新型固定床生物反应器在细胞源猪伪狂犬病毒生产中的应用The application of novel fixed-bed bioreactor in cell-based pseudorabies virus production
聂简琪;孙杨;杨艳坤;刘秀霞;李业;白仲虎;
摘要(Abstract):
针对目前固定床生物反应器结构复杂、易产生泡沫等缺点,研发结构简单且可无泡通气的新型固定床生物反应器,并对该反应器的混合时间、k_La、细胞分布均匀性和细胞捕获能力进行测定。并进一步利用该反应器实施低密度接种Vero细胞进行细胞培养并扩增猪伪狂犬病毒。试验结果表明,该新型固定床反应器的混合时间、k_La与传统STR反应器相当,细胞捕获能力良好且细胞在反应器中分布均匀。采用低密度接种时Vero细胞可在该反应器中进行良好的生长,接毒5 d后累计收获的病毒总颗粒数达到10.37 log_(10) TCID_(50),相当于2.3×10~5剂量疫苗。该研究为细胞源疫苗的生产提供了新的反应器与技术。
关键词(KeyWords): 固定床生物反应器;Vero细胞;猪伪狂犬病毒;低密度接种;疫苗
基金项目(Foundation): 国家自然科学基金(No.21878124,31570034);; 111计划(No.111-2-06);; 泰山产业领军人才(No.tscy20160307)
作者(Author): 聂简琪;孙杨;杨艳坤;刘秀霞;李业;白仲虎;
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参考文献(References):
- [1]GALLO-RAMIREZ L E,NIKOLAY A,GENZEL Y,et al.Bioreactor concepts for cell culture-based viral vaccine production [J].Expert Review of Vaccines,2015,14(9):1181-1195.
- [2]陈金华,徐葛林.固定床反应器在病毒性疫苗研究中的应用[J].中国生物制品学杂志,2014,27(5):732-736.
- [3]HANS N,SARAH G,HERMAN F,et al.Cell biological and molecular characteristics of pseudorabies virus infections in cell cultures and in pigs with emphasis on the respiratory tract [J].Veterinary Research,2007,38(2):229-241.
- [4]DONG B,ZARLENGA D S,REN X.An overview of live attenuated recombinant pseudorabies viruses for use as novel vaccines [J].Journal of Immunological Research,2014,2014:824630.
- [5]杜丽飞,刘森林,危婷,等.猪伪狂犬病的研究进展[J].现代畜牧兽医,2019,1:54-59.
- [6]ZHOU J Z,LI S,WANG X B,et al.Bartha-k61 vaccine protects growing pigs against challenge with an emerging variant pseudorabies virus [J].Vaccine,2017,35(2017):1161-1166.
- [7]FREULING C M,MULLER T F,METTENLEITER T C.Vaccines against pseudorabies virus (PrV) [J].Veterinary Microbiology,2017,206:3-9.
- [8]刘涛,赵京媛,孙晓成.猪伪狂犬疫苗研究进展[J].兽医导刊,2012,12:72-74.
- [9]NOEL B P,TERPENING S J,SNOW D,et al.Vero cell technology for rapid development of inactivated whole virus vaccines for emerging viral diseases [J].Expert Review of Vaccines,2017,16(9):883-894.
- [10]XU P,CLARK C,RYDER T,et al.Characterization of TAP Ambr 250 disposable bioreactors,as a reliable scale-down model for biologics process development [J].Biotechnology Progress,2017,33(2):478-489.
- [11]许峰.基于工业化生产的治疗用单克隆抗体上游制备及放大工艺研究[D].苏州:苏州大学,2014.
- [12]XING Z,KENTY B M,LI Z J,et al.Scale-up analysis for a CHO cell culture process in large-scale bioreactors [J].Biotechnology and Bioengineering,2009,103(4):733-746.
- [13]RU A L,JACOB D,TRANSFIGURACION J,et al.Scalable production of influenza virus in HEK-293 cells for efficient vaccine manufacturing[J].Vaccine,2010,28(21):3661-3671.
- [14]LIU C C,GUO M S,LIN F H,et al.Purification and characterization of enterovirus 71 viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system [J].PLoS One,2011,6(5):e20005.
- [15]NUNEZ E G,LEME J,DE ALMEIDA PARIZOTTO L,et al.Influence of aeration-homogenization system in stirred tank bioreactors,dissolved oxygen concentration and pH control mode on BHK-21 cell growth and metabolism [J].Cytotechnology,2014,66(4):605-617.
- [16]SLIVAC I,GAURINA SRCEK V,RADOSEVIC K,et al.Aujeszky′s disease virus production in disposable bioreactor [J].Journal of Biosciences,2006,31(3):363-368.
- [17]ZHANG X,STETTLER M,DE SANCTIS D,et al.Use of orbital shaken disposable bioreactors for mammalian cell cultures from the milliliter-scale to the 1,000-liter scale [J].Advance in Biochemical Engineering Biotechnology,2009,115:33-53.
- [18]NIENOW A W.Reactor engineering in large scale animal cell culture [J].Cytotechnology,2006,50(1/2/3):9-33.
- [19]郑兵兵,动物细胞培养用生物反应器内流体混合及气液传质特性研究[D].上海:华东理工大学,2013.
- [20]刘岩松,王华,孙宏亮,等.应用篮式生物反应器制备森林脑炎灭活疫苗(Vero细胞)[J].中国生物制品学杂志,2015,28(12):1324-1331.