B型流感疫苗候选株在MDCK悬浮细胞上培养工艺优化研究
Optimization of Influenza B Vaccine Candidates Cultured in MDCK Suspension Cells
武毓姝 1孙伟洋 2赵梦琳 2夏叶婷 3冯越 1罗敏 4任娟 4高鸿博 2王铁成 2夏咸柱 2高玉伟 1闫芳5
作者信息
- 1. 山西农业大学,晋中 030801;中国农业科学院长春兽医研究所,长春 130122
- 2. 中国农业科学院长春兽医研究所,长春 130122
- 3. 中国农业科学院长春兽医研究所,长春 130122;河南大学,开封 475001
- 4. 中国农业科学院长春兽医研究所,长春 130122;山东师范大学,济南 250014
- 5. 山西农业大学,晋中 030801
- 折叠
摘要
为了大规模生产重组B型流感疫苗候选株rIBV,本研究探索了 rIBV在无血清MDCKs细胞上的最佳培养条件,再扩大到生物反应器中优化工艺参数,经过连续传代15代后测定了 MDCKs细胞源毒种rIBV的遗传稳定性和病毒滴度.通过对病毒培养条件进行优化,我们确定了 rIBV在MDCKs细胞上增殖的最佳培养条件是TOI为 72 h、MOI=0.001、CCI 为 4.0 × 106 细胞、TPCK胰酶浓度为 2μg/mL、50％DO 和 pH=7.2±0.5o rIBV在MDCKs细胞上连续传代15代后仍然具有高度遗传稳定性.相比于鸡胚源rIBV株,适应MDCKs细胞生长15代后rIBV株的EID50由10625/mL提高至109,5/mL,TCID50由104.5/mL提高至10825/mL.本研究对重组B型流感疫苗候选株rIBV在MDCKs细胞上的培养工艺进行了优化,获得一株具有高感染力和高病毒滴度的MDCKs细胞源疫苗候选株rIBV,为下游流感疫苗生产及纯化工艺奠定了基础.
Abstract
In order to produce recombinant influenza B vaccine candidate strain rIBV on a large scale,this study explored the optimal culture conditions of rIBV on MDCKs cells in serum-free medium,and then expanded to a bioreactor to optimize the process parameters.After 15 successive passsages,the genetic stability and viral titer of MDCKs cell derived viral strain rIBV were determined.By optimizing the virus culture conditions,we determined that the optimal culture conditions for rIBV proliferation on MDCKs cells were TOI of 72 hpi,MOI=0.001,CCI of 4.0 × 106 cells/mL,TPCK trypsin concentration of 2 μg/mL,50％DO and pH of 7.2±0.5.rIBV showed high genetic stability after 15 successive passages on MDCKs cells.Compared with the chicken embryo derived rIBV strain,the EID50 and TCID50 of rIBV strain were increased from 106 25/mL to 1095/mL and 108,25/mL respectively after 15 passages of adaptation to MDCKs cell culture.In this study,the culture process of recombinant influenza B vaccine candidate rIBV on MDCKs cells was optimized,and a MDCKs cell-derived vaccine candidate rIBV with high infectivity and high viral titer was obtained,which laid a foundation for downstream influenza vaccine production and purification process.
关键词
流感病毒/MDCKs细胞/悬浮培养/生物反应器/工艺优化Key words
Influenza virus/MDCKs cells/Suspension culture/Bioreactor/Process optimization引用本文复制引用
出版年
2024
NETL
NSTL
万方数据