重组炭疽保护性抗原的表达、纯化与生物活性分析

Expression, Purification and Characterization of the Recombinant Anthrax Protective Antigen

构建分泌型表达质粒 ,在大肠杆菌中实现了重组炭疽保护性抗原 (rPA)的分泌型表达。重组蛋白位于细菌外周质 ,表达量约占菌体总蛋白的 10 %。以离子交换、疏水层析和凝胶过滤为基础 ,建立了rPA的纯化工艺 ,每升培养物可获得约 15mgrPA ,纯度可达 95 %以上。体外细胞毒性试验显示rPA具有较好的生物学活性。用rPA免疫家兔产生的抗血清在体外可抑制炭疽致死毒素的活性 ,表明rPA可诱导机体产生保护性免疫。

An expression plasmid carrying anthrax protective antigen (PA) gene was constructed, which has an OmpA signal sequence attached to the 5′ end of PA gene. The plasmid was transformed into E.coli and induced to express recombinant PA (rPA). The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion exchange, hydrophobic interaction chromatography, and gel filtration, about 15 mg of 95% pure rPA was obtained from 1 liter culture. The bioactivity of rPA was proved by in vitro cytotoxicity assay. The polyclonal antiserum from rabbits immunized with rPA could inhibit the action of anthrax lethal toxin in vitro , which suggests that antibodies against rPA can provide high passive protection against anthrax. The results reported here may be helpful to develop a safe and efficacious recombinant PA vaccine against anthrax.