一种快速精确编辑疱疹病毒基因组的方法

A rapid and accurate method for herpesviral gnome editing

摘要:

为了快速且准确地对疱疹病毒基因组进行基因敲除、插入或者点突变等修饰，通过同源重组将马立克氏病病毒 (MDV) 超强毒株Md5基因组克隆到细菌人工染色体 (BAC)。将筛选的阳性重组体DNA电转进DH10B菌株，用PCR及限制性片段多态分析 (RFLP) 方法鉴定含Md5全基因组的BAC克隆。将阳性重组体DNA转染入鸡胚成纤维细胞 (CEF)，拯救出重组病毒，命名为Md5BAC。进一步利用Red酶介导的两步法基因重组技术构建MDVlorf10基因敲除毒株。为了验证被敲除基因功能的特异性，将lorf10插入原位点以构建基因复原毒株。将构建的重组毒株分别感染CEF细胞，用间接免疫荧光试验确认重组病毒均包装成功；病毒生长曲线结果表明，lorf10敲除不影响病毒的体外增殖。总之，这为其他疱疹病毒的基因组编辑提供了技术参考。

Abstract:

To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek’s disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Furthermore, a lorf10 deletion mutant was constructed by two step Red-mediated homologous recombination. To confirm the specific role of gene deletion, the lorf10 was reinserted into the original site of MDV genome to make a revertant strain. All the constructs were rescued by transfection into CEF cells, respectively. The successful packaging of recombinant viruses was confirmed by indirect immunofluorescence assay. The results of growth kinetics assay and plaques area measurement showed that the lorf10 is dispensable for MDV propagation in vitro. Overall, this study successfully constructed an infectious BAC clone of MDV and demonstrated its application in genome manipulation; the knowledge gained from our study could be further applied to other hepesviruses.