rSalmosin-Mel融合蛋白在毕赤酵母中表达及活性研究

发布时间：2018-06-08 03:30

  本文选题：基质金属蛋白酶 + 整合素　； 参考：《生物技术》2017年03期

【摘要】：[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。
[Abstract]:[objective] to construct the eukaryotic expression vector of rSalmosin-Mel fusion protein Pichia pastoris, to establish the expression and purification process and to evaluate the antitumor activity of rSalmosin-Mel fusion protein. [methods] the rSalmosin-Mel gene was linked to pPIC9K, and the eukaryotic expression vector pPIC9K / rD-M was constructed and electrotransformed into Pichia pastoris GS115. The fermentation time, methanol concentration and pH of culture medium were optimized. The recombinant protein was purified by QSepharose HP and Sephadex G-75 chromatography. The inhibitory effect of rD-M on the proliferation of MCF-7 cells was detected by MTT colorimetry. [results] yeast engineering strain secreting and expressing rD-M fusion protein was obtained, and the fermentation conditions were determined at 28 鈩，

本文编号：1994237