摘要
目的建立狂犬病病毒固定株PV2061在人二倍体细胞MRC-5上的适应株,为狂犬病疫苗研究提供生产用毒株。方法将狂犬病病毒固定株PV2061接种于MRC-5细胞并进行连续性传代,通过对感染剂量(1∶100、1∶200、1∶300、1∶400、1∶500)、收获天数(4、7、10、13、16 d)及感染方式(混种感染、吸附感染、带毒传代)的优化,确定在MRC-5细胞上的培养条件,并分析不同层析介质(Sepharose 4FF、Sepharose 6FF)对狂犬病病毒分离纯化效果的影响。结果狂犬病病毒固定株PV2061能较好地适应MRC-5细胞,通过连续性传代,在第4代病毒滴度可达6.0 lgLD_(50)/mL以上,11~19代病毒滴度稳定在6.5 lgLD_(50)/mL。感染剂量在1∶100~1∶500范围内,病毒滴度均能达6.0 lgLD_(50)/mL以上;病毒峰值主要集中在8~13 d;混种感染和带毒传代优于吸附感染,病毒滴度均在5.5 lgLD_(50)/mL以上。与Sepharose 6FF相比,Sepharose 4FF能有效去除总蛋白质,两种介质对抗原回收率均在85%~120%范围内,牛血清白蛋白残留量均低于50 ng/mL。结论建立了MRC-5细胞传代适应的狂犬病病毒适应株MPV2061,为后续人用狂犬病疫苗生产工艺的研究提供了数据支持。
Objective To establish a rabies virus fixed strain PV2061 adpated to human diploid cell MRC-5 and provide a strain for production of rabies vaccine for research use.Methods Rabies virus fixed strain PV2061 was inoculated to MRC-5 cells for continuous subculture,of which the culture conditions including the MOI(1∶100,1∶200,1∶300,1∶400 and 1∶500),harvesting time(4,7,10,13 and 16 d)and infection mode(mixed infection,adsorption infection and subculture)were optimized.The effects of different chromatographic media(Sepharose 4FF and Sepharose 6FF)on isolation and purification of rabies virus were analyzed.Results The rabies virus fixed strain PV2061 was adapted to MRC-5 cells well,of which the titer of passage 4 was more than 6.0 lg LD_(50)/mL,while those of the passages 11~19 were maintained at 6.5 lgLD_(50)/mL.The titer of virus inoculated at MOIs of 1∶100~1∶500 reached more than 6.0 lgLD_(50)/mL,while the virus peak mainly appeared 8~13 d after inoculation.Mixed infection and subculture were superior to adsorption infection,of which the virus titers were both more than 5.5 lgLD_(50)/mL.Compared with Sepharose 6FF,Sepharose 4FF effectively removed the total protein,by which the antigen recovery rates were both 85%~120%and residual BSA contents were both less than 50 ng/mL.Conclusion The rabies virus strain MPV2061 adapted to subculture in MRC-5 cells was established,which provided a data support for the subsequent study on production process of human rabies vaccine.
作者
廖辉
吴琼
焦龙
甄祖刚
杨文腰
周荔葆
LIAO Hui;WU Qiong;JIAO Long;ZHEN Zu-gang;YANG Wen-yao;ZHOU Li-bao(Liaoning Chengda Bio-Tech.Company Limited,Shenyang 110179,Liaoning Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2022年第7期785-789,共5页
Chinese Journal of Biologicals
基金
沈阳市科技计划项目(F12-144-9-00)。
关键词
狂犬病病毒
固定株
二倍体细胞
连续传代
病毒滴度
感染方式
层析纯化
Rabies virus
Fixed strain
Diploid cells
Continuous subculture
Virus titer
Infection mode
Chromatographic purification
