乙型肝炎病毒(hepatitis B virus，HBV)共价闭合环状DNA(covalently closed circular DNA，cccDNA)是病毒慢性感染的分子基础。本课题组前期研究通过Cre/loxP介导的位点特异性DNA重组策略，在细胞核内由前体质粒诱导重组cccDNA(rcccDNA^loxP)产生，首次建立了HBV cccDNA的体外培养细胞和小鼠实验模型。本研究基于大肠埃希菌ZYCY10P3S2T PhiC31重组酶诱导表达系统，建立了一种体外诱导HBV rcccDNA(rcccDNA^attR)微环产生和纯化的策略。纯化的rcccDNA^attR微环具有超螺旋结构，细胞培养实验证实其能支持功能性的HBV复制和抗原表达。与普通的线性HBV复制子编码质粒相比，rcccDNA^attR尾静脉高压注射小鼠模型能诱导显著延长的病毒抗原血症。因此，本研究在原核表达系统和实验小鼠水平提供了一种更为简化的HBV cccDNA实验模型系统，并再次显示rcccDNA具有显著的稳定性，能作为一种基本策略在小鼠模型中诱导病毒持续感染。

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 (rcccDNA^loxP). rcccDNA^loxP 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 (rcccDNA^attR). Purified rcccDNA^attR minicircles were supercoils which demonstrated to support functional HBV expression and replication in the cell culture. Using the technique of hydrodynamic injection, rcccDNA^attR 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.