牛型布鲁氏菌外膜蛋白bp26基因的克隆与原核表达

摘要

根据GenBank中登录的牛型布鲁氏菌外膜蛋白bp26基因序列,设计了1对引物,采用PCR技术,牛流产病例分泌物为模板,扩增bp26的编码基因,得到1条753 bp的片段.将其连入Simple-T载体中进行酶切和测序鉴定.测序正确后,将该基因插入到pET-28a(+)载体中构建原核表达载体,将此重组质粒转化到Rosetta(DE3)感受态细胞中,并用IPTG诱导,将诱导产物用SDS-PAGE和Western-blot检测.结果显示bp26基因可以在大肠杆菌中获得表达,表达产物分子质量约为29 ku,与预计的蛋白分子质量一致.Western-blot试验证明,该蛋白可以与患牛布氏杆菌的阳性血清产生特异性结合反应,具有良好的抗原性.%The bp26 protein gene of Bovine brucella, 753 bp in length, was amplified by poly-merase chain reaction (PCR) using a pair of specific primers designed according to the published DNA sequence from GenBank, and was cloned into the vetor Simple-T. Then the recom-binant fragment in Simple-T was subcloned into the eukaryotic expression vector pET-28a ( + ). After being induced by isopropyl-b-D-thiogalactopyranoside (IPTG), the recombinant bp26 protein was expressed. The result of SDS-PAGE and Western-blot showed that the recombinant bp26 protein was 29 ku and had strong positive reactions with Bovine brucella-spe-cific antibody.