应用抑制性差减杂交技术构建副猪嗜血杆菌基因组差减文库

Construction of a subtracted DNA library by suppression subtracted hybridization for differentially expressed genes in Haemophilus parasuis

为构建副猪嗜血杆菌(Haemophilus parasuis,HPS)有毒力菌株SW124(血清4型)和无毒力菌株H465(血清11型)间的差异表达基因文库,采用抑制性差减杂交技术(Suppression subtractive hybridization,SSH)分析SW124和H465菌株细菌基因组的表达差异,并进行两轮差减杂交和两次PCR扩增,将第2次PCR产物与pMD19-T载体相连,转化大肠杆菌E.coli DH5α感受态细胞并进行文库扩增和蓝白斑筛选,RT-PCR鉴定差异表达文库。结果共获得327个阳性克隆,筛选100个克隆制备质粒进行PCR检测,均扩增出100～2 000 bp大小的片段。差异DNA差减文库的构建为进一步筛选、克隆HPS特异的未知新基因、建立分子鉴别新体系奠定了基础。

In order to constructed the genomic subtracted library between of Haemophilus parasuis（HPS） virulent strain SW124 and non-virulent strain H465,suppression subtractive hybridization（SSH） was used to identify the genetic difference between SW124 and H465.After twice subtractive hybridization and two times of PCR,the products of last PCR amplification were inserted into pMD19-T simple vector and be transformed into the E.coli DH5α and screened of blue and white clones of the transformants.The subtracted genomic DNA library of differentially expressed genes were identified by RT-PCR.The results were as followed： 327 positive clones were obtained and analysis of 100 clones with PCR showed that all plasmids in the clones contain about 100～2 000 bp inserts.The DNA subtractive library of HPS SW124 and H465 were successfully constructed using SSH technique,which laied foundation for screening and cloning new specific differentially expressed genes in them,and further establishing new system for molecular differentiation.