布鲁菌VirB12基因的分泌表达及鉴定

Secretory expression and identification of Brucella VirB12 gene

为获得高纯度易纯化的布鲁菌VirB12蛋白,根据GenBank中的VirB12的基因序列设计引物并进行扩增,将其连接pBIC质粒后转化到短小芽孢杆菌中,得到了VirB12蛋白的分泌型表达菌株。诱导后对其进行了PAGE和WesternBlot-ting鉴定。结果表明,成功构建了VirB12的分泌表达菌株,诱导后在培养上清中大量表达,蛋白含量高达80%,其能与布鲁菌标准阳性血清、6×His单克隆抗体发生反应,与A19-ΔVirB12菌株免疫血清不发生反应,有望用来作为抗原建立区分野毒感染血清和A19-ΔVirB12免疫血清的检测方法,更好地进行布病的防控。

In order to obtain high purity and easy purification VirB12 protein of brucella, a pair of primers based on the GenBank VirB12 gene sequence weve designed and the VirB12 gene was amplified and cloned into pBIC plasmid to obtain VirB12 expression strains. The expressed product was identified by PAGE and Western Blotting. The results showed that Brucella VirB12 protein was successfully expressed up to 80% in the supernatant of culture medium. The results of mmunoblot analysis with brucella positive ser- um, 6 × His monoclonal antibody and A19-ΔVirB12-immunoreactive sera showed that VirB12 protein had good antigen specificity and could be used to differentiate wild-type sera from A19-ΔVirB12-immunoreactive sera.