不同MDCK单克隆细胞株对H5亚型禽流感病毒增殖条件和放大工艺的研究

Research on Multiplication of H5 Subtype Avian Influenza Virus in Different MDCK Sub-clonal Cells and Scaled Production Technology

为了解H5亚型禽流感病毒rFJ56株在MDCK全悬浮细胞上的增殖规律,确定100 L生物反应器工艺的最适接毒量和收获时间,对H5亚型禽流感病毒rFJ56株敏感的MDCK全悬浮细胞进行单克隆筛选和全悬浮驯化,筛选出对该毒株最敏感的MDCK单克隆细胞株;在摇瓶工艺的基础上,将该细胞在100 L生物反应器中接种H5亚型禽流感病毒rFJ56株,检测接种后不同时间病毒HA效价和病毒滴度(EID 50),确定最适接毒量和收获时间。共筛选出22株MDCK全悬浮细胞单克隆细胞株,其中5株对H5亚型禽流感病毒rFJ56株最敏感,接种后病毒HA效价均可达到9 log2;对MDCK单克隆细胞株按0.01%体积比接种H5亚型禽流感病毒rFJ56株,在100 L生物反应器中培养60 h,HA效价可达10 log2,病毒含量可达108.17 EID 50/0.1 mL,可为H5亚型禽流感病毒无血清全悬浮培养放大工艺提供参考。

In order to understand the multiplication of H5 subtype avian influenza virus rFJ56 strain in MDCK suspension cells,and determine the optimal viral dose and the optimal harvest time of the 100L bioreactor,the MDCK suspension cells sensitive to rFJ56 strain were monoclonally screened and fullly suspended,and the most sensitive MDCK sub-clonal cell lines were selected.On the basis of the shake flask process,the cell line was inoculated with rFJ56 strain in 100 L bioreactors,and HA titer and EID 50 at different times after inoculation were detected to determine the optimal viral dose and the optimal harvest time.The results showed that a total of 22 MDCK sub-clonal cell lines were selected,and 5 of them were most sensitive to rFJ56 strain.The optimal culture process of avian influenza virus rFJ56 strain in 100L bioreactors was inoculated with the virus into MDCK sub-clonal cell line in a volume ratio of 0.01%,and cultured for 60 hours.The HA titer was up to 10 log2,and the virus titer was up to 108.17 EID 50/0.1 mL.This study provided parameter indexes for serum-free full suspension culture for H5 subtype avian influenza virus.