School of Life Sciences, Institute of Physical Science and Information Technology, Anhui University, 111 Jiulong Road, Hefei, 230601, Anhui, People's Republic of China.
Biotechnol Lett. 2023 Oct;45(10):1327-1337. doi: 10.1007/s10529-023-03412-9. Epub 2023 Aug 1.
Viruses, such as Ebola virus (EBOV), evolve rapidly and threaten the human health. There is a great demand to exploit efficient gene-editing techniques for the identification of virus to probe virulence mechanism for drug development.
Based on lambda Red recombination in Escherichia coli (E. coli), counter-selection, and in vitro annealing, a high-efficiency genetic method was utilized here for precisely engineering viruses. EBOV trVLPs assay and dual luciferase reporter assay were used to further test the effect of mutations on virus replication.
Considering the significance of matrix protein VP24 in EBOV replication, the types of mutations within vp24, including several single-base substitutions, one double-base substitution, two seamless deletions, and one targeted insertion, were generated on the multi-copy plasmid of E. coli. Further, the length of the homology arms for recombination and in vitro annealing, and the amount of DNA cassettes and linear plasmids were optimized to create a more elaborate and cost-efficient protocol than original approach. The effects of VP24 mutations on the expression of a reporter gene (luciferase) from the EBOV minigenome were determined, and results indicated that mutations of key sites within VP24 have significant impacts on EBOV replication.
This precise mutagenesis method will facilitate effective and simple editing of viral genes in E. coli.
病毒（如埃博拉病毒[EBOV]）进化迅速，威胁人类健康。因此，迫切需要开发有效的基因编辑技术来鉴定病毒，以探究其毒力机制，从而开发药物。
基于大肠杆菌（E. coli）中的 lambda Red 重组、反选择和体外退火，我们在这里利用一种高效的遗传方法来精确地对病毒进行工程改造。我们使用 EBOV trVLPs 测定法和双荧光素酶报告基因测定法进一步测试突变对病毒复制的影响。
考虑到基质蛋白 VP24 在 EBOV 复制中的重要性，我们在 E. coli 的多拷贝质粒上生成了 vp24 内的多种突变类型，包括几个单碱基取代、一个双碱基取代、两个无缝缺失和一个靶向插入。此外，我们还优化了重组和体外退火同源臂的长度、DNA 盒和线性质粒的数量，以创建比原始方法更精细、更经济高效的方案。我们还确定了 VP24 突变对 EBOV 小基因组报告基因（荧光素酶）表达的影响，结果表明 VP24 内关键位点的突变对 EBOV 复制有显著影响。
这种精确的诱变方法将有助于在大肠杆菌中有效且简单地编辑病毒基因。
