副猪嗜血杆菌种特异性基因的筛选与鉴定

摘要

为筛选副猪嗜血杆菌（HPS）种特异性的基因，采用抑制消减杂交（SSH）技术，以HPS DNA为Tester，以猪胸膜肺炎放线杆菌（APP）DNA为Driver，通过2轮消减杂交和2次PCR扩增，将第2次PCR产物连接pMD19-T载体，转化大肠杆菌DH5α感受态细胞，筛选阳性克隆后测序，经Southern Blot验证和BLAST分析，获得6个HPS特异基因片段。研究为建立副猪嗜血杆菌基因鉴别诊断方法及ELISA抗原靶标的鉴定奠定基础。%In order to screen species-specific genes of Haemophilus parasuis (HPS), suppression subtractive hybridization (SSH) was employed by using HPS DNA as tester and APP DNA as driver. After twice subtractive hybridization and two times of PCR amplification, the products were cloned into pMD19-T simple vector and transformed int E.coli DH5α. Then, the positive clones were screened and sequenced. Six differential fragments of HPS were obtained by Southern Blot verification and BLAST analysis. This study laid a good foundation for establishing a diagnostic method for discriminating the infection of Haemophilus parasuis and identification ELISA antigen targets.