Ⅰ型登革病毒D06063株人二倍体细胞适应株的筛选及纯化  

Screening and purification of Dengue-Ⅰ virus D06063 strain adapted in human diploid cells

作　　者：赵玉娇[1] 潘玥[1] 陈俊英[1] 杨丽娟[1] 岳耀斐 施海晶 马绍辉[1] 孙强明[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,昆明650118

出　　处：《中国生物制品学杂志》2012年第12期1611-1614,共4页Chinese Journal of Biologicals

基　　金：云南省科技厅社会发展科技计划项目(2011CA016);中国医学科学院病原生物学研究所中央级公益性科研院所科研业务费项目(2009IPB102);中国医学科学院医学生物学研究所引进人才项目(IMB2009RC01)

摘　　要：目的筛选Ⅰ型登革病毒(Dengue virus,DV)中国株D06063人胚肺二倍体细胞KMB17的适应株,经噬斑纯化后,分析其生物学特性。方法将Ⅰ型DV中国株D06063在C6/36细胞上进行扩增,采用微量滴定法测定病毒的感染性滴度;RT-PCR法鉴定DV型别;病毒连续以4.0 MOI的量感染KMB17细胞,传代至病毒完全适应在细胞内扩增,再连续传10代,筛选出KMB17细胞适应株,并进行3轮噬斑纯化;免疫荧光法检测纯化病毒,电镜观察病毒颗粒的形态。结果在C6/36细胞上扩增的Ⅰ型DV D06063株的滴度为6.0 lg CCID50/ml,经RT-PCR可扩增出511 bp的DV特异基因和482 bp的Ⅰ型DV型特异性基因;病毒感染KMB17细胞后,可产生明显的细胞病变,至第10代,病毒的感染性滴度达峰值,为6.75 lg CCID50/ml;纯化的病毒经免疫荧光检测呈阳性,电镜观察显示病毒形态正常。结论成功筛选出传代稳定性好、病毒扩增量高的Ⅰ型DVKMB17细胞适应株,纯化的病毒株保持了原始毒株的基本生物学特性。Objective To screen the adaptive strain of Dengue-Ⅰ virus D06063 strain（China strain） in human diploid KMB17 cells,subject to plaque purification and analyze its biological characteristics.Methods Dengue-Ⅰvirus D06063 strain was propagated in C6 / 36 cells,determined for infectious titer by microtitration,and identified for type by RT-PCR,then subcultured in KMB17 cells at a MOI of 4.0 till the virus was completely adapted to propagation in cell,and further subcultured for 10 passages.The adapted strain was screened and subjected to three rounds of plaque purification.The purified strain was analyzed by IFA and observed for morphology of virus particles by electron microscopy.Results The Dengue-Ⅰvirus D06063 strain propagated in C6/36 cells reached a titer of 6.0 lgCCID50 / ml,from which the Dengue virus（DV）specific gene at a length of 511 bp,and DV type Ⅰ specific gene at a length of 482 bp were amplified by RT-PCR.The virus caused obvious CPE in infected KMB17 cells,of which the titer reached the maximum of 6.75 lgCCID50 / ml after subculture for 10 passages.The purified virus was positive in IFA,and showed normal morphology under electron microscope.Conclusion The Dengue-Ⅰvirus D06063 strain adapted in KMB17 cells was successfully screened,which was high stable during subculture and reached a high propagation level.The purified strain maintained the basic biological characteristics of original strain.

关 键 词：登革病毒 人胚肺二倍体细胞 噬斑纯化 生物学特性 

分 类 号：R373.33[医药卫生—病原生物学] R392.33[医药卫生—基础医学]