摘要
目的 建立乙型肝炎病毒（HBV）复制状态模型。方法 体外连接HBV DNA获得6.4 kb的双拷贝DNA片段，然后将其插入pcDNA3载体的Eco RV位点，通过聚合酶链反应（PCR）和酶切筛选获得头尾串联双拷贝HBV全基因重组真核表达质粒，再通过脂质体介导法转染肝癌细胞。结果 通过G418筛选，对阳性克隆细胞进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western免疫印迹分析，证明已将HBV全基因重组真核表达质粒转染至肝癌细胞并获得稳定表达。结论 通过脂质体介导法将HBV全基因转染至肝癌细胞，成功建立了HBV复制状态的细胞模型。
： Objective To establish hepatitis B virus (HBV) replication models in hepatoma cells. Methods The DNA fragment of 2 copies of HBV(HBsAg: ayw subtype) genome was inserted into EcoRV site of plasmid pcDNA3. After selection with polymerase chain reaction ( PCR) and restriction enzyme analysis, eukaryotic expression recombinant plasmid pAKS containing tandem copies of HBV genome in head-to tail connection was obtained Transfection of HepG2 cells with pAKS was performed subsequently via lipofectamine. Result After detection with ELISA, SDS-PAGE and Western-blot analysis, a hepatoma cell line in which HBV genome replication took place was identified. Conclusion We have successfully transfected HBV into HepG2 cells via lipofectamine and established HBV replication models in hepatoma cells.
出处
《第一军医大学学报》
CSCD
北大核心
2001年第7期501-502,共2页
Journal of First Military Medical University
基金
广州市科委科技项目资助(99-K-007-03)
关键词
乙型肝炎病毒
基因重组
基因转染
基因表达
肝癌
： hepatitis B virus
gene rearrangement
gene, viral
transfection
gene expression
