重叠PCR法构建副猪嗜血杆菌外源打靶基因

Construction of Foreign Targeting Gene of Haemophilus parasuis by Overlapping PCR

利用重叠PCR法构建了包含副猪嗜血杆菌血清5型hhdA基因上、下游同源臂(H-up、H-down)和卡那霉素抗性基因(Kan)的外源打靶基因Hup-kan-Hdown。通过对反应条件进行优化,发现退火温度为50.5℃,加入H-up、H-down和Kan各为5μL(450 ng)时得到Hup-kan-Hdown的量最多。将此外源打靶基因的两端引入PstI和SacI酶切位点,克隆至具有迁移作用的自杀质粒pDS132,构建了可在大肠杆菌E.coliSM10-λpir中稳定遗传的打靶载体,为下一步建立副猪嗜血杆菌的高效基因打靶系统奠定基础。

The foreign targeting gene,Hup-kan-Hdown,which includes upstream and downstream homologous genes of hhdA（H-up,H-down）of Haemophilus parasuis serotype 5 and kanamycin resistant gene（Kan）,was constructed by overlapping PCR for the first time.The optimized condition for the maximum production of overlapping PCR was that annealing temperature of the first reaction was 50.5℃,the amounts of H-up,Kan and H-down products was equal in 5 μL（450 ng）.The fusion fragment was then cloned to a suicide plasmid pDS132 and a recombinant plasmid for gene targeting was constructed.The recombinant plasmid could stably duplicate in E.coli SM10-λpir.The results provide the basis for establishment of an efficient gene targeting operating system for Haemophilus parasuis.