摘要
目的:为诱导HBV特异性CTL,构建HBV全基因真核表达载体,并对其在细胞中的表达进行了初步研究。方法:以EcoRⅠ酶切pBR322-2HBV和pcDNA3,回收完整的HBV基因片段及线性化的载体pcDNA3,T4DNA连接酶连接HBV和pcDNA3,转化、筛选,酶切鉴定HBV的插入方向。以脂质体将构建的pcDNA3-HBV转染HepG2肝癌细胞,经G418筛选,ELISA检测细胞培养上清中HBsAg、HBeAg的表达,RT-PCR检测转染细胞中PreS1mRNA的表达。结果:经酶切鉴定获得正向插入的pcDNA3-HBV,转染HepG2后,建立了新的肝癌细胞系HepG2.02G,细胞培养上清中可检测到高水平的HBsAg,PreS1mRNA在转染细胞中表达。结论:经体外重组,获得携带全长HBV基因片段的pcDNA3-HBV表达载体,并可在肝癌细胞中稳定表达。
Objective:To construct eukaryotic cell expression vector carrying HBV full length gene,and study its replication,transcription and expression in human hepatoma cell line.Methods:pBR322-2HBV and pcDNA3were lined by EcoRⅠdigestion,then HBV full length DNA and linear pcDNA3were extracted.HBV entire genome was inserted at the EcoRⅠsite in the MCS of pcDNA3with T4ligase.Hu-man hepatoma cell HepG2was transfected with pcDNA3-HBV by using lipofectamine transfection reagent and screened with antibiotic G418.HBsAg and HBeAg were measured by ELISA kits.Pre-S1mRNA expres-sion was tested by RT-PCR.Results:We got pcDNA3-HBV expression vector carring HBV full length DNA and identified its sense direction by endonucleases digestion.After transfection,HepG2.02G cell line was established with G418screening and passaged for10months.HepG2.02G cells could express HBsAg constantly.PreS1mRNA was also measured by RT-PCR.Conclusion:pcDNA3-HBV expression vector was constructed,which could be effectively replicated,transcribed and expressed in human hepatoma cell HepG2.And a new cell line HepG2.02G which could express HBV antigen was established with this expression vector.
出处
《山东大学学报（医学版）》
CAS
2003年第4期351-354,共4页
Journal of Shandong University:Health Sciences
基金
国家自然科学基金委海外青年学者合作研究基金资助项目(30128023)
国家自然科学基金资助项目(30070341)
关键词
肝炎病毒
乙型
表达载体
真核细胞
Hepatitis B virus
Expression vector
Eukaryotic cell
