EXPRESSION OF CLONED HEPATITIS-B VIRUS-DNA IN HUMAN CELL-CULTURES
EXPRESSION OF CLONED HEPATITIS-B VIRUS-DNA IN HUMAN CELL-CULTURES
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DOI：
10.1073/pnas.77.9.5507
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发表时间：
1980-01-01
期刊：
影响因子：
--
通讯作者：
ACS, G
中科院分区：
文献类型：
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作者：
HIRSCHMAN, SZ;PRICE, P;ACS, G
从B型肝炎病毒（HBV）ayw亚型中分离DNA，将其与所有4种脱氧核苷三磷酸体外孵育，以通过内源性DNA聚合酶完成环状病毒基因组。用EcoRI限制性内切酶切割纯化的病毒DNA，插入质粒pBR 322的EcoRI位点，并克隆到大肠杆菌chi中。1776.用EcoRI切割来自含有3200个碱基对HBV DNA插入片段的克隆pHBV-1的DNA，并在有利于分子内连接的条件下与噬菌体T4连接酶孵育。HeLa [人宫颈癌]细胞培养物暴露于该DNA后，在传代培养后11-14天显示出显著的细胞病变变化，伴随着B型肝炎核心和表面抗原的产生。从这些细胞的培养基中的抗肝炎B表面抗原免疫沉淀物的EM检查显示具有中心核的42-nm颗粒和20-nm圆形颗粒。虽然没有完整的环形或EcoRI切割的线性pHBV-1 DNA诱发这些影响在HeLa细胞，细胞病变的变化和核内B型肝炎核心抗原检测在HeLa细胞感染Dane颗粒。
DNA was isolated from the ayw subtype of hepatitis B virus (HBV) that was incubated in vitro with all 4 deoxynucleoside triphosphates to complete the circular viral genome by the endogenous DNA polymerase. The purified viral DNA was cleaved with EcoRI restriction endonuclease, inserted into the EcoRI site of plasmid pBR322, and cloned in Escherichia coli .chi. 1776. DNA from a clone, pHBV-1, that contained a 3200-base-pair insert of HBV DNA was cleaved with EcoRI and incubated with phage T4 ligase under conditions favoring intramolecular ligation. HeLa [human cervical carcinoma] cell cultures exposed to this DNA showed marked cytopathic changes, accompanied by production of hepatitis B core and surface antigens, 11-14 days after subculture. EM examination of anti-hepatitis B surface antigen immunoprecipitates from culture media of these cells revealed both 42-nm particles with central cores and 20-nm round particles. Although neither intact circular nor EcoRI-cleaved linear pHBV-1 DNA evoked these effects in HeLa cells, both cytophathic changes and intranuclear hepatitis B core antigen were detected in HeLa cells infected with Dane particles.