孟淑芳;林林;冯建平;李修兰;王佑春;李德富;

• 1:中国药品生物制品检定所卫生部国家生物技术产品质量标准化重点实验室

摘要(Abstract)：

目的建立鼠细小病毒(MMV)感染性滴度测定方法,并制备高滴度的MMV,为生物制品病毒清除/灭活工艺的验证奠定基础。方法通过分析NB324K细胞接种量、培养时间及病毒吸附时间等参数,建立MMV病毒滴度测定的半数细胞感染剂量法(CCID5)0,并分析其精密性;以A9细胞制备MMV,分析感染复数(MOI)、收获时间及收获样本对病毒滴度的影响,并检测病毒的稳定性。结果NB324K细胞的最佳接种浓度、培养时间及病毒吸附时间分别为5×103个/孔、48h及2h,所建立的检测方法精密性较好;A9细胞沉淀中病毒滴度高于上清,且随病毒MOI值的升高而逐渐增加,在MOI为10时,感染后48h收获,病毒滴度最高。制备的病毒液的平均滴度为8.69CCID50/ml,4℃和37℃保存7d及反复冻融5次,稳定性良好。结论已建立了MMV感染性滴度测定方法,并制备了高滴度的病毒,为以MMV为指示病毒进行病毒清除/灭活工艺验证奠定了基础。

关键词(KeyWords)： 鼠细小病毒;感染性滴度;NB324K细胞;A9细胞;半数细胞感染剂量;感染复数

Abstract:

Keywords:

基金项目(Foundation): “国家科技基础条件平台建设-实验细胞资源的整理整合与共享”项目资助(2005DKA21502)

作者(Authors): 孟淑芳;林林;冯建平;李修兰;王佑春;李德富;

DOI: 10.13200/j.cjb.2009.08.85.mengshf.020

参考文献(References)：

• [1]Fields BN,Knipe DM,Howley PM,et al.Fields virology.3rd ed.Philadelphia:Lippincott-Raven Publishers,1996.
• [2]Linser P,Bruning H,Armentrout RW.Specific binding sites for a parvovirus,minute virus of mice,on cultured mouse cells.J Virol,1977,24(1):211-221.
• [3]Williams WP,Tamburic L,Astell CR.Increased levels of B1and B2SINE transcripts in mouse fibroblast cells due to minute virus of mice infection.Virology,2004,327(2):233-241.
• [4]Astell CR.Replication of minute virus of mice in Chinese hamster ovary fibroblasts.J Gene Virol,1977,35(3):587-591.
• [5]Boschetti N,Wyss K,Mischler A,et al.Stability of minute virus of mice against temperature and sodium hydroxide.Biologicals,2003,(3):181-185.
• [6]Boschetti N,Niederhauser I,Kempf C,et al.Different susceptibility of B19virus and mice minute virus to low pH treatment.Transfu-sion,2004,44(7):1079-1086.
• [7]Garnick RL.Experience with viral contamination in cell culture.Dev Biol Stand,1996,88:49-56.
• [8]Chang A,Havas S,Borellini F,et al.A rapid and simple procedure to detect the presence of MVM in conditioned cell fluids or culture media.Biologicals,1997,25(4):415-419.
• [9]Zhan D,Roy MR,Valera C,et al.Detection of minute virus of mice using real time quantitative PCR in assessment of virus clearance during the purification of mammalian cell substrate derived bio-therapeutics.Biologicals,2002,30(4):259-270.
• [10]Wickramasinghe SR,Han B,Carlson JO,et al.Clearance of minute virus of mice by flocculation and microfiltration.Biotech-nol Bioeng,2004,86(6):612-621.
• [11]黄祯详.医学病毒学基础及实验技术.北京:科学出版社,1990:897.
• [12]Paglino J,Burnett E,Tattersall P.Exploring the contribution of distal P4promoter elements to the oncoselectivity of minute virus of mice.Virology,2007,361(1):174-184.
• [13]Tattersall F,Bratton J.Reciprocal productive and restrictive virus-cell interactions of immunosuppressive and prototype strains of minute virus of mice.J Virol,1983,46(3):944-955.