the purified virus with the simply manipulability, high repeatability and efficiency.