应用微环DNA技术体外诱导和制备重组的乙型肝炎病毒共价闭合环状DNA

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。

Preparing recombinant cccDNA of hepatitis B virus in vitro using minicircle DNA vector technology

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.