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

作　　者：张念章[1] 储岳峰[1] 赵萍[1] 高鹏程[1] 贺英[1] 逯忠新[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部草食动物疫病重点开放实验室农业部畜禽病毒学重点开放实验室,兰州730046

出　　处：《生物技术通报》2011年第7期186-190,共5页Biotechnology Bulletin

基　　金：国家重点基础研究发展计划("973")项目(2006CB504403);"十一五"科技支撑计划项目(2006BAD06A01;2006BAD06A12);家畜疫病病原生物学国家重点实验室基金项目(SKLVEB2008ZZKT009);广东省兽医公共卫生公共实验室开放基金项目(GSKJ090202)

摘　　要：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.

关 键 词：副猪嗜血杆菌 重叠PCR 外源打靶基因 

分 类 号：S852.61[农业科学—基础兽医学]