Liu Qinfang, Wang Shuai, Ma Guangpeng, Pu Juan, Forbes Nicole E, Brown Earl G, Liu Jin-Hua
J Mol Genet Med. 2009 Dec 1;3(2):225-31. doi: 10.4172/1747-0862.1000039.
Typical reverse genetics systems for generating influenza viruses require the insertion of each genome segments by DNA ligation into vectors for genome synthesis and expression. Herein is described the construction and use of a novel pair of plasmid vectors for cloning all eight genome segments of influenza A virus by homologous recombination for influenza virus reverse genetics. Plasmids, pLLBA and pLLBG, were constructed to possess opposing RNA polymerase I and RNA polymerase II transcription units for generating influenza genomic and messenger RNAs, respectively. In addition these promoters flanked a recombination cassette which comprised the conserved 5' (13bp) and 3' (12bp) terminal promoters of influenza virus. These vectors differed due to the presence of an A or a G (plus sense) to correspond to differences at nucleotide position 4 among negative-sense influenza virus promoters. The cloning approach involved homologous recombination of each influenza gene segment and the appropriate linearized pLLBA or pLLBG vectors in E. coli. Direct cloning by recombination was simpler and faster than conventional restriction digestion and ligation methods. This new vector system was successfully used to clone and rescue various influenza viruses and thus has the potential to promote the rapid analysis and vaccine development of novel influenza strains.
用于产生流感病毒的典型反向遗传学系统需要通过DNA连接将每个基因组片段插入用于基因组合成和表达的载体中。本文描述了一种新型质粒载体对的构建和使用，该载体对通过同源重组克隆甲型流感病毒的所有八个基因组片段，用于流感病毒反向遗传学研究。构建了质粒pLLBA和pLLBG，使其分别具有相反的RNA聚合酶I和RNA聚合酶II转录单元，用于产生流感病毒基因组RNA和信使RNA。此外，这些启动子位于一个重组盒两侧，该重组盒包含流感病毒保守的5'（13bp）和3'（12bp）末端启动子。这些载体因存在A或G（正链）而有所不同，以对应负链流感病毒启动子中核苷酸位置4处的差异。克隆方法涉及每个流感基因片段与适当的线性化pLLBA或pLLBG载体在大肠杆菌中的同源重组。通过重组直接克隆比传统的限制性消化和连接方法更简单、更快。这种新的载体系统已成功用于克隆和拯救各种流感病毒，因此有潜力促进新型流感毒株的快速分析和疫苗开发。
