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| Preparing recombinant cccDNA of hepatitis B virus in vitro using minicircle DNA vector technology |
| ZHU Yuanfei1, 2, LI Gaiyun2, CHANG Hao2, YU Kangkang2, GAO Yueqiu1, DENG Qiang1, 2 |
| 1.Laboratory of Cellular Immunity, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; 2.Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200032, China |
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Abstract Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is essential to establish and sustain viral replication. We recently reported a technique involving HBV recombinant cccDNA (rcccDNA) using Cre/loxP-mediated DNA recombination (rcccDNAloxP). rcccDNAloxP could be substantially induced in the nuclei of hepatocytes, which thus represents a useful surrogate for HBV cccDNA-related investigations. In the present study, we described an approach to prepare rcccDNA in an Escherichia coli (E. coli) strain ZYCY10P3S2T based on PhiC31-mediated site-specific recombination (rcccDNAattR). Purified rcccDNAattR minicircles were supercoils which demonstrated to support functional HBV expression and replication in the cell culture. Using the technique of hydrodynamic injection, rcccDNAattR induced significantly prolonged HBV antigenemia in immunocompetent mice, as compared to a regular plasmid encoding linear HBV replicon. Collectively, our study provided a simplified method to surrogate HBV cccDNA in the prokaryotic expression system and in the mouse model. Our results suggested again that rcccDNA is intrinsically stable and could act as a prototype for modeling HBV persistence in mouse livers.
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Received: 28 February 2017
Published: 25 August 2017
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Corresponding Authors:
GAO Yueqiu,LI Gaiyun
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2017, Vol. 12 
