Targeted transfection and expression of hepatitis B viral DNA in human hepatoma cells.
Targeted transfection and expression of hepatitis B viral DNA in human hepatoma cells.
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乙型肝炎病毒 DNA 在人肝癌细胞中的靶向转染和表达。
DOI：
10.1172/jci116287
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Wu,GY
中科院分区：
文献类型：
--
作者：
Liang,TJ;Makdisi,WJ;Sun,S;Hasegawa,K;Zhang,Y;Wands,JR;Wu,CH;Wu,GY
由共价连接到聚-L-赖氨酸的脱唾液酸糖蛋白组成的可溶性DNA载体系统用于结合DNA并将B型肝炎病毒（HBV）DNA构建体递送到脱唾液酸糖蛋白受体阳性的人肝癌细胞。用表面或核心基因表达构建体转染后4天，测得培养基中的HBsAg和HBeAg分别为16 ng/ml和32 U/ml/10（7）个细胞。抗原的产生被完全抑制的添加过量的无唾液酸orosomucoid。另一方面，去唾液酸糖蛋白受体阴性的人肝癌细胞，SK-Hep 1，在与HBV DNA孵育后，在相同的条件下不产生任何病毒抗原复合物组成的无唾液酸orosomucoid和聚-L-赖氨酸。使用完整的HBV基因组构建体，HBsAg和HBeAg水平分别达到16 ng/ml和16 U/ml/10（7）个细胞。北方印迹显示特征性的HBV RNA转录物包括3.5-、2.4-和2.1-kb片段。Southern印迹杂交检测细胞内和细胞外HBV DNA序列，包括松弛环状、线性和单链形式。最后，通过电子显微镜观察从用过的培养基中纯化的42 nm Dane颗粒。这项研究表明，靶向DNA载体系统可以在体外转染HBV DNA，从而产生完整的HBV病毒体。图片
A soluble DNA carrier system consisting of an asialoglycoprotein covalently linked to poly-L-lysine was used to bind DNA and deliver hepatitis B virus (HBV) DNA constructs to asialoglycoprotein receptor-positive human hepatoma cells. 4 d after transfection with surface or core gene expression constructs, HBsAg and HBeAg in the media were measured to be 16 ng/ml and 32 U/ml per 10(7) cells, respectively. Antigen production was completely inhibited by the addition of an excess of asialoorosomucoid. On the other hand, asialoglycoprotein receptor-negative human hepatoma cells, SK-Hep1, did not produce any viral antigens under identical conditions after incubation with HBV DNA complexed to a conjugate composed of asialoorosomucoid and poly-L-lysine. Using a complete HBV genome construct, HBsAg and HBeAg levels reached 16 ng/ml and 16 U/ml per 10(7) cells, respectively. Northern blots revealed characteristic HBV RNA transcripts including 3.5-, 2.4-, and 2.1-kb fragments. Intracellular and extracellular HBV DNA sequences including relaxed circular, linear and single stranded forms were detected by Southern blot hybridization. Finally, 42-nm Dane particles purified from the spent cultures medium were visualized by electron microscopy. This study demonstrates that a targetable DNA carrier system can transfect HBV DNA in vitro resulting in the production of complete HBV virions.Images