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生物反应器悬浮培养Vero细胞制备水貂犬瘟热活疫苗
冯二凯,程悦宁,罗国良,易立,王振军,郭利,陈立志,程世鹏
0
(中国农业科学院特产研究所)
摘要:
为建立水貂犬瘟热Vero活疫苗的生物反应器悬浮培养制备工艺并制备疫苗,在7L生物反应器中悬浮培养Vero细胞,并考察培养基、细胞初始接种密度、培养方式、病毒感染复数和感染时间等参数对细胞增殖、病毒滴度以及细胞代谢的影响。 结果显示,在含有5 g/L微载体的DMEM培养基中接种Vero细胞(30-40 cells/球),设定pH、温度和溶氧值分别为7.2,37℃,50%和55 rpm,培养方式为批次培养(0-48 h)+灌流培养组合方式(48-96 h),培养Vero细胞3-4 d或细胞密度达到200 cells/球以上时;按照MOI=0.0001-0.001吸附接毒,调低温度(33℃)继续培养100-120 h,即可获得高滴度病毒液(106.5-107.2 TCID50/0.1 mL),经无菌检验合格后,配制水貂犬瘟热Vero细胞活疫苗(CDV3-CL株,悬浮培养),疫苗符合《中国兽药典》三部(2015版)的规定。结论:建立了7L生物反应器悬浮培养Vero细胞制备水貂犬瘟热活疫苗的新工艺,为进一步规模化生产奠定了基础。
关键词:  非洲绿猴肾细胞  大规模培养  微载体  悬浮培养  生物反应器  
DOI:
投稿时间:2019-04-22修订日期:2019-06-11
基金项目::吉林省科技发展计划(20150204073NY);吉林省“双十”重大科技攻关(20150201006NY)作者冯二凯,在读博士,主要从事毛皮动物疫苗悬浮培养工艺开发,tcsfek@126.com*通讯作者程世鹏,博士,研究员,主要从事毛皮动物疫苗、诊断制剂开发及疾病机理研究tcscsp@126.com ,程悦宁1,罗国良1,易 立1,王振军1,郭 利1,陈立志2,
Suspension culture of Vero cells in bioreactor and preparation of mink canine distemper attenuated live vaccine
(Institution of Special Animal and Plant Science of Chinese Academy of Agriculture Science,Jilin Changchun)
Abstract:
To develop a new process for preparing of mink canine distemper attenuated live vaccine (CDV3-CL Zhu) in a bioreactor, Vero cell as a cellular matrix were cultured in a 7L bioreactor. The pH, temperature,dissolved oxygen (DO) and stirring speed were mainted at the value of 7.2, 37℃, 50% and 55 rpm, respectively. The effect of culture mode, initial inoculum density and culture medium on growth and density of cell were investigated. In addition, the effect of multiplicity of infection (MOI) and time of infection (TOI) on the titer of virus liquids were also studied. The results indicated that culture Vero cell in DEME containing 5g/L microcarriers with batch plus perfusion culture mode for 3-4 d or the cell density reached above 200 cells per microcarrier, inoculated canine distemper virus under a MOI of 0.0001-0.001, turn down temperature and continue to culture for another 100-120 h, the semi-product with highest virus titer of 107.2 TCID50/0.1 mL were collected, which was almost 30 times higher than that in rolling bottle. So, the procedure for production of mink canine distemper attenuated live vaccine (CDV3-CL Zhu) in Vero cells on microcarriers in a bioreactor was successfully developed, which laid a foundation of further large-scale culture of CDV and development of vaccine.
Key words:  African Green Monkey Kidney  Large scale culture  Microcarriers  Suspension

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