日本脑炎病毒受体功能缺陷细胞的筛选和鉴定

目的筛选并鉴定日本脑炎病毒（JEV）受体功能缺陷的BHK-21细胞突变株。方法用化学诱变剂ICR-191人工诱变JEV易感细胞系BHK-21,经JEV攻击,对存活细胞克隆化,RT-PCR筛选JEV RNA阴性细胞,用流式细胞术,免疫荧光和空斑实验鉴定其对JEV的敏感性。结果筛选到1株JEV RNA阴性细胞3A10-3F。流式细胞术检测3A10-3F细胞与JEV的结合率为2.10%,免疫荧光和空斑实验证实其对JEV感染有相当程度的抵抗,在JEV感染复数MOI 1时3A10-3F细胞培养上清中JEV最高滴度比BHK-21细胞中JEV最高滴度下降2个数量级。结论用化学诱变法筛选到1株JEV受体功能缺陷细胞,为进一步鉴定JEV受体,深入研究JEV致病机理提供了有益线索。

Selection and identification of a BHK-21 cell mutant lacking the function of Japanese encephalitis virus receptor

Objective To select and characterize a BHK-21 cell mutant lacking the function of Japanese encephalitis virus(JEV) receptor.Methods ICR-191,a DNA alkylating agent,was used to introduce random mutations in BHK-21 cells that were susceptible to JEV.After challenged with JEV,the surviving cells were cloned by limiting dilution.Individual clones were detected by RT-PCR for JEV RNA,and the JEV RNA-negative mutant cell line was further analyzed by flow cytometry,IFA and viral plaque assay.Results A JEV RNA-negative mutant cell line 3A10-3F was selected.Flow cytometry indicated that the binding was 2.10% between 3A10-3F cell and JEV.The results of IFA and viral plaque assay suggested 3A10-3F cell was considerably resistant to JEV infection.At MOI 1,the highest titer for JEV from 3A10-3F cells is reduced by 2 logs compared to the highest titer for JEV from BHK-21 cells. Conclusion A mutant cell line lacking the function of JEV receptor was selected.This cell line may facilitate characterization of JEV receptor molecule(s) on BHK-21 cells,which will surely deepen our understanding on JEV pathogenesis.