乙型流感病毒在无血清悬浮培养MDCK细胞上增殖条件的优化  

Optimization of condition for proliferation of influenza B virus in MDCK cells in serum-free suspension culture

作　　者：王笑天 李爽[1] 席莉 丁瑞远 刘玮彤 姚为民[1] 柴文 张雪梅[1] 吴业红[1] WANG Xiao-tian;LI Shuang;XI Li;DING Rui-yuan;LIU Wei-tong;YAO Wei-min;CHAI Wen;ZHANG Xue-mei;WU Ye-hong(Changchun Institute of Biological Products,Changchun 130012,Jilin Province,China)

机构地区：[1]长春生物制品研究所,吉林长春130012

出　　处：《中国生物制品学杂志》2020年第5期544-548,共5页Chinese Journal of Biologicals

基　　金：国家科技重大专项(2015ZX09102016);国家科技重大专项(2018ZX09737008-003);吉林省科技发展计划项目(201603052YY);长春市科技计划项目(18YJ024)。

摘　　要：目的优化乙型流感病毒在无血清悬浮培养MDCK细胞上的增殖条件。方法以乙型流感病毒感染无血清悬浮培养的MDCK细胞,对培养条件中的MOI(10^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7)、胰酶终浓度(2.5、5、7.5、10、15、20μg/mL)、细胞接毒密度(50×10^4、100×10^4、300×10^4、500×10^4、700×10^4个/mL)及病毒收获时间(24、48、72、96、120 h)进行优化。同时对流感病毒在贴壁培养及无血清悬浮培养MDCK细胞上的增殖情况进行比较。结果乙型流感病毒在无血清悬浮培养MDCK细胞上最适增殖条件为:MOI为10^-2、胰酶终浓度为10μg/m L、细胞接毒密度为300×10^4个/mL、病毒收获时间为72 h。流感病毒在无血清悬浮培养MDCK细胞上增殖的HA血凝滴度及病毒滴度(CCID50)均明显高于贴壁培养的MDCK细胞(P<0.05)。结论优化了乙型流感病毒在无血清悬浮培养MDCK细胞上的增殖条件,为后续采用生物反应器大规模生产流感疫苗奠定了基础。Objective To optimize the condition for proliferation of influenza B virus in MDCK cells in suspension culture.Methods MDCK cells in serum-free suspension culture was infected with influenza B virus,and the MOI(10^-1,10^-2,10^-3,10^-4,10^-5,10^-6 and 10^-7),final concentration of trypsin(2.5,5,7.5,10,15 and 20μg/mL),cell density(50×10^4,100×10^4,300×10^4,500×10^4 and 700×10^4 cells/mL)and virus harvest time(24,48,72,96 and 120 h)were optimized.Results The optimal MOI,final concentration of trypsin,cell density and virus harvest time for proliferation of influenza B virus in MDCK cells in serum-free suspension culture were 10^-2,10μg/mL,300×10^4 cells/mL and 72 h respectively,while the HA titer and CCID50 of virus were significantly higher than those in MDCK cells in adherent culture(P<0.05).Conclusion This condition for proliferation of influenza B virus in MDCK cells in suspension culture was optimized,which laid a foundation of large-scale production of influenza vaccine by using bioreactor.

关 键 词：MDCK细胞 悬浮培养 流感病毒 HA血凝滴度 

分 类 号：R373.13[医药卫生—病原生物学]