Mailly Laurent, Boulade-Ladame Charlotte, Orfanoudakis Georges, Deryckere François
Unité Mixte de Recherche 7175, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France.
Virol J. 2008 Jun 6;5:73. doi: 10.1186/1743-422X-5-73.
The construction of expression vectors derived from the human adenovirus type 5 (Ad5), usually based on homologous recombination, is time consuming as a shuttle plasmid has to be selected before recombination with the viral genome. Here, we describe a method allowing direct cloning of a transgene in the E3 region of the Ad5 genome already containing the immediate early CMV promoter upstream of three unique restriction sites. This allowed the construction of recombinant adenoviral genomes in just one step, reducing considerably the time of selection and, of course, production of the corresponding vectors. Using this vector, we produced recombinant adenoviruses, each giving high-level expression of the transgene in the transduced cells.
源自人5型腺病毒（Ad5）的表达载体构建通常基于同源重组，由于在与病毒基因组重组之前必须选择穿梭质粒，因此耗时较长。在此，我们描述了一种方法，可将转基因直接克隆到Ad5基因组的E3区域，该区域已在三个独特的限制性酶切位点上游包含立即早期巨细胞病毒（CMV）启动子。这使得只需一步即可构建重组腺病毒基因组，大大减少了筛选时间，当然也减少了相应载体的生产时间。使用该载体，我们生产了重组腺病毒，每种重组腺病毒在转导细胞中均能使转基因高水平表达。
