摘要
比较17D 213-77黄热病病毒株(17D)在不同基质细胞中的培养,观察病毒增殖的情况,探索一种替代目前黄热病疫苗生产的方法。以17D在经典的全鸡胚卵黄囊中传代培养作为对照方法,分别研究其在鸡胚成纤维细胞(DF-1细胞)和Vero细胞上传代培养条件,优化病毒感染复数(Multiplicity of Infection,MOI)和培养时间;将17D在DF-1细胞上连续传代,观察子代病毒的滴度变化;同时将17D在原代鸡胚细胞(Primary Chicken Embryonic Fibroblasts,CEF)上传代培养。17D在DF-1细胞传代培养最适MOI为0.0001,最佳培养时间为3 d,且连续传代后可以保持稳定的病毒滴度(>8 Lg PFU/m L);17D在Vero细胞传代培养最适MOI为0.01,最佳培养时间为4 d;4种方式传代17D在接种量、培养时间和收获病毒量上有一定区别。DF-1细胞在培养17D上存在一定优势,有成为生产17D黄热病疫苗的基质细胞的可能。
The traditional production methodfor 17D 213-77 Yellow Fever Virus(17D),which 17D inoculated EV-7 chick embryos,wastime consuming and labor intensive.Here we described a method using chicken embryo fibroblast(DF-1)to produce 17D.We compared differentways to propagate 17D and to explore an alternative for currentmode of yellow fever vaccine production by observing the proliferation of 17D.When normal cells in 6-wellplates grew to over 95%,17D was subcultured on Vero cells or DF-1 cells,of which the conditions for propagation were different,including MOI and harvest time.Based on optimized virus passaging methods,17D was serially passaged 30 times on DF-1 cells culture.The dense monolayer of CEF cells was inoculated 17 Dat MOI 0.001 for3 days.The titers of harvest virus were all detected by plaque formation assays.Under the optimum conditions,17D was propagated on DF-1 cells at MOI 0.0001 and the virus liquid was harvested on the third day after inoculation.The stable viral titer can be maintained after continuous passages on DF-1 cells,which was more than 8 Lg PFU/m L.On the contrast,optimized inoculative condition for Vero cells were to inoculate 17D at optimal MOI 0.01,and harvest time was on the forth day.There were some differencesin inoculation size,harvest time,harvest size and virus titer between these four passage ways to propagate 17D.The process of vaccine production on a simple cellular environment was more profitable than that on chick embryos or CEF cells.By comparison,DF-1 cells had its advantages on the propagation of 17D and it has the potentialto be a host cell of 17D.
作者
钱依韵
许定花
熊文典
刘孝民
金坚
陈蕴
QIAN Yi-yun;XU Ding-hua;XIONG Wen-dian;LIU Xiao-min;JIN Jian;CHEN Yun(Jiangnan University,Collage of Pharmacy Wuxi 214122,China;Wuxi Xinlianxin Biomedical Technology Co.,Ltd,Wuxi 214142,China)
出处
《药物生物技术》
CAS
2020年第3期201-204,共4页
Pharmaceutical Biotechnology
