一种体外初筛抗乙型肝炎病毒药物实验方法

An assay for screening anti-HBV in vitro

目的 :建立一个操作简便 ,灵敏度高且可半定量的体外初筛抗HBV药物实验方法。方法 :实验利用 2 .2 .15细胞、终点稀释PCR和MTT实验等技术。结果 :用标准HBV质粒PTHBV 1DNA证实该方法检测HBVDNA灵敏度达 0 .0 1pg级 ,利用终点稀释PCR检测未用药处理的 4× 10 62 .2 .15细胞上清中HBVDNA水平约有 1.2× 10 6拷贝 ;利用已知有效抗HBV药物(- )FTC验证了该方法可靠性 ,结果显示 (- )FTC抑制 2 .2 .15细胞内及上清HBVDNA的EC50 分别为 0 .5 μm和0 .0 45 μm ,选择抑制指数 (SI)分别为 >80 0和 >8888.9。结论 :能敏感区分药物作用效果 。

OBJECTIVE A safe,convenient and semi quantitative technique was developed to screen anti HBV in vitro.METHODS The screening system was consisted of cell culture of 2.2.15 . PCR with end point dilution and MTT assay. RESULTS The sensitivity of the system to test HBV DNA was 0.01 pg which was identified by using a standard HBV plasmid DNA. By using the PCR with end-point dilution, the content of HBV DNA tested in supernatant of 4×10 6 2.2.15 cells was around 1.2 ×10 6 copies. To confirm the reliability of the method, the anti HBV effects of (-)FTC known as an effective anti HBV compound was detected and the results showed that the EC 50 of the compound against HBV DNA in the intro and extro cellular were 0.5 μm and 0.045 μm,respectively. And the seletive index(SI) were >800 and > 8888.9 .CONCLUSIONS The results revealed that the technique for screening anti HBV compounds was specific and sensitive.