潮汐式生物反应器CelCradle^TM培养轮状病毒工艺的建立  

Development of culture process for rotavirus by using a novel tidal bioreactor CelCradle^TM

作　　者：尹娜[1] 周艳[1] 刘洋 胡晓青 张光明[1] 孙茂盛[1] 李鸿钧[1] YIN Na;ZHOU Yan;LIU Yang;HU Xiao-qing;ZHANG Guang-ming;SUN Mao-sheng;LI Hong-jun(Institute of Medical Biology,Chinese Academy of Medical Science,Kunming 650118,Yunnan Province,China)

机构地区：[1]中国医学科学院医学生物学研究所,云南昆明650118

出　　处：《中国生物制品学杂志》2020年第5期554-557,562,共5页Chinese Journal of Biologicals

基　　金：中国医学科学院与健康科技创新工程协同创新团队项目(中国医学科学院基金)(2016-12M-1-019);云南省重大科技专项(生物医药)(云南省自然科学基金)(2018ZF006)。

摘　　要：目的建立潮汐式生物反应器CelCradle^TM培养轮状病毒的工艺。方法利用500 mL的潮汐式生物反应器CelCradle^TM培养Vero细胞,通过葡萄糖含量监测及活细胞计数进行换液,待活细胞数目达2.75×10^9个时,分别按0.01、0.05、0.1和0.2 MOI接种轮状病毒ZTR-68株,于37℃,5%CO2培养箱中培养7 d,每天取样,聚丙烯酰胺凝胶电泳检测病毒基因组核酸带型,免疫荧光法检测病毒增殖情况,透射电镜观察病毒的形态结构,蚀斑法检测病毒滴度,确定生物反应器CelCradle^TM培养轮状病毒的最适MOI。结果在CelCradle^TM反应器中接种3×108个Vero细胞,经7 d培养,活细胞数目可达2.75×10^9个;以0.05 MOI轮状病毒ZTR-68接种Vero细胞的病毒基因组带型与转瓶培养的保持一致,感染后3~5 d,轮状病毒荧光阳性细胞数目最多,电镜下可见形态结构完整的轮状病毒颗粒;以0.05 MOI接种轮状病毒ZTR-68株96 h病毒滴度最高,为7.7 LogPFU/mL。结论初步建立了潮汐式生物反应器CelCradle^TM培养轮状病毒的工艺,为进一步大规模生产轮状病毒灭活疫苗奠定了实验基础。Objective To develop a process for culture of rotavirus in a novel tidal bioreactor CelCradle^TM.Methods Vero cells were cultured in 500 mL tidal bioreactor CelCradle^TM.The media were changed by monitoring the glucose content and the viable count.Rotavirus ZTR-68 strain was inoculated to Vero cells at MOIs of 0.01,0.05,0.1 and 0.2 respectively when the viable count reached 2.75×10^9 and incubated at 37℃for 7 d,from which samples were taken daily,observed for genomic DNA band pattern by polyacrylamide gel electrophoresis and for morphology and structure by transmission electron microscopy,and determined for infectious titer by plaque assay,based on which the MOI was optimized.Results A portion of 3×108 Vero cells were inoculated into 500 mL tidal bioreactor CelCradle^TM and cultured for 7 d when the viable count reached 2.75×10^9.The genomic DNA band pattern of rotavirus ZTR-68 strain inoculated at a MOI of 0.05 was consistent with that in roller culture.The count of fluorescent positive cells reached the peak value 3~5 d after infection,while the rotavirus particles with intact structure was observed under electron microscope.The infectious titer of rotavirus ZTR-68 strain reached a peak value of 7.7 LogPFU/mL 96 h after inoculation at a MOI of 0.05.Conclusion The culture process of rotavirus in a novel tidal bioreactor CelCradle^TM was preliminarily developed,which laid an experimental foundation of further large-scale production of inactivated rotavirus vaccine.

关 键 词：潮汐式生物反应器 轮状病毒 VERO细胞 

分 类 号：R373.2[医药卫生—病原生物学]