4型流感病毒疫苗株在MDCK细胞中增殖条件的优化

Optimization of condition for propagation of four subtypes of influenza virus in MDCK cells

目的优化4型流感病毒疫苗株H1N1(IVR-215)、H3N2(NIB-121)、BV(BVR-11)、BY(BVR-1B)在稳定表达牛胰蛋白酶原的MDCK细胞(MTY6)和MDCK细胞中的增殖条件,并比较MTY6和MDCK细胞培养不同型别流感病毒的差异。方法以血凝滴度和病毒滴度[半数组织感染剂量(median tissue culture infective dose,TCID_(50))]为指标,通过棋盘法对4型流感病毒疫苗株在MTY6和MDCK细胞上种毒的MOI(10^(-1)、10^(-2)、10^(-3)、10^(-4))、胰酶终浓度(0.5、1、1.5、2μg/mL)及收毒时间(0、24、48、72、96 h)进行优化;以接毒后细胞病变效应(cytopathic effect,CPE)为指标,分析两种细胞对不同型别流感病毒的敏感性。结果 H1N1、H3N2、BV和BY株病毒在MTY6细胞中培养的最适MOI分别为10^(-4)、10^(-4)、10^(-2)、10^(-2),胰酶终浓度分别为0.5、1、0.5、0.5μg/mL,收毒时间分别为96、72、48、72 h。H1N1、H3N2、BV和BY株病毒在MDCK细胞中培养的最适MOI均为10^(-4),胰酶浓度为0.5、1、0.5和1.5μg/mL,收毒时间为96、48、72和72 h。4型流感病毒疫苗株在MTY6细胞中培养比在MDCK细胞中具有更高的血凝滴度和TCID50。在相同时间内,H1N1、H3N2、BY株病毒在MTY6细胞中比在MDCK细胞中能够引起更明显的CPE。结论成功优化了4型流感病毒疫苗株在MTY6细胞中的增殖条件,MTY6细胞与MDCK细胞相比,培养4型流感病毒疫苗株能够获得更高的病毒滴度,并增加病毒产量。本研究为MTY6细胞用于流感疫苗生产奠定了基础。

Objective To optimize the condition for propagation of influenza virus of subtypes H1N1 (IVR-215),H3N2(NIB-121),BV(BVR-11)and BY(BVR-1b)in MTY6(the MDCK cells for stable expression of bovine trypsinogen)and MDCK cells,and compare the differences of various subtypes of virus cultured in the two cell lines.Methods Using hemagglutination titer and virus titer(median tissue culture infective dose,TCID_(50))as indexes,the MOI(10^(-1),10^(-2),10^(-3) and 10^(-4)),final trypsin concentration (0.5,1,1.5 and 2μg/mL)and harvest time (0,24,48,72 and 96 h)of four subtypes of influenza virus were optimized by chessboard assay.The sensitivity of the two cell lines to various subtypes of virus was analyzed by using the cytopathic effect (CPE) after virus inoculation as index.Results The optimal MOIs for culture of subtypes H1N1,H3N2,BV and BY of influenza virus in MTY6 cells were 10^(-4),10^(-4),10^(-2) and 10^(-2),while the final trypsin concentrations were 0.5,1,0.5 and 0.5μg/mL,and the harvest times were 96,72,48 and 72 h,respectively.However,all the optimal MOIs for culture of the four subtypes in MDCK cells were 10^(-4),while the final trypsin concentrations were 0.5,1,0.5 and 1.5μg/mL,and the harvest time weres 96,48,72 and 72 h,respectively.Both the hemagglutination titer and TCID50 of the four subtypes cultured in MTY6 cells were significantly higher than those in MDCK cells.Within the same time duration,subtypes H1N1,H3N2 and BY caused significant CPEs in MTY6 cells as compared with those in MDCK cells.Conclusion The condition for propagation of four subtypes of influenza virus in MTY6 cells was successfully optimized.Compared with those cultured in MDCK cells,high virus titer and yield were obtained in MTY6 cells.It laid a foundation of apllication of MTY6 cells in the production of influenza vaccine.