猪胸膜肺炎放线杆菌血清7型抑制消减杂交文库的构建和分析

为了研究猪传染性胸膜肺炎放线杆菌(APP)不同血清型之间的差异表达基因,采用抑制消减杂交(SSH)法,以APP血清7型DNA作为测试方(Tester)、APP血清1型DNA作为驱动方(Driver),进行2轮杂交和2轮选择PCR扩增,将PCR扩增产物连接pMD18-T载体,筛选阳性克隆后进行测序和序列比对。结果显示,15个阳性克隆中的13个为已知序列,其同源性为88%~100%,2个为未知序列,其结构和功能还需进一步研究。首次构建了APP7的抑制消减杂交文库,为APP感染和致病机制的研究奠定了基础。

Construction and analysis of suppression subtractive hybridization library of Actinobacillus pleuropneumoniae serotype 7

Suppression subtractive hybridization(SSH) was used to screen and analyze differential expression genes in different serotypes of Actinobacillus pleuropneumoniae(APP) using APP7 genome as tester and APP1 genome as driver.PCR products were ligated into pMD18-T Vector after two-round hybridization and two-round selective PCR amplification.The positive clones were screened,sequenced and compared.In consequence,13 sequences out of 15 positive clones were known,and the identity among the sequences varied from 80% to 100%.The other 2 were proved to be new genes.The result demonstrated that the SSH library of APP7,which were firstly constructed,provided a theoretical base for studies on the infection and pathogenesis of APP.