克隆乙型肝炎病毒DNA在肝与肾细胞中复制与表达的研究  

Studies on replication and expression of cloned hepatitis B virus DNA in transfected kidney cells and hepatocytes

作　　者：张蕾青[1] 张德强[1] 姚忻[1] 马张妹[1] 何丽芳[1] 

机构地区：[1]上海医科大学医学分子病毒学卫生部重点实验室,200032

出　　处：《上海医学》1999年第10期585-588,共4页Shanghai Medical Journal

基　　金：国家自然科学基金

摘　　要：目的　对比研究在体外细胞培养系统中乙型肝炎病毒（ Ｈ Ｂ Ｖ） 在肝、肾细胞中的复制与表达。方法　用克隆 Ｈ Ｂ Ｖ Ｄ Ｎ Ａ（ｐ１７７ 、ｐ３ ．８ Ⅱ、ｐ Ｃ Ｍ Ｖ３ ．９） 分别转染 Ｈｅｐ Ｇ２ 与２９３ 细胞系；ｐ１７７ 与ｐ３ ．８ Ⅱ还转染了原代人胚肾细胞。培养后收集转染细胞培养上清液，用 Ｅ Ｌ Ｉ Ｓ Ａ 法检测 Ｈ Ｂｓ Ａｇ 与 Ｈ Ｂｅ Ａｇ ；提取转染细胞的 Ｄ Ｎ Ａ 与 Ｒ Ｎ Ａ，进行 Ｓｏｕｔｈｅｒｎ 与 Ｎｏｒｔｈｅｒｎ 印迹杂交检测细胞内 Ｈ Ｂ Ｖ Ｄ Ｎ Ａ 与 Ｈ Ｂ Ｖ Ｒ Ｎ Ａ。结果　转染 Ｈｅｐ Ｇ２ 细胞， Ｈ Ｂｓ Ａｇ 与 Ｈ Ｂｅ Ａｇ 均有高水平表达； Ｓｏｕｔｈｅｒｎ 与 Ｎｏｒｔｈｅｒｎ 印迹杂交显示特异的 Ｈ Ｂ Ｖ Ｄ Ｎ Ａ 与 Ｈ Ｂ Ｖ Ｒ Ｎ Ａ 杂交信号。而转染２９３ 细胞后， Ｈ Ｂｓ Ａｇ 与 Ｈ Ｂｅ Ａｇ 仅出现很低水平表达； Ｓｏｕｔｈｅｒｎ 和 Ｎｏｒｔｈｅｒｎ 印迹杂交除ｐ Ｃ Ｍ Ｖ３ ．９ 转染细胞出现明显的 Ｈ Ｂ Ｖ 特异杂交信号外，其他为阴性或微弱阳性。转染原代人胚肾细胞均无 Ｈ Ｂｓ Ａｇ 与 Ｈ Ｂｅ Ａｇ 表达。结论　 Ｈ Ｂ Ｖ Ｄ Ｎ Ａ 在肝细胞中可以复制与表达，在肾细胞中仅有低水平的病毒抗原表达而无复制，只?Objective To compare the replication and expression of HBV DNA in kidney cells and hepatocytes. Methods Cloned recombinant plasmid HBV DNAs (p177,p3.8Ⅱ,pCMV3.9) were used to transfect HepG2 and 293 cell lines respectively. Besides, p177 and p3.8Ⅱ were also used to transfect the primary kidney cells. Supernatant from transfected cells were collected and assayed for HBsAg and HBeAg. DNAs and RNAs were extracted from transfected cell lines and Southern and Northern blots were hybridized with HBV probes.Results After being transfected, HepG2 cells expressed high levels of HBsAg and HBeAg, and specific HBV DNA signals were detected by Southern and Northern blots. However, expression of HBsAg and HBeAg were very low in transfected 293 cells. By Southern and Northern blots hybridization signals could only be detected in pCMV 3.9 transfected 293 cells, while no replication of HBV was detected under its native promoters.Conclusion Cloned HBV DNA replicated and expressed HBsAg and HBeAg in HepG2 cells, while only very low levels of HBsAg and HBeAg could be detected in transfec ted 293 cells. No replication of HBV DNA was detected in 293 cells except when HBV genome in the recombinant plasmid was driven by a strong promoter (pCMV) used in transfection study. Our data do not support that HBV can replicate in kidney tissue.(Shanghai Med J, 1999,22∶585 588)

关 键 词：乙型肝炎病毒 抗原表达 复制 DNA 克隆 

分 类 号：R512.62[医药卫生—内科学]