ST细胞全悬浮培养的驯化及其培养伪狂犬病毒的工艺研究

将Siat7e基因转染ST细胞,筛选,驯化得到一株可悬浮培养的ST细胞株,此株细胞能够稳定连续传代,适应无血清、高密度培养,最高密度可达6×10⁶/mL,从摇瓶放大至生物反应器,生长稳定;采用驯化的全悬浮ST细胞培养伪狂犬病毒,从接毒时细胞密度、接毒量、收获时间三个方面优化了伪狂犬病毒的培养参数,确定接毒时细胞密度为2.0×10⁶/mL~3.0×10⁶/mL,接毒量为0.1 MOI~1 MOI,收毒时间为接毒后24 h~36 h。经过50 L生物反应器3个批次的工艺验证,培养的病毒含量均不低于109.0TCID50/mL,说明驯化的ST全悬浮细胞适合伪狂犬病毒的培养。

Generation of ST Cells to Suspension Culture and its process of culturing pseudorabies virus

Siat7e gene was transfected into ST cells,through screening,successfully conversioned the ST cell to suspension culture.This cell is stable and subcultured,can adapt to high density culture within serum-free medium,the maximum density of cell growth was 6×10⁶/mL.The cells grew steadily when scale up from the shaking flask to the bioreactor.In order to study the culturing procedure of Pseudorabies virus using this suspended ST cells,the cell density,amount of virus inoculation,and harvest time were optimized,which displayed that the best results could get when using the initial seeding density of 2.0×10⁶/mL~3.0×10⁶/mL,infected with 0.1 MOI~1 MOI,and harvested between post-infection 24 h~36 h.After three batches of 50 L bioreactor suspension cultures,the virus titers was not less than 109.0 TCID₅₀/mL,indicating that this ST suspended cells were suitable for Pseudorabies virus culture.