丁型肝炎病毒复制包装载体的构建

发布时间：2018-04-09 15:23

本文选题：丁型肝炎病毒　切入点：载体构建　出处：《中国现代医学杂志》2017年11期

【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 HDV transposon vector for hepatitis D virus (HDV) replication packaging.Methods the HDV replicon and hepatitis B virus surface antigen (HBs) expression frame PB126I3 were constructed by molecular cloning method. The constructed vectors were transfected into HuH-7 cells and the expression of GFP was observed by fluorescence microscope after 48 h transfection.After 48 hours of transfection, the supernatant was collected to detect the expression of HBsAg by Elisa, and the RNA content of HDV RNA was detected by reverse transcription-fluorescence quantitative polymerase chain reaction (RT-qPCR) on 8.5 days after transfection.At the same time, the supernatant virus was condense and infected with HBV receptor sodium ion / taurocholic acid cotransporter Na ~ + / NTCP. the expression of HDV 未 antigen was detected by immunofluorescence on the 7th day after infection.Results the expression of HBsAg and the higher titer of HDV particles could be detected in the supernatant of HDV replication-packaging vector PB126I3 after transient transfection of HuH-7, and the HDV 未 antigen could be detected in HepG2.N9 cells 7 days after the infection.Conclusion HDV replication packaging transposon vector is successfully constructed, which provides a preliminary basis for large-scale replication and packaging of HDV and attempts to establish stable transfection cell lines of HDV.
【作者单位】：河北北方学院研究生部;中国人民解放军第309医院肾脏内科;军事医学科学院放射与辐射医学研究所;
【基金】：国家863青年科学家项目(No:2015AA020946) 北京市科技新星项目(No:Z171100001117119)
【分类号】：R373.21

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