大鼠细小病毒H-1株培养方法的建立  

Establishment of a culture method for Toolan virus(H-1)

作　　者：刘先菊 佟巍 张丽芳 王艳蓉 高子琪 仉惠敏 荣蓉 刘云波 

机构地区：[1]中国医学科学院医学实验动物研究所卫生部实验动物检测中心,北京100021

出　　处：《中国实验动物学报》2011年第6期495-498,I0001,共5页Acta Laboratorium Animalis Scientia Sinica

基　　金：中央级公益性科研院所基本科研业务费专项资金项目;项目编号:DWS200906

摘　　要：目的建立用大鼠胶质瘤细胞系(Rat glial cell line C6)替代大鼠原代胚细胞(primary rat embryocells,RE)培养大鼠细小病毒(Toolan virus,H-1)的方法。方法 将0.8 mL H-1病毒接种C6细胞(75T培养瓶,细胞接种量为2×105/mL,培养过夜),待细胞病变CPE达++～+++时,用免疫荧光(FITC)鉴定所培养病毒的抗原,用血球凝集试验(HA)测定培养物上清效价,用DNA测序鉴定所培养的病毒,用96孔板培养法测定H-1病毒的TCID50。结果H-1在接种到C6细胞的第3～4天,细胞发生明显的病变,CPE可达++++,FITC鉴定呈H-1抗原阳性,病毒的培养上清中HA效价为1∶320。测序结果表明:该病毒序列与NCBI中H-1序列同源性达99%,确定为H-1病毒。收获的H-1的TCID50为103.2/0.1 mL。结论用大鼠胶质瘤细胞系(C6)可以替代大鼠原代胚细胞(RE)进行大鼠细小病毒的培养。Objective To establish a culture method for Toolan virus（H-1） using the rat glioma cell line C6 cells instead of primary rat embryo cells（RE）.Methods C6 cells were diluted to 2×105/mL and then cultured overnight before infection.H-1 virus was inoculated into the C6 cells.The infected cells were cultured until the cytopathic effect（CPE） reached ＂＋ ＋ to ＋ ＋ ＋＂.The viral antigen was identified by immunofluorescence（FITC） assay and the viral titer in the culture supernatant was detected by hemagglutination test（HA）.In addition,the virus was identified by DNA sequencing of the conserved regions of H-1 genome.The virulence of viral culture（TCID50） was determined by 96-wells cell culture.Results The CPE of C6 cells reached ＂＋ ＋ ＋＂ on the 3rd to 4th day post H-1 virus infection.The results of FITC staining indicated that the viral antigen was positive for H-1 virus,and HA titer of the viral culture reached 1∶320.The cultivated virus was identified as H-1 virus,as its sequence had 99% identity with that of H-1 virus,and the virulence of H-1 virus obtained by infection in C6 cells was 103.2/mL.Conclusions The infection of H-1 virus in C6 cells and subsequent identification test indicate that H-1 virus can be maintained in C6 cells instead of rat embryo cells.

关 键 词：细小病毒 大鼠胶质瘤细胞 大鼠原代胚细胞 免疫荧光试验 血球凝集试验 

分 类 号：Q95-33[生物学—动物学]