化学发光Southern blot法检测HBV体外复制及其应用于药物对HBV的抑制分析

目的：建立化学发光Southern blot检测细胞内HBV DNA的方法，同时检测3种不同靶点抗乙肝药物的体外作用。方法：用地高辛标记HBV探针，优化杂交条件，检测来自HepG2及HepG2.2.15 HBV DNA复制中间体；利用建立的化学发光Southern blot检测HBV DNA的方法检测经拉米夫定、Bay41-4109、α-Galcer以不同药物浓度处理的HepG2.2.15HBV DNA复制中间体的水平。结果：（1）标记的HBV探针的检测灵敏度为0.1pg，杂交系统的检测灵敏度为1pg，可检测到HepG2.2.15细胞内的HBV DNA特异性信号；（2）以该法检测胞内HBV DNA可见3种药物都有明显的抑制作用，其半数有效量（IC50）分别为1.53μmol/L、0.41μmol/L、0.01μmol/L。结论：胞质HBV DNA的水平能准确地反映不同靶点抗HBV药物的抗病毒效果，建议在观察药物特别是中药抗病毒研究中采用。

Chemiluminescent Southern blot System for Detection of HBV Replication in vitro and Its Application for Analysis the Drugs Inhibition to HBV

Objective:To stablish of chemiluminescent Southern blot detection system for examining HBV DNA replication intermediates in HepG2.2.15,and analyse the inhibition of HBV replication with three kind of drugs with different targets.Methods:The HBV DNA replication intermediates were extracted and analyzed by Southern blot with HBV probe,which(pTHBV1047) was labelling with digoxigenin.The results of the hybridization were detected by chemiluminescent,and the condition of hybridization was optimized.After treated with lamivudine,Bay41-4109, α-Galcer in different concentration,the HBV DNA from the HepG2215 cells were detected with the system.Results:the sensitivity of the system was 1pg of pTHBV1047,and HBV specific positive signals was detected with the DNA from HepG2.2.15.The three kinds of drugs can inhibit the HBV replication obviously with chemiluminescent Southern blot detection system,the IC50 were 1.53μmol/L,0.41μmol/L,0.01μmol/L. Conclusion:The HBV replication intermediates from the cell of HepG2.2.15 can reflect the antiviral effect accurately with different targets drugs and this mothod would be used in the study of Chinese midicine.