Construction and identification of the cell line for detecting Enterovirus 71
10.3760/cma.j.issn.1003-9279.2016.04.016
- VernacularTitle:EV71检测细胞系的构建与鉴定
- Author:
Jianghong YAN
1
;
Xinjun LYU
;
Xiaoyan TAO
;
Pengcheng YU
;
Weichen WU
;
Shuying LI
;
Wuyang ZHU
Author Information
1. 063000 唐山,华北理工大学基础医学院,河北省慢性疾病重点实验室,唐山市慢性病临床基础研究重点实验室
- Keywords:
Enterovirus;
Cell lines
- From:
Chinese Journal of Experimental and Clinical Virology
2016;30(4):402-405
- CountryChina
- Language:Chinese
-
Abstract:
Objective To select and identify the cell line for detecting Enterovirus 71 (EV71).Methods pWSK-T7-EV71-GFP containing green fluorescent protein (GFP) gene is an infectious clone for EV71,based on which the UGFP cassette was constructed by inserting GFP gene into 5' and 3' untranslated region (UTR) of the genome of EV71.The lentiviral expression plasmid pLV-UGFP containing UGFP was constructed on the basis of pLV-Puro,a lentiviral vector.To obtain lentivirus,pLV-UGFP plasmids were transfected together with the packaging plasmids into HEK293T cells by liposomes.Then,the target cells BHK-21 were infected with the lentivirus particles.Puromycin-resistant cell colonies were detached from the 6 well-plate and sub-cloned by use of 96 well-plate.Finally,we selected the packaging cell lines that could express the defective replicons stably,named BHK/UGFP cells.Results GFP expression assays indicated that BHK/UGFP cells infected with EV71 could express GFP at 48 hours post-infection,while no green fluorescence was observed after BHK/UGFP cells were infection with Sindbis virus (SINV,XJ-160) or Japanese encephalitis virus (JEV,P3),demonstrating that the selected cells could specifically detect EV71 infection.The sensitive assay results indicated that this method on the basis of BHK/UGFP cells could at least detect 10 PFU/ml of EV71 in tissue culture.Conclusion This result indicated that the BHK/UGFP cells selected in this study were specific and effective to detect EV71 from tissue culture.
