生物反应器微载体系统培养Sabin株脊髓灰质炎病毒条件的优化  

Optimization of condition for culture of poliovirus Sabin strain by using microcarrier system in bioreactor

作　　者：章孟学 周健[1] 杨耀云[1] 胡文著 杨璐洁 王虓宇[1] 赖超[1] 彭佳[1] 孙明波[1] 杨卉娟[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,云南昆明650118

出　　处：《中国生物制品学杂志》2017年第5期530-535,共6页Chinese Journal of Biologicals

基　　金："重大新药创制"科技重大专项(2012ZX09101-318);云南省重点新产品生物重大科技专项(生物疫苗)(2012ZA008)

摘　　要：目的优化生物反应器微载体系统培养Sabin株脊髓灰质炎病毒(poliovirus,PV)的条件,以提高Sabin株PV产量。方法应用搅拌式生物反应器培养PV,对其进行Vero细胞培养密度、病毒培养温度及病毒接种MOI条件的优化,收获病毒液后,检测病毒D抗原含量和感染性滴度,并进行全基因组深度测序。结果 9.0 g/L微载体培养Vero细胞得到病毒D抗原含量显著高于低微载体浓度培养结果(P<0.01);病毒D抗原含量在34℃培养时最高(P<0.01);病毒D抗原含量在MOI 0.050接种时最高(P<0.05),MOI 0.010和MOI 0.005时无明显差异。SabinⅠ型480、525和Ⅲ型Pfizer株472、2 493位点突变率随着细胞培养密度(微载体浓度)的增加而增加;提高病毒培养温度以及改变病毒MOI,大多数毒力相关位点突变率无明显变化。结论提高细胞培养密度、以MOI 0.050接种病毒以及34℃培养病毒均有利于提高病毒产量。Objective To optimize the condition for culture of poliovirus（PV）Sabin strain by using microcarrier system in bioreactor so as to increase the yield of the strain. Methods PV was cultured in stirred bioreactors, and the cell density,virus culture temperature and multiplicity of infection（MOI）were optimized. The harvested virus liquid was determined for D-antigen content and infectious titer, the complete genome was sequenced in depth. Results The D-antigen content of Vero cells cultured with 9. 0 g/L microcarriers was significantly higher than that with microcarriers at lower concentrations（P〈0. 01）. The content was the highest at 34 ℃（P〈0. 01）. However, the content of virus inoculated at a MOI of 0. 050 was the highest（P〈0. 05）, which showed no significant difference at MOIs of 0. 005 and 0. 010. The mutation rate at sites 480 and 525 of Sabin Ⅰ strain and at sites 472 and 2 493 of Pfizer Ⅲ strain increased with the increasing cell density（microcarrier concen-tration）. The mutation rates at most of virulence-associated sites showed no significant change after rise of temperature for virus culture and change of MOI. Conclusion Increasing cell culture density, inoculation of virus at a MOI of 0. 050, and culture of virus at 34 ℃ were beneficial to increase of the virus yield.

关 键 词：生物反应器 脊髓灰质炎病毒 细胞培养密度 病毒培养温度 MOI 全基因组深度测序 

分 类 号：R373.22[医药卫生—病原生物学] R392-33[医药卫生—基础医学]