狂犬病毒在摇摆式反应器的培养工艺摸索  

Grope of a cultivation procedure for Rabies virus in a swing bioreactor

作　　者：李慧 周维 洪玮琪 陈楚如 吴灼斌 王雪云 LI Hui;ZHOU Wei;HONG Weiqi;CHEN Churu;WU Zhuobin;WANG Xueyun(Shenzhen Weiguang Biological Procedures Co.,Ltd,Shenzhen,Guangdong 518107,China)

机构地区：[1]深圳市卫光生物制品股份有限公司,广东深圳518107

出　　处：《医学动物防制》2024年第7期694-699,共6页Journal of Medical Pest Control

基　　金：广东省基础与应用基础研究基金会(2021A1515010848)。

摘　　要：目的探讨狂犬病毒采用鸡胚细胞在摇摆式反应器培养的工艺,为狂犬病毒的培养提供一种简便的新工艺。方法2021年4月选取发育良好的9~11d鸡胚,在无菌环境下将其制备成原代鸡胚细胞后,将狂犬病毒CTNCEC25株和原代鸡胚细胞接种于一次性反应袋中,置于摇摆式反应器进行培养,通过对细胞密度(2.0×10^(6)、3.0×10^(6)、4.0×10^(6)和5.0×10^(6)个/ml)、感染复数(multiplicity of infection,MOI)(0.001、0.010、0.030、0.050、0.100)、溶氧(50%、70%、30%)、温度(32℃、33℃和34℃)等关键参数进行摸索,从而获得狂犬病毒最适的培养条件,并按照最适的培养条件培养3批病毒液验证该培养条件的稳定性。结果狂犬病毒CTNCEC25株在摇摆式反应器培养的最佳细胞密度为4.0×10^(6)~5.0×10^(6)个/ml、MOI为0.010~0.100、培养48h后的溶氧为50%、48h后的培养温度为32~34℃,采用该条件培养获得的病毒收获液滴度均在8.0lgFFU/ml以上,远高于中国药典规定的6.0lgFFU/ml。结论通过本研究,建立了狂犬病毒CTNCEC25株在摇摆式反应器的培养工艺,为鸡胚细胞狂犬病疫苗产业化生产提供了参考。Objective To discuss the cultivation procedure for Rabies virus in chicke embryo cells by a swing bioreactor to provide a convenient new procedure for the cultivation of Rabies virus.Methods In April 2021,well-developed 9-11 days chicken embryos were selected and prepared into primary chicken embryo cells in a sterile environment.The Rabies virus CTNCEC25 strain and primary chicken embyo cells were inoculated in disposable reaction bags and placed in a swing bioreactor for cultivation.The optimal cultivation conditions for Rabies virus were groped by exploring key parameters such as cells density(2.0×10^(6),3.0×10^(6),4.0×10^(6) and 5.0×10^(6) cells/ml),multiplicity of infection(MOI)(0.001,0.010,0.030,0.050,and 0.100),dissolved oxygen(50%,70%,and 30%),and temperature(32℃,33℃,and 34℃),and three batches of viral fluids were cultured according to the optimal cultivation conditions to verify the stability of the cultivation conditions.Results The optimal cell density of the Rabies virus CTNCEC25 strain cultured in the swing bireactor was 4×10^(6)-5×10^(6) cell/ml.MOI was 0.010-0.100.Dissolved oxygen was 50% after 48 hours of culture.Culture temperature was 32-34℃ after 48 hours of culture.The titers of the virus harvests obtained by using the cultivation under these conditions were all above 8.0lgFFU/ml,which was much higher than the 6.0lgFFU/ml specified in the Chinese Pharmacopoeia.Conclusion Through this study,the cultivation procedure for the Rabies virus CTNCEC25 strain in the swing bioreactor is established,which provided a reference for the industrialization of rabies vaccine in chicken embryo cells.

关 键 词：狂犬病毒 鸡胚细胞 摇摆式反应器 滴度 抗原 

分 类 号：R373.9[医药卫生—病原生物学]