弓形虫GRA1基因在毕赤酵母菌中的表达、纯化与鉴定

Expression,purification and identification of GRA1 gene from Toxoplasma gondii in Pichia pastoris

目的研究弓形虫致密颗粒抗原-1(GRA1)在毕赤酵母菌中的表达,获得高效表达的具有生物学活性的目的蛋白。方法将已构建重组的pPIC9K-GRA1质粒转化入酵母菌GS115,筛选His+Muts表型转化子,摇瓶培养,1%甲醇诱导表达。表达产物经高效液相色谱法纯化后,测定其生物学活性。结果诱导4d的蛋白表达量达到2.5g/L。SDS-PAGE显示表达蛋白的分子质量单位为24ku,Westernblot鉴定表达产物具有抗原性。表达蛋白经高效液相色谱纯化产物后纯度达到90%以上。结论弓形虫GRA1基因在毕赤酵母中成功分泌表达目的蛋白,表达产物具有抗原性。

Objective To study the expression of the GRA1 gene of Toxoplasma gondii in Pichia pastoris and to obtain high level expressed protein of GRA1 with good biological activity. Methods Recombinant expression plasmids pPIC9K-GRA1 was constructed and transformed into GS115.The His＋Muts phenotype transformants were screened, fermented and induced by 1% methanol. The expression products were purified by high-efficiency liquid chromatography, and the biological activity was determined. Results After 4 days of methanol induction,the expressed GRA1 come up to 2.5 g/L. The GRA1 was successfully expressed a specific 24 ku protein in P. pastoris showed by SDS-PAGE. Western blot further proved it having good antigenicity. The purity of the product by High Performance Liquid Chromatography System （HPLCS） was 90%. Conclusion The GRA1 protein with good antigenicity is successfully expressed in P. pastoris.