犬细小病毒YA-16-C66株纸片载体培养工艺优化及其与转瓶培养工艺的比较分析

Optimization of Flake disks culture of Canine Parvovirus YA-16-C66 strain and Comparative analysis of its culture technology with spinner bottle

关键词:  犬细小病毒  生物反应器  纸片载体  转瓶  培养工艺  

Key words:  Canine Parvovirus  Bioreactor  flake disks  spinner bottle  cultivation process  

摘要:

采用微型生物反应器确定纸片载体上F81细胞最佳的接种密度和最佳接毒剂量,在NBS反应器中验证温度对犬细小病毒YA-16-C66株增殖的影响。经过三次重复试验,确定利用NBS生物反应器培养犬细小病毒YA-16-C66株最佳工艺为：采用同步接毒法,培养基为含1%新生牛血清的199溶液,细胞接种密度为1.5×10_⁷cells/g,接毒剂量为0.05 MOI,在pH7.2条件下,37.0℃培养48h后降温到35.0℃继续培养24-48小时,细胞病变达到80%~90%时收获上清液。三批次反应器与转瓶工艺产品比较,结果表明：反应器比转瓶收获液病毒含量高1.1~1.9 lg TCID₅₀/100μL；相同培养面积,反应器所得病毒总量是转瓶所得病毒总量的2.8-18.5倍。

Abstract:

The optimal inoculation density and dose of F81 cells on Flake disks were determined in a mini-bioreactor, and the effect of temperature on the proliferation of YA-16-C66 strain of Canine Parvovirus was verified in the NBS reactor. After three time of repeated experiments,the optimal culture process for the cultivation of YA-16-C66 strain of Canine Parvovirus by NBS bioreactor was determined:In the synchronous inoculation method, the culture medium was 199 solution containing 1% newborn bovine serum, the cell inoculation density was 1.5×10_⁷cells/g, and the inoculation dose was 0.05 MOI. Under the condition of pH7.2, the cells were cultured at 37.0℃ for 48h, then the temperature was lowered to 35.0℃ for 24-48 hours, and the supernatant was harvested when the cytopathod reached 80%~90%. The results showed that the virus content in the reactor was 1.1~1.9 lg TCID₅₀/100 μL higher than that in the reactant. With the same culture area, the total amount of virus obtained from the reactor was 2.8-18.5 times that of the total amount of virus obtained from the spinner bottle.