中科院深圳先进技术研究院医药所基因与细胞工程研究室陈志英团队，成功利用微环DNA技术生产出乙肝病毒共价闭合环状DNA。这一技术为开发根治慢性乙肝病毒感染的治疗药物提供了重要条件。相关成果日前发表于《Scientific Reports》杂志。

据世界卫生组织报道，全球有超过3亿人感染慢性乙肝病毒，其在亚洲和非洲的发病率尤其高。尽管现有抗病毒药物可控制乙型肝炎病毒的复制，却无法将其彻底清除。

共价闭合环状DNA是乙肝病毒复制的模板，其在肝细胞核内持续、稳定的存在被认为是乙肝病毒感染慢性化及抗病毒治疗停止后复发的最重要因素。因此，彻底清除残余共价闭合环状DNA是治愈慢性乙肝病毒感染的关键。然而，此前一直缺乏生产大量共价闭合环状DNA的便捷技术。

在最新研究中，陈志英团队利用微环DNA技术，在常规实验室环境中生产出毫克级别的重组共价闭合环状DNA。其无论在结构还是功能上均跟野生型非常接近：重组共价闭合环状DNA能介导病毒基因组复制、病毒基因表达，并能产生完整的感染性病毒颗粒。该技术有望助力乙肝病毒共价闭合环状DNA的生物学研究及抗共价闭合环状DNA药物的研发。（来源：中国科学报 丁宁宁 谌平 冯春）

The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely

Abstract  HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing tool in vitro. In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus.

原文链接：http://www.nature.com/articles/srep25552