【摘要】 为获得结核杆菌热休克蛋白 70在毕赤酵母中的分泌表达。构建了酵母表达质粒pPIC9K hsp70 ,并将其线性化后用电穿孔法导入PichiapastorisGS115 ,经PCR方法筛选出阳性菌落 ,在 0 .5 %甲醇诱导下分泌表达。所得产物经离心收集上清、超滤浓缩脱盐、亲和层析后 ,分别用SDS PAGE、Westernblot和动物免疫实验对上清中的重组Hsp70进行鉴定 ,并考察产物对DC的作用。经SDS PAGE、Westernblot分析表明表达的Hsp70表观分子量为 70kD并能特异性地与抗 Mt.Hsp70单抗结合 ,动物实验表明重组的Hsp70能在体诱导免疫应答。重组Hsp70能够诱导DC成熟并释放Th1型细胞因子。摇瓶发酵表达量达 12 0mg L ,占培养上清 30 %以上。这为研究结核杆菌热休克蛋白70的生理功能提供了必要的物质条件更多还原

【Abstract】 To obtain the expression of Mycobacterium tuberculosis heat shock protein 70 in methylotropic yeast. The expression vector pPIC9K hsp 70 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℃ 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 70kD 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 70kD protein was about 120mg/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.更多还原