A southern blot assay for detection of hepatitis B virus covalently closed circular DNA from cell cultures.
A southern blot assay for detection of hepatitis B virus covalently closed circular DNA from cell cultures.
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DOI：
10.1007/978-1-62703-484-5_13
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Guo, Haitao
中科院分区：
文献类型：
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作者：
Cai, Dawei;Nie, Hui;Yan, Ran;Guo, Ju-Tao;Block, Timothy M;Guo, Haitao
慢性乙肝仍然是一个严重的公共卫生负担，影响着全球约3.5亿人，导致肝硬化和肝癌，每年约有100万人死于乙肝及其并发症。乙肝是由乙肝病毒(乙肝病毒)感染引起的。作为病毒生命周期的重要组成部分，乙肝病毒共价闭合环状DNA(CcDNA)在感染的肝细胞中合成并维持在低拷贝数，是所有病毒RNA的转录模板。因此，cccDNA负责病毒感染和持久性的建立。CccDNA的存在和寿命可能也解释了目前抗病毒治疗的局限性。因此，了解cccDNA形成和调控的机制对于了解乙肝的发病机制和寻找治疗乙肝的方法至关重要。我们详细描述了一种提取和检测HBVcccDNA的方法。该方法包括两个主要步骤：(1)用Hirt无蛋白DNA抽提法提取HBVcccDNA；(2)用Southern杂交分析检测HBVcccDNA。该方法简便、可靠，可用于细胞培养样品的ccDNA检测，对乙肝病毒分子生物学和抗病毒研究均有一定的参考价值。
Chronic hepatitis B remains a substantial public health burden affecting approximately 350 million people worldwide, causing cirrhosis and liver cancer, and about 1 million people die each year from hepatitis B and its complications. Hepatitis B is caused by hepatitis B virus (HBV) infection. As an essential component of the viral life cycle, HBV covalently closed circular DNA (cccDNA) is synthesized and maintained at low copy numbers in the nucleus of infected hepatocytes, and serves as the transcription template for all viral RNAs. Therefore, cccDNA is responsible for the establishment of viral infection and persistence. The presence and longevity of cccDNA may also explain the limitations of current antiviral therapy for hepatitis B. Thus, understanding the mechanisms underlying cccDNA formation and regulation is critical in understanding the HBV pathogenesis and finding a cure for hepatitis B. Here we describe a protocol for HBV cccDNA extraction and detection in detail. The procedure includes two major steps: (1) HBV cccDNA extraction by Hirt protein-free DNA extraction method and (2) HBV cccDNA detection by Southern blot analysis. The method is straightforward and reliable for cccDNA assay with cell culture samples, and it is useful for both HBV molecular biology and antiviral research.