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[目的]构建rSalmosin-Mel融合蛋白毕赤酵母真核表达载体,建立表达纯化工艺并初步评价其抗肿瘤活性。[方法]rSalmosin-Mel基因同pPIC9K相连,构建pPIC9K/rD-M真核表达载体,电转化至毕赤酵母菌GS115中。对重组菌的发酵时间、甲醇浓度及培养基pH进行优化。采用QSepharose HP和Sephadex G-75层析技术纯化重组蛋白rD-M。通过MTT比色法检测rD-M对MCF-7细胞增殖的抑制作用。[结果]获得了高效分泌表达rD-M融合蛋白的酵母工程菌株,确定了在28℃、pH 6.0、浓度为1.5%甲醇诱导96 h发酵条件。通过层析纯化出纯度大于95%的重组蛋白。MTT实验结果表明,0.25、0.5、1、2和4 nmol/L的rD-M对MCF-7细胞增殖具有明显的抑制作用。[结论]实现了rSalmosin-Mel融合蛋白在毕赤酵母中最优条件下的分泌型表达,为其作为基因工程抗肿瘤药物奠定基础。
[Objective] To construct eukaryotic expression vector of rSalmosin-Mel fusion protein Pichia pastoris and to establish an expression purification process and evaluate its anti-tumor activity. [Method] The rSalmosin-Mel gene was ligated with pPIC9K to construct the eukaryotic expression vector pPIC9K / rD-M, which was electroporated into Pichia pastoris GS115. The recombinant bacteria fermentation time, methanol concentration and medium pH optimization. Recombinant protein rD-M was purified using Q Sepharose HP and Sephadex G-75 chromatography. The inhibitory effect of rD-M on the proliferation of MCF-7 cells was detected by MTT assay. [Result] The yeasts engineered to efficiently express rD-M fusion protein were obtained. The fermentation conditions of 96 h induction at 1.5% methanol at 28 ℃, pH 6.0 were determined. Purified by chromatography more than 95% purity of the recombinant protein. MTT assay showed that rD-M at 0.25, 0.5, 1, 2 and 4 nmol / L significantly inhibited the proliferation of MCF-7 cells. [Conclusion] The secreted expression of rSalmosin-Mel fusion protein was achieved under the optimal conditions in Pichia pastoris, which laid the foundation for its application as a genetically engineered antitumor drug.