Sf9细胞的高效扩增及类病毒颗粒高效自组装的培养工艺

High efficiency proliferation culture of Sf9 cells and high efficiency self-assembly of virus-like particles

目的优化SFX-Insect培养基及类病毒颗粒(virus like particles,VLPs)的自组装培养工艺,提高Sf9细胞的扩增效率及VLPs的自组装效率。方法以SFX-Insect培养基作为基础培养基,添加不同营养物(HyPep^(TM) 1510及葡萄糖),优化培养基。分别在摇瓶及WAVE生物反应器中培养Sf9细胞,同时补加不同倍数(1、2、5、8倍)的补料浓缩液,观察细胞培养效果;将重组戊型肝炎杆状病毒分别加入SFX-Insect及优化培养基培养的Sf9细胞中,分析目的蛋白表达量;根据细胞的不同生长阶段、接毒时是否补料及不同接毒MOI值,将优化培养基培养的Sf9细胞进行分组,分析各组细胞中目的蛋白的表达量并观察VLPs的形成。结果 SFX-Insect基础培养基中同时添加10 g/L葡萄糖和10 g/L HyPep^(TM)1510为Sf9细胞最佳培养基。优化培养基摇瓶培养Sf9细胞,活细胞密度高达2×10~7个/ml以上;在细胞对数生长中后期进行1及2倍补料可延长细胞平台生长期至20 d以上;将摇瓶培养的细胞放大至WAVE生物反应器中培养,密度高达107个/ml以上,且传代培养后,可维持10 d以上的平台生长期。优化培养基培养的Sf9细胞目的蛋白的表达量比SFX-Insect培养基提高了6倍;优化培养基培养Sf9细胞至对数生长中前期,进行补料接毒(MOI=2),VLPs的组装效率大幅提高,镜下可见形态均一的VLPs。结论成功优化了SFX-Insect培养基及VLPs的自组装培养工艺,提高了Sf9细胞的扩增效率及VLPs产量。

Objective To optimize SFX-Insect medium and the self-assembly process of virus-like particles（VLPs） to increase the proliferation efficiency of Sf9 cells and the self-assembly efficiency of VLPs. Methods SFX-Insect medium as a basal medium was optimized by addition with Hy Pep^（TM）1510 or glucose, with which Sf9 cells were cultured in shake flask and WAVE bioreactor respectively. The optimized medium was supplemented with Hy Pep^（TM）1510 and glucose to 1,2, 5 and 8 times of the original concentrations, and observed for the cell culture effect. The Sf9 cells cultured in original and optimized SFX-Insect media were inoculated by recombinant baculovirus with hepatitis E virus（HEV）, and analyzed for the expression level of target protein. The Sf9 cells cultured in optimized medium were grouped according to the growth phase, MOI and whether fed-batch or not when the virus was inoculated. The expression levels of target protein in various groups were determined, while the formations of VLPs were observed. Results SFX-Insect medium added with 10 g/L glucose and 10 g/L Hy PepTM1510 was optimal for culture of Sf9 cells, with which the cell density reached more than 2 ×107 cells/ml in shake flask, while the platform growth phase was prolonged to more than 20 d after fed-batch to 1 and 2 times of original concentrations in the middle and late stages of logarithmic growth phase. However, the cell density was more than 10~7 cells/ml in WAVE Bioreactor. After subculture, the platform growth phase was more than 10 d. The expression level of target protein in Sf9 cells cultured by optimized medium increased by 6 folds compared with that by SFX-Insect medium. When the optimized medium was supplemented with nutrient component and inoculated with virus（MOI = 2） at early and middle logarithmic growth phases of Sf9 cells, the assembly efficiency of VLPs increased remarkably, and the VLPs even in shape were observed under microscope. Conclusion SFX-Insect commercial medium and the self-assembly process of VLPs were successfully optimized, while the proliferation efficiency of Sf9 cells and yield of VLPs increased.