流感病毒在MDCK细胞上灭活验证方法的筛选及应用  

Screening and application of a verification method for influenza virus inactivation in MDCK cells

作　　者：吕原 李臣锋 杨越 隋丽娜 李爽 吴业红 王清爽 张雪梅 LV Yuan;LI Chenfeng;YANG Yue;SUI Lina;LI Shuang;WU Yehong;WANG Qingshuang;ZHANG Xuemei(Changchun Institute of Biological Products,Co.,Ltd.,Changchun 130012,Jilin Province,China;不详)

机构地区：[1]长春生物制品研究所有限责任公司,吉林长春130012 [2]长春理工大学生命科学技术学院生物工程系,吉林长春130022 [3]北京生物制品研究所有限责任公司,北京100176

出　　处：《中国生物制品学杂志》2025年第2期197-203,共7页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划项目(20210204082YY);中央引导地方科技发展资金项目(YDZX20202200004719-1)。

摘　　要：目的筛选流感病毒在MDCK细胞上的灭活验证方法,并应用于流感病毒灭活前后样品的检测,以期为流感疫苗质量检测提供可靠的方法。方法将适应MDCK细胞的B/Yamagata(BY)、B/Victoria(BV)、H1N1、H3N24种型别的流感病毒分别稀释为10、1、0.1、0.01、0.001、0.0001 lgCCID50/mL后,接种于MDCK细胞,分别采用换液法(吸附1.5 h后弃掉病毒培养液,再加入病毒培养液,每3 d传代1次,共传3代)、补液法(吸附1.5 h后直接补加病毒培养液,每3 d传代1次,共传3代)及《欧洲药典》法(每4 d传代1次,共传3代)培养,每天观察细胞病变(cytopathic effect,CPE)情况,收获各代次病毒液,检测血凝素(hemagglutinin,HA)滴度,确定最佳灭活验证方法。采用最佳灭活验证方法检测流感病毒灭活前后样品。结果换液法和补液法均可在0.1~1 lgCCID50/mL滴度范围内检测到流感病毒,《欧洲药典》法可在1~10 lgCCID50/mL滴度范围内检测到流感病毒,且补液法测得的HA滴度高于其他两种方法,因此,确定补液法为最佳灭活验证方法。采用补液法检测流感病毒灭活前样品均为阳性,灭活后样品均为阴性。结论补液法是流感病毒在MDCK细胞上的最佳灭活验证方法,可用于流感疫苗质量的检测。Objective To screen a verification method for influenza virus inactivation in MDCK cells,and apply it to the sample detection before and after influenza virus inactivation,so as to provide a reliable method for the quality detection of influenza vaccines.Methods Four types of influenza viruses adapted to MDCK cells,B/Yamagata(BY),B/Victoria(BV),H1N1 and H3N2,were diluted to 10,1,0.1,0.01,0.001,and 0.0001 lgCCID_(50)/mL separately,which were then inoculated into MDCK cells and cultured by changing the medium(discarding after adsorption for 1.5 h and adding virus culture medium,passaging every 3 d for a total of 3 generations),rehydration method(adding virus culture medium directly after adsorption for 1.5 h,passaging every 3 d for a total of 3 generations),and method in European Pharmacopoeia(passaging every 4 d for a total of 3 generations).The cytopathic effect(CPE)was observed every day,the virus solutions of different passages were harvested,and the hemagglutinin(HA)titers were detected to determine the optimum inactivation verification method.Finally,the optimum inactivation verification method was used to detect samples before and after inactivation of influenza virus.Results Influenza virus could be detected at the titer of 0.1-1 lgCCID_(50)/mL by both changing medium method and rehydration method,while influenza virus could be detected in the titer range of 1-10 lgCCID_(50)/mL by test protocol in European Pharmacopoeia,and the titer of HA detected by rehydration method was higher than those by the other two methods.Therefore,rehydration method was determined as the optimum inactivation verification method.The samples detected by rehydration method were all positive before influenza virus inactivation and negative after virus inactivation.Conclusion Rehydration method is the optimum method to verify the influenza virus inactivation in MDCK cells,which can be used to detect the quality of influenza vaccines.

关 键 词：流感病毒 流感疫苗 MDCK细胞 病毒灭活 

分 类 号：R373.13[医药卫生—病原生物学]