乙型肝炎病毒共价闭合环状 DNA 染色质免疫沉淀检测法。
Hepatitis B Virus Covalently Closed Circular DNA Chromatin Immunoprecipitation Assay.
机构信息
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.
出版信息
Methods Mol Biol. 2024;2837:23-32. doi: 10.1007/978-1-0716-4027-2_3.
Hepatitis B virus (HBV) is an obligate human hepatotropic DNA virus causing both transient and chronic infection. The livers of chronic hepatitis B patients have a high risk of developing liver fibrosis, cirrhosis, and hepatocellular carcinoma. The nuclear episomal viral DNA intermediate, covalently closed circular DNA (cccDNA), forms a highly stable complex with host and viral proteins to serve as a transcription template and support HBV infection chronicity. Thus, characterization of the composition and dynamics of cccDNA nucleoprotein complexes providing cccDNA stability and gene regulation is of high importance for both basic and medical research. The presented method for chromatin immunoprecipitation coupled with qPCR (ChIP-qPCR) allows to assess provisional physical interaction of the protein of interest (POI) with cccDNA using POI-specific antibody, the level of enrichment of a POI on cccDNA versus control/background is characterized quantitatively using qPCR.
乙型肝炎病毒（HBV）是一种专性嗜肝 DNA 病毒，可导致急性和慢性感染。慢性乙型肝炎患者的肝脏有很高的风险发展为肝纤维化、肝硬化和肝细胞癌。核附加体病毒 DNA 中间体，共价闭合环状 DNA（cccDNA），与宿主和病毒蛋白形成高度稳定的复合物，作为转录模板并支持 HBV 感染的慢性。因此，对 cccDNA 核蛋白复合物的组成和动态进行表征，为基础和医学研究提供 cccDNA 的稳定性和基因调控具有重要意义。本研究采用染色质免疫沉淀结合 qPCR（ChIP-qPCR）的方法，使用 POI 特异性抗体评估感兴趣蛋白（POI）与 cccDNA 的暂定物理相互作用，使用 qPCR 定量表征 POI 在 cccDNA 上相对于对照/背景的富集程度。
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