EV71检测细胞系的构建与鉴定

摘要

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.%目的 构建71型肠道病毒(enterovirus 71,EV71)检测细胞系.方法 以含有绿色荧光蛋白(green fluorescent protein,GFP)基因的EV71感染性克隆pWSK-T7-EV71-GFP为分子基础,构建GFP基因嵌入病毒基因组两端非编码区的表达盒UGFP,并将其克隆入质粒pLV-Puro获得慢病毒表达载体pLV-UGFP.将质粒pLV-UGFP和包装质粒Helper用脂质体转染法共转染入HEK293T细胞中,转染48 h后,用收集到的慢病毒颗粒感染BHK-21细胞,经嘌呤霉素压力筛选和系列稀释法获得用于外源EV71检测的工程细胞系BHK/UGFP.结果 BHK/UGFP细胞感染EV71 48 h后,可检测到GFP的表达,作为对照组细胞感染甲病毒属的辛德毕斯病毒(XJ-160)和黄病毒属的乙脑病毒(P3)等单股正链RNA病毒,则检测不到绿色荧光.该检测细胞系用不同滴度的EV71感染,至少可以检测到10 PFU/ml的EV71.结论 构建的BHK/UGFP检测细胞系可用于外源EV71的检测,且该检测方法特异性和灵敏性均良好.