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

作　　者：武毓姝 孙伟洋 赵梦琳 夏叶婷 冯越 罗敏 任娟 高鸿博 王铁成 夏咸柱[2] 高玉伟 闫芳[1] WU Yushu;SUN Weiyang;ZHAO Menglin;XIA Yeting;FENG Yue;LUO Min;REN Juan;GAO Hongbo;WANG Tiecheng;XIA Xianzhu;GAO Yuwei;YAN Fang(Shanri Agricultural University,Jinzhong 030801,China;Key Laboratory of Jilin Province for Zoonosis Prevention and Control,Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Changchun 130122,China;Henan University,Kaifeng 475001,China;Shandong Normal University,Jinan 250014,China)

机构地区：[1]山西农业大学,晋中030801 [2]中国农业科学院长春兽医研究所,长春130122 [3]河南大学,开封475001 [4]山东师范大学,济南250014

出　　处：《病毒学报》2024年第2期283-292,共10页Chinese Journal of Virology

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72h、MOI=0.001、CCI为4.0×10^(6)细胞、TPCK胰酶浓度为2μg/m L、50%DO和pH=7.2±0.5。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EID_(50)由10^(6.25)/mL提高至10^(9.5)/mL,TCID_(50)由10^(4.5)/mL提高至10^(8.25)/mL。本研究对重组B型流感疫苗候选株r IBV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r IBV,为下游流感疫苗生产及纯化工艺奠定了基础。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 4.0×10^(6)cells/mL,TPCK trypsin concentration of 2μg/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 EID_(50) and TCID_(50) of rIBV strain were increased from 10^(6.25)/mL to 10^(9.5)/mL and 10^(8.25)/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 

分 类 号：R373.1[医药卫生—病原生物学]