优化Ⅲ型脊髓灰质炎病毒在Vero细胞中的增殖条件  

Optimizing the Multiplication Conditions for Type 3 Poliovirus in Vero Cells

作　　者：沈武玲[1] 蔡玮[1] 平玲 张名[1] 牛杰 高娜[1] 彭佳[1] 葛进 唐聪[1] 周健[1] SHEN Wuling;CAI Wei;PING Ling;ZHANG Ming;NIU Jie;GAO Na;PENG Jia;GE Jin;TANG Cong;ZHOU Jian(Institute of Medical Biology,Chinese Academy of Medical Sciences,Kunming 650032,China)

机构地区：[1]中国医学科学院医学生物学研究所,昆明650032

出　　处：《病毒学报》2020年第6期1138-1142,共5页Chinese Journal of Virology

基　　金：云南省重大科技专项(生物医药)(项目号:2017ZF008),题目:Sabin株脊髓灰质炎灭活疫苗提质增效新工艺研究。

摘　　要：型脊髓灰质炎病毒经下游灭活工艺后抗原损失量较大,可以通过优化上游培养工艺来提高病毒产量。为研究病毒接种时间、感染复数(MOI)和病毒收获时间对Ⅲ型脊髓灰质炎病毒感染性滴度(CCID50)和D抗原的影响,优化Ⅲ型脊髓灰质炎病毒在Vero细胞中的增殖条件,为生产及毒种制备提供指导依据,本研究以接种胞龄和MOI为病毒接种的两个变量条件,以病毒滴度和D抗原含量为病毒收获液中病毒产量的判定指标。先固定MOI,在不同胞龄的Vero细胞上以相同MOI接种Ⅲ型脊髓灰质炎病毒,根据病毒收获液中病毒产量的结果筛选出产毒量最高的接种胞龄范围。然后以最佳产毒胞龄为固定条件,暨Vero细胞生长至96h^144h,以不同MOI接种病毒,进一步筛选出产毒量最高的MOI范围。结果显示,当MOI=0.003时,Vero细胞培养48h、72h、96h、120h和144h种毒组均在种毒后70h完全病变,Vero细胞培养120h种毒所得收获液D抗原含量和病毒滴度均为最高,分别为(331±31)DU/mL、(8.04±0.14)lgCCID50/mL。接种胞龄固定在96h^144h范围,当MOI为0.001、0.01和0.1时,细胞完全病变时间分别为81h、66h和55h,所得病毒收获液的D抗原含量分别为(301.1±13.8)DU/mL、(303.7±15.6)DU/mL和(272.6±19.6)DU/mL,滴度分别为(7.88±0.13)lgCCID50/mL、(7.58±0.14)lgCCID50/mL和(7.75±0.13)lgCCID50/mL。本研究提示,相同MOI下,不同细胞胞龄种毒后细胞达到完全病变时间相同;细胞培养至平台期前后接种Ⅲ型脊髓灰质炎病毒所获得的收获液D抗原含量和感染性滴度均最高;MOI影响Ⅲ型病毒病变进程,但对病毒产量影响不大;90%以上Vero细胞发生病变、脱落时为最佳收获病毒时间点。Relatively high losses of D-antigen concentration of type 3 poliovirus were observed after inactivation.It was feasible to optimize the upstream processing to improve virus yields.To explore the effects of time of infection(TOI),multiplicity of infection(MOI)and harvest time on virus titer and D-antigen concentration to assist process optimization for type 3 poliovirus,the cell culture time and MOI were variable in the study.Infectious poliovirus particle concentration was quantified using virus titer assay and the quality was assessed using D-antigen ELISA assay.Firstly,type 3 poliovirus was used to infect Vero cells at different culture time with same MOI.Secondly,Vero cells were cultured to the same phase and then infected with type 3 poliovirus at different MOI.Results showed that at^70 h post infection,Vero cells inoculated with virus at MOI of 0.003 when cultured to 48 h,72 h,96 h,120 h and 144 h,respectively,appeared complete cytopathic effect.Both the titer and D-antigen concentration of the harvest were the highest at TOI of 120 h.The titer was(8.04±0.14)lgCCID50/mL and D-antigen concentration was(331±31)DU/mL.The harvest time was 81 h,66 h and 55 h,respectively,at MOI of 0.001,0.01,0.1.The D-antigen concentrations of the harvest at MOI of 0.001,0.01,0.1 were(301.1±13.8)DU/mL,(303.7±15.6)DU/mL and(272.6±19.6)DU/mL,whereas the titers were(7.88±0.13)lgCCID50/mL,(7.58±0.14)lgCCID50/mL and(7.75±0.13)lgCCID50/mL,respectively.The present study suggested that infection at same MOI but different TOI showed no significant difference in infection progress.The maximum virus yields reached when infection at late exponential phase and stable phase of the cell growth.MOI showed obvious influence on infection progress but no effects on virus yields.It was the best harvest time to get maximum virus yield when more than 90%cells shrank and fell off.

关 键 词：VERO细胞 Ⅲ型脊髓灰质炎病毒 感染性滴度 D抗原 

分 类 号：R373.22[医药卫生—病原生物学]