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目的建立HBV HepG2肝细胞株,使HBV能长期稳定地表达抗原并复制。方法将含完整转录单位的1.2倍体HBV DNA经Sal (?)位点克隆入真核表达载体pREP10,构建的重组载体pREP-HBV以Lipofectamine2000转染HepG2细胞,250μg/mL潮霉素筛选抗性细胞克隆。ELISA检测细胞上清液中的HBsAg和HBeAg。电子显微镜下观察细胞上清液HBV颗粒。制备HBV特异性探针,以Southern印迹法检测细胞株内HBV核心颗粒DNA。结果获得含1.2倍体HBV DNA的重组载体,即pREP-HBV,该重组载体转染HepG2细胞后,获得5株潮霉素抗性细胞RHBV1～RHBV5,均能表达HBsAg和HBeAg。Southern印迹结果显示各株细胞均可见明显的杂交拖带,即存在HBV DNA复制中间体。细胞培养上清液浓缩后在电子显微镜下可见成簇的、直径约42 nm的HBV颗粒及直径为22～26 nm的球形颗粒。结论成功建立了HBV稳定复制及表达的HepG2细胞株,目前细胞已传代50次,每3天1次。
Objective To establish a HepG2 hepatocyte cell line that allows HBV to express and replicate stably for a long time. Methods The 1.2 ploid HBV DNA containing the complete transcriptional unit was cloned into the eukaryotic expression vector pREP10 by the Sal (?) Site. The recombinant vector pREP-HBV was transfected into HepG2 cells with Lipofectamine 2000 and screened with 250 μg / mL hygromycin Cell cloning. ELISA was used to detect HBsAg and HBeAg in the cell supernatant. The cell supernatant HBV particles were observed under an electron microscope. HBV specific probes were prepared and HBV core particle DNA was detected by Southern blotting. Results The recombinant vector containing 1.2-fold HBV DNA, pREP-HBV, was obtained. Five recombinant hygromycin-resistant cells, RHBV1-RHBV5, were transfected into HepG2 cells, both expressing HBsAg and HBeAg. Southern blot results showed that each cell line can be seen obvious hybrid tow, that is, there HBV HBV replication intermediates. After the cell culture supernatant was concentrated, clusters of HBV particles with a diameter of about 42 nm and spherical particles with a diameter of 22-26 nm were observed under an electron microscope. Conclusion HepG2 cells stably replicated and expressed in HBV were established successfully. At present, the cells have been passaged 50 times every three days.