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

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.