中国现代医学杂志2017，Vol.27Issue(11)：31-35,5.DOI:10.3969/j.issn.1005-8982.2017.11.006
丁型肝炎病毒复制包装载体的构建
Construction of hepatitis D virus replication vector
摘要
Abstract
Objective To construct a transposon vector for hepatitis D virus (HDV) replication and packaging. Methods The piggyBac (PB) transposon vector PB126I3 containing HDV replicon and hepatitis B virus surface antigen (HBsAg) expression cassette was constructed by using standard molecular cloning methods. HuH-7 cells were transfected with the vector PB126I3. The expression of green fluorescent protein (GFP) was observed by fluorescence microscopy, and the HBsAg expression level in cell medium was detected by ELISA 48 hours after transfection. HDV RNA in cell supernatants was extracted and detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) 8.5 days after transfection. Meanwhile, HDV particles in cell medium were concentrated and infected the HepG2.N9 cells which were stably transfected with HBV receptor NTCP. The cellular expression of HDV δ antigen was detected by immunofluorescence 7 days after infection. Results HuH-7 cells were successfully transfected by the vector PB126I3, and secreted a large number of HBsAg and HDV particles in cell medium. HDV δ antigen was detected obviously in HepG2.N9 cells 7 days after HDV infection. Conclusions HDV replicon vector PB126I3 were successfully constructed for HDV replication and packaging, which will promote the large-scale of HDV production and HDV transgenic hepatocyte cell line establishment in the future.关键词
丁型肝炎病毒/载体构建/复制/包装/瞬时转染Key words
hepatitis D virus/vector construction/replication/packaging/transient transfection分类
医药卫生引用本文复制引用
李颖,张五星,周伟,王学军..丁型肝炎病毒复制包装载体的构建[J].中国现代医学杂志,2017,27(11):31-35,5.基金项目
国家863青年科学家项目(No:2015AA020946) （No:2015AA020946）
北京市科技新星项目(No:Z171100001117119) （No:Z171100001117119）
