【摘要】 建立了用毕赤酵母表达系统表达皮蝇素A(HypodermotinA,HA)的技术,并对表达条件进行了优化筛选。将皮蝇素A基因连接到质粒pPIC9k上,构建了重组转移质粒pPIC9k-HA。重组质粒通过电激转化入酵母细胞GS115后,用组氨酸缺陷培养基和G418分别进行筛选。重组酵母细胞在含0.5%甲醇的培养基中诱导产生目的蛋白,培养3d后收取上清,用阴离子交换层析柱分离纯化。经SDS-PAGE检测,所表达的蛋白相对分子质量约为30000。Western-blotting表明,该蛋白能与兔抗HA血清特异性结合。明胶电泳检测显示,该蛋白具有酶活性。单因素改变表达条件的结果表明,甲醇浓度在0.5%~1%、溶氧量在70%以上、诱导2~3d的情况下表达产量最高,达1500mg/L左右。该研究结果对防治牛皮蝇蛆病疫苗的深入研究及毕赤酵母在寄生虫领域的应用有一定价值。更多还原

【Abstract】 An approach of expression and optimize of Hypodermin A (HA) in Pichia Pastoris system was established. At first, HA gene and plasmid pPIC9k was combined together to construct the recombinant plasmid pPIC9k-HA. After electroporated to the GS115 cells, the recombinant plasmid was screened out by His-medium and G418. The protein was induced by the medium containing 0.5% methanol. The supernatant was collected 3 days after, and then purified by FPLC. SDS-PAGE indicated that the target protein is around 30 000 . Western-blotting shows it can react with rabbit-anti HA serum. Gelatin SDS-PAGE shows its enzyme activity. The optimization experiments indicated that higher yield can be gotten by 0.5%-1%methanol, 70% cubage, till 2 to 3 days. The yield is around 1 500 mg/L. The study above facilitates the further research on HA as a vaccine and the use of Pichia Pastoris in parasite domain.更多还原