Peng Ming-Li, Ling Ning, Xu Hong-Mei, Qing Yu-Ling, Ren Hong
Institute for ViraL Hepatitis, Chongqing University of Medical Sciences, Chongqing 400010, China.
Sheng Wu Gong Cheng Xue Bao. 2003 May;19(3):286-90.
To obtain the expression of Mycobacterium tuberculosis heat shock protein 70 in methylotropic yeast. The expression vector pPIC9K-hsp70 was constructed, linearized and introduced into Pichia pastoris GS115 by electroporation. The result protein was secreted into the supernatant induced by 0.5% methanol at 30 degrees C and purified by centrifugation, ultrafiltration and ATP-agarose. The recombinant Hsp70 was identified by SDS-PAGE, Western blot, mice experiment and effect on the immature DC. The SDS-PAGE and Western blot analysis showed that the apparent molecular weight of expressed Hsp70 was about 70 kD and the expressed protein could specifically react with anti-Mt. Hsp70 IgG. And mice immunization indicated the expressed hsp70 had immunogenicity. Hsp70 could induce DC maturation and release Th1 cytokine. The secreted 70 kD protein was about 120 mg/L which accounted for more than 30% of the total supernatant protein and was purified to electrophoretic purity. The Hsp70, which had the biological activity, is successfully secretorily expressed in the Pichia pastoris GS115.
为了在甲基营养型酵母中获得结核分枝杆菌热休克蛋白70的表达。构建表达载体pPIC9K-hsp70，线性化后通过电穿孔法导入毕赤酵母GS115。表达的蛋白在30℃下用0.5%甲醇诱导分泌到上清液中，通过离心、超滤和ATP-琼脂糖进行纯化。重组Hsp70通过SDS-PAGE、Western印迹、小鼠实验以及对未成熟树突状细胞的作用进行鉴定。SDS-PAGE和Western印迹分析表明，表达的Hsp70表观分子量约为70 kD，表达的蛋白能与抗结核分枝杆菌Hsp70 IgG特异性反应。小鼠免疫表明表达的hsp70具有免疫原性。Hsp70可诱导树突状细胞成熟并释放Th1细胞因子。分泌的70 kD蛋白约为120 mg/L，占总上清液蛋白的30%以上，并纯化至电泳纯。具有生物活性的Hsp70在毕赤酵母GS115中成功实现了分泌表达。
