Faculty of Medicine, Institute of Virology, Philipps University Marburg, Hans-Meerwein-Str. 2, 35043, Marburg, Germany,
Arch Virol. 2013 Oct;158(10):2049-58. doi: 10.1007/s00705-013-1697-4. Epub 2013 Apr 25.
The reverse genetics system for influenza A viruses described by Hoffmann et al. (Virology 267(2):310-317, 2000, Proc Natl Acad Sci USA 97(11):6108-6113, 2000, ArchVirol 146(12):2275-2289, 2001) is one of the most commonly used. However, this cloning strategy is rather time-consuming and lacks a selection marker to identify positive clones carrying viral genes. We report here the optimization of the cloning protocol of viral genes into pHW2000 by (i) introducing a selection marker and (ii) simplifying the cloning strategy: now the cloning reaction takes only a few minutes and, in addition, is independent of internal restriction sites for BsmBI/Esp3I, BsaI or AarI. In order to accelerate the whole cloning protocol for the generation of recombinant viruses, we first introduced a lacP/Z-element (lac-promoter/lacZα-fragment) between the two BsmBI sites of pHW2000 to allow selection of positive clones by blue/white screening. Then we optimized the digestion/ligation-protocol: In our system, enzymatic digestion and ligation of PCR products into the vector is performed in a single "one-tube" reaction. Due to this strategy, time and material consumption is reduced by a great amount, as vector and cDNA do not have to be digested and purified prior to the ligation. Therefore, this one-tube reaction yields positive clones with high efficiency and fidelity, again saving time and material, which were formerly required for screening and analyzing clones. Finally, to add more versatility to the system, we also created an entry vector based on TA-cloning. This entry vector provides several advantages: inserted genes can easily be modified, e.g., by site-directed mutagenesis or tag attachment, and then subcloned into pHW2000 or other plasmids containing a similar cloning site (e.g., our modified pCAGGS-Esp-blue) by the same rapid and reliable one-tube reaction protocol described here. In fact, the presented protocol is suitable to be adapted to other reverse genetics systems (e.g., those for members of the order Mononegavirales or the family Bunyaviridae) or cloning of genes in general.
由 Hoffmann 等人描述的流感病毒反向遗传学系统（Virology 267(2):310-317, 2000, Proc Natl Acad Sci USA 97(11):6108-6113, 2000, ArchVirol 146(12):2275-2289, 2001）是最常用的系统之一。然而，这种克隆策略相当耗时，并且缺乏用于鉴定携带病毒基因的阳性克隆的选择标记。我们在此报告了通过（i）引入选择标记和（ii）简化克隆策略对 pHW2000 中病毒基因的克隆方案进行优化：现在克隆反应仅需几分钟，并且此外，它不受内部限制位点 BsmBI/Esp3I、BsaI 或 AarI 的限制。为了加速用于生成重组病毒的整个克隆方案，我们首先在 pHW2000 的两个 BsmBI 位点之间引入 lacP/Z 元件（lac 启动子/lacZα 片段），以通过蓝/白筛选来选择阳性克隆。然后我们优化了酶切/连接方案：在我们的系统中，PCR 产物的酶切和连接到载体在单个“一管”反应中进行。由于这种策略，大大减少了时间和材料的消耗，因为在连接之前不需要对载体和 cDNA 进行酶切和纯化。因此，该一管反应以高效率和保真度产生阳性克隆，再次节省了以前用于筛选和分析克隆的时间和材料。最后，为了增加系统的多功能性，我们还基于 TA 克隆创建了一个入口载体。该入口载体提供了多个优势：可以轻松修饰插入的基因，例如，通过定点突变或标签附着，然后通过相同的快速可靠的一管反应方案将其亚克隆到 pHW2000 或其他含有类似克隆位点的质粒中（例如，我们修改的 pCAGGS-Esp-blue）。实际上，所提出的方案适合于适应其他反向遗传学系统（例如，那些用于单股负链 RNA 病毒目或布尼亚病毒科的系统）或一般的基因克隆。
