BHK-21细胞规模化培养鸡新城疫病毒工艺的研究和初步应用  

Research and Preliminary Application of Large-scale Cultivation Process of Newcastle Disease Virus in BHK-21 Cells

作　　者：周欣 丁丽萍 李庆东 侯乐孜 沈丹丹 李双双 姬星宇 张先锋 ZHOU Xin;DING Li-ping;LI Qing-dong;HOU Yue-zi;SHEN Dan-dan;LI Shuang-shuang;JI Xing-yu;ZHANG Xian-feng(Shangqiu Meilan Bioengineering Co.,Ltd.,Shangqiu 476200,China;Henan Puhua Gene Technology Co.,Ltd.,Zhengzhou 450045,China;Shangqiu Animal Quarantine and Disease Prevention and Control Center,Shangqiu 476000,China;College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]商丘美兰生物工程有限公司,河南商丘476200 [2]河南普华基因科技有限公司,河南郑州450045 [3]商丘市动物检疫和疫病预防控制中心,河南商丘476000 [4]河南农业大学动物医学院,河南郑州450046

出　　处：《中国兽医杂志》2023年第7期54-61,共8页Chinese Journal of Veterinary Medicine

摘　　要：为了实现鸡新城疫病毒HN2018株(基因Ⅶ型)在乳仓鼠肾(BHK-21)细胞上的无血清规模化培养,本试验采用悬浮培养技术驯化和筛选了1株能够稳定传代的BHK-21-xh悬浮细胞株;使用该细胞以初始密度为100×10^(4)个/mL接种摇瓶进行培养,并对摇瓶培养鸡新城疫病毒HN2018株的接毒细胞密度、培养温度、接毒量、收毒时间等工艺参数进行摸索和优化;利用摇瓶优化的病毒培养工艺,在10和100 L生物反应器中逐级放大培养BHK-21-xh悬浮细胞,接种鸡新城疫病毒;采用生物反应器悬浮培养的鸡新城疫病毒HN2018株细胞毒与鸡胚毒分别制备成灭活疫苗,免疫SPF鸡进行免疫效力的比较。结果显示,在摇瓶中培养72 h细胞密度均不低于800×10^(4)个/mL,细胞活率均不低于96%;按照BHK-21-xh细胞密度不低于800×10^(4)个/mL,病毒感染复数(MOI)为0.216进行接毒,同时添加终浓度为20μg/mL的胰蛋白酶,于35℃温度条件下培养64~72 h收获病毒液,鸡新城疫悬浮培养细胞毒红细胞凝集(HA)效价最高能够达到10log 2。在生物反应器中接种鸡新城疫病毒,其HA效价均能达到10log 2。细胞苗与鸡胚苗免疫效力相当,攻毒后均能获得100%保护,且免疫血清红细胞凝集抑制(HI)抗体效价均远高于新城疫国家标准。结果表明,应用细胞无血清全悬浮培养技术规模化培养鸡新城疫病毒HN2018株,生产工艺稳定,获得的抗原效价和纯度高,制备的灭活疫苗免疫效力良好,能够应用于新城疫疫苗的规模化生产。In order to achieve serum-free large-scale cultivation of Newcastle disease virus(NDV)HN2018 strain(Genotype VII)in baby hamster kidney-21(BHK-21)cells,this study employed suspension culture techniques to domesticate and screen a stable passaging BHK-21-xh suspension cell line.The cultivated BHK-21-xh cells were inoculated in shake flasks with an initial density of 100×10^(4) cells/mL,and process parameters such as cell inoculation density,cultivation temperature,virus inoculation dosage,and harvesting time for the cultivation of NDV HN2018 strain in shake flasks were explored and optimized.The optimized virus cultivation process in shake flasks was further scaled up to 10 L and 100 L bioreactors for gradually amplifying the cultivation of BHK-21-xh suspension cells and inoculating them with NDV.The cytolytic and chicken embryo-derived NDV HN2018 strain cultivated in bioreactors using suspension culture was used to prepare inactivated vaccines,and their immunogenicity was compared by immunizing SPF chickens.The results showed that the cell density in shake flasks after 72 hours of cultivation was not less than 800×10^(4) cells/mL,with a cell viability of not less than 96%.Inoculating BHK-21-xh cells at a density of not less than 800×10^(4) cells/mL and a multiplicity of infection(MOI)of 0.216,with the addition of trypsin at a final concentration of 20μg/mL,the viral fluid was harvested at a temperature of 35°C for 64-72 hours,the hemagglutination(HA)titer of NDV suspension culture reached a maximum of 10log 2.In the bioreactor,the HA titer of the inoculated NDV also reached 10log 2.Both the cell-based and the chicken embryo-based vaccines exhibited equivalent immunogenicity,providing 100%protection after challenge,and the hemagglutination inhibition(HI)antibody titers were significantly higher than the national standard for Newcastle disease.These results demonstrate the feasibility of serum-free suspension cultivation techniques for large-scale production of NDV HN2018 strain,with stable production processe

关 键 词：BHK-21-xh细胞 新城疫病毒 生物反应器 悬浮培养 

分 类 号：S851.3[农业科学—预防兽医学]