猪胸膜肺炎放线杆菌血清1型信号标签诱导突变体库的构建  

CONSTRUCTION AND EVALUATION OF SIGNATURE-TAGGED MUTAGENESIS LIBRARIES OF ACTINOBACILLUS PLEUROP NEUMONIAE SEROTYPE

作　　者：赫明雷[1,2] 刘慧芳[1] 刘思国[1] 司薇[1] 王春来[1] 杨金国[1] 杜艳芬[1] 王聃[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,哈尔滨150001 [2]东北农业大学动物医学院,哈尔滨150030

出　　处：《中国动物传染病学报》2009年第1期38-44,共7页Chinese Journal of Animal Infectious Diseases

基　　金：中国农业科学院哈尔滨兽医研究所中央级公益性科研院所基本科研业务专项资金项目(2008-14)资助

摘　　要：本研究以自杀性质粒pUT携带含有信号标签的Mini-Tn5转座子对猪胸膜肺炎放线杆菌血清1型菌进行转座诱变,构建了带有12对特异性信号标签的Mini-Tn5转座子的pUT自杀质粒,转化到供体菌E.coliβ2155后,利用双亲本滤膜杂交法与受体猪胸膜肺炎放线杆菌血清1型菌(APP1)进行接合转移,构建并优化了接合转移体系。利用抗性和营养缺陷培养平板筛选得到接合突变体,通过抗性通用引物与12个特异标签引物和胸膜肺炎放线杆菌毒素IV(ApxIV)鉴定引物分别对这些突变体进行了PCR鉴定和测序验证。结果表明,经过加入标签的Mini-Tn5转座子可以通过接合转移的方式从供体菌E.coliβ2155中插入到APP1基因组当中,并成功构建了12个含有特异信号标签的重组质粒,获得了APP1的12个转座突变体库,经筛选鉴定后得到561个突变株。这为研究APP1的功能基因和筛选特定突变株提供了必要的基础。In this study , a signature- tagged mutagenesis（STM） system was used to identify genes in pathogens that were required for growth and replication of Actinobacillus pleuropneumoniae serotype 1 （APP1） in pigs, and signature- tagged mutagenesis libraries of APP1 were obtained. Twelve suicide plasmids pUT- MiniTn5 - Kin2 which include specific signature tags were constructed, and transformed into E. coli β 2155 respectively. Filter hybridization was used to construct and optimize the conjugal transfer system. Conjugation mutants were first screened using culture plates which have resistance and heterotrophia and further identified by PCR using universal primer of resistance and 12 specific tags primer and ApxIV toxins identification primer respectively, and then sequenced. The results showed E. coli β2155 and APP1 can be transposed and conjugated, and 12 libraries of transposon mutagenesis were got which had 561 mutants in all. These results provided essential basis to research the pathogenic mechanism, discover virulence and drug resistance gene, and develop attenuated live vaccine of APP1.

关 键 词：胸膜肺炎放线杆菌 转座子 信号标签 

分 类 号：S852.619[农业科学—基础兽医学]