丁型肝炎病毒复制包装载体的构建

Construction of hepatitis D virus replication vector

摘要:

目的  构建一种可以实现丁型肝炎病毒（HDV）复制包装的HDV转座子载体。方法  采用分子克隆方法构建含HDV复制子和乙型肝炎病毒表面抗原（HBsAg）表达框的转座子（PB）载体PB126I3，将构建好的载体转染HuH-7细胞，转染48 h后用荧光显微镜观察绿色荧光蛋白（GFP）的表达，转染48 h后收集细胞上清用酶联免疫吸附法（ELISA）检测HBsAg的表达，质粒转染细胞8.5 d后提取病毒用逆转录荧光定量聚合酶链反应（RT-qPCR）检测HDV核糖核酸（RNA）的含量；同时细胞上清病毒浓缩后感染HBV受体钠离子/牛磺胆酸共转运蛋白（Na+/NTCP）稳定转染的HepG2.N9细胞系并于感染后第7天用免疫荧光检测细胞内HDVδ抗原的表达。结果  HDV复制包装载体PB126I3瞬时转染HuH-7后细胞上清可检测到HBsAg的表达和较高滴度的HDV颗粒，并且该病毒颗粒感染HepG2.N9细胞7 d后细胞内可检测到HDVδ抗原。结论  HDV复制包装转座子载体构建成功，为未来HDV的大规模复制包装以及尝试建立HDV稳定转染细胞系提供前期基础。

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.