猪传染性胸膜肺炎放线杆菌毒素Ⅲ基因克隆、序列分析及表达

Cloning, Sequencing and Expression of the ApxⅢA Gene fromActinobacillus pleuropneumoniae

参照已知猪传染性胸膜肺炎放线杆菌血清2型菌株毒素 (Apx A)序列设计1对特异性引物,经PCR扩增得到目的片段约1096bp,然后克隆入pMD18\|T载体中,经测序比较,与标准序列的核苷酸同源性达98.5%。从T-载体中切下目的片段后,定向克隆入pET32a(+)中,转化BL21(DE3),经诱导后,SDS-PAGE结果显示,毒素 得到高效表达,Western-blotting检测呈阳性。这为研制猪传染性胸膜肺炎新型疫苗和建立有效的诊断方法奠定了基础。

The ApxⅢA gene from serotype 2 strain of Actinobacillus pleuropneumoniae was amplified by PCR with a size of 1()096 bp. The amplified DNA fragment was cloned into pMD18-T, and sequenced. The result of sequencing showed that the consistency was 98.5% compared with that of standard strains. Inserted in pET-32a, and transformed in Escherichia coli BL21 (DE3), the fragment was expressed after induced, and it could be detected by Western blotting. These results become the base of researching and producing novel vaccine and developing effective diagnostic method.