BACKGROUND Recombinant Modified Vaccinia Virus Ankara has been employed as a safe and potent viral vector vaccine against infectious diseases and cancer. We generated recMVAs encoding norovirus GII.4 genotype capsid protein by using a marker-based approach and a BAC-based system. In the marker-based approach, the capsid gene together with a reporter gene was introduced into the MVA genome in DF-1 cells. Several rounds of plaque purification were carried out to get rid of the WT-MVA. In the BAC-based approach, recMVA-BAC was produced by en passant recombineering in E. coli. Subsequently, the recMVAs were rescued in DF-1 cells using a helper rabbit fibroma virus. The BAC backbone and the helper virus were eliminated by passaging in DF-1 cells. Biochemical characteristics of the recMVAs were studied. RESULTS We found the purification of the rare spontaneous recombinants time-consuming in the marker-based system. In contrast, the BAC-based system rapidly inserted the gene of interest in E. coli by en passant recombineering before virion production in DF-1 cells. The elimination of the reporter gene was found to be faster and more efficient in the BAC-based approach. With Western blotting and electron microscopy, we could prove successful capsid protein expression and proper virus-assembly, respectively. The MVA-BAC produced higher recombinant virus titers and infected DF-1 cells more efficiently. CONCLUSIONS Comparing both methods, we conclude that, in contrast to the tedious and time-consuming traditional method, the MVA-BAC system allows us to quickly generate high titer recMVAs.

中文翻译：

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重组MVA诺如病毒的产生：基于细菌人工染色体和标记的系统的比较研究。

背景技术重组修饰的痘苗病毒安卡拉已被用作针对传染病和癌症的安全有效的病毒载体疫苗。我们通过使用基于标记的方法和基于BAC的系统生成了编码诺如病毒GII.4基因型衣壳蛋白的recMVA。在基于标记的方法中，衣壳基因与报道基因一起被引入DF-1细胞的MVA基因组中。进行了几轮噬菌斑纯化以去除WT-MVA。在基于BAC的方法中，recMVA-BAC是通过在大肠杆菌中进行伴随重组而产生的。随后，使用辅助兔纤维瘤病毒将recMVA挽救在DF-1细胞中。通过在DF-1细胞中传代，可以消除BAC主干和辅助病毒。研究了recMVA的生化特性。结果我们发现在基于标记的系统中纯化罕见的自发重组体非常耗时。相比之下，基于BAC的系统通过在DF-1细胞中产生病毒体之前进行传代重组，将感兴趣的基因快速插入大肠杆菌中。发现在基于BAC的方法中消除报道基因更快，更有效。借助蛋白质印迹和电子显微镜，我们可以分别证明衣壳蛋白的成功表达和正确的病毒组装。MVA-BAC产生更高的重组病毒滴度，更有效地感染DF-1细胞。结论比较这两种方法，我们得出结论，与乏味且耗时的传统方法相反，MVA-BAC系统使我们能够快速生成高滴度的recMVA。