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目的 探讨HBVDNA复制和表达的跨种属特异性 ,为HBVDNA转染跨种属原代肝细胞模型的建立提供实践基础和理论依据。方法 分离培养原代鸭肝细胞 (PDH) ,电转线性HBVDNA(转染组 ,1.19× 10 12 拷贝 / 1× 10 7PDH)或急性感染DHBV (感染组 ,4× 10 8病毒颗粒 / 1× 10 7PDH) ,分别于转染 /感染后 1d、3d、5d等时点检测PDH培养上清或裂解液中HBsAg、HBeAg与DHBsAg (采用IMX系统或ELISA检测 ) ;并采用DotBlotting检测PDH裂解液中总HBVDNA。于转染 /感染后 2d提取PDH总DNA采用SouthernBlotting分析HBVDNA与DHBV复制型式。以单纯电击或未感染的PDH为对照组。结果 转染后 1d、3d和 5d各时点转染组PDH裂解液中HBsAg分别为 15 .2 4、14.5 5和5 13 (P/N值 ,阳性≥ 2 .1) ,HBeAg均为阴性 ;PDH培养上清中HBsAg与HBeAg均阴性 ;感染组各时点PDH上清中DHBsAg分别为 14.6、31.5 3、34.73(S/N值 ,阳性≥ 2 .1) ;上述各项指标于对照组均为阴性。DotBlotting显示转染组与感染组PDH各时点的裂解液中HBVDNA与DHBVDNA均为阳性 ,对照组为阴性 ;SouthernBlot分析显示 :转染组HBVDNA与感染组DHBV均为游离复制型 ,可见 4.0kb以下分子涂抹带 ,包括rcDNA、cccDNA(scDNA )和ssDNA等复制中间体 ;未见整合型HBVDNA 高分子区(4～ 2 4kb)?
Objective To investigate the cross-species specificity of HBVDNA replication and expression and to provide a practical basis and a theoretical basis for the establishment of a cross-species primary hepatocyte cell line transfected with HBV DNA. Methods Primary cultured duck hepatocytes (PDH), linear HBVDNA (transfected group, 1.19 × 10 12 copies / 1 × 10 7 PDH) or acute infected DHBV (infected group, 4 × 10 8 virions / 1 × 10 7 PDH ), HBsAg, HBeAg and DHBsAg in PDH culture supernatant or lysate were detected at 1d, 3d, 5d, respectively, after transfection / infection. The detection of total HBVDNA in PDH lysate was carried out by DotBlotting . Total DNA of PDH was extracted 2 days after transfection / infection. Southern blotting was used to analyze the pattern of HBVDNA and DHBV replication. A simple electric shock or uninfected PDH as control group. Results HBsAg in PDH lysates at 1, 3, and 5 d after transfection were 15.24, 14.55 and 5 13 (P / N values, positive ≥ 2.1), respectively. PDH culture supernatant HBsAg and HBeAg were negative; infection group at each time point PDH supernatant DHBsAg were 14.6,31.5,34.73 (S / N value, positive ≥ 2.1); the above indicators in the control group Negative. DotBlotting showed that HBVDNA and DHBVDNA in the lysates of PDH transfected and infected groups were all positive and negative in the control group. SouthernBlot analysis showed that HBVDNA in transfected group and DHBV in infected group were both free- Molecular smear zone, including rcDNA, cccDNA (scDNA) and ssDNA and other replication intermediates; no integrated HBVDNA polymer region (4 ~ 24kb)?