猪传染性胸膜肺炎放线杆菌转铁结合蛋白B基因的克隆与表达

Cloning and expression of the tbpB gene of Actinobacillus pleuropneumoniae

参照猪传染性胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)血清1型菌株,设计1对特异性引物,PCR扩增转铁结合蛋白B(tbpB)全基因,克隆到pMD19-T Simple载体中,经测序比较,与参考序列的核苷酸同源性达99.72%。试验将tbpB基因定向克隆到pET-32a(+)中,转化BL21(DE3),经诱导后,SDS-PAGE结果显示转铁结合蛋白B得到表达,Western blot检测呈阳性。

The tbpB gene from serotype 1 strain of Actinobacillus pleuropneumoniae was amplified by PCR. The amplified DNA fragment was cloned into pMD19-T simple, and sequenced. The result showed that the gene was 1 812 bp in length and shared 99.72 % sequence identity with that of standard strains. The gene was subcloned into in pET-32a （＋） and successfully expressed in Escherichia coli BL21 （DE3） after induced, which was confirmed by Western blot.