rSalmosin-Mel融合蛋白在毕赤酵母中表达及活性研究

Expression of fusion protein r Salmosin-Mel in Pichia pastoris and characterization of its biologic activities

[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 the eukaryotic expression vector of rSalmosin - Mel fusion protein, establish the expres- sion and purification process and evaluate its anti - tumour activity. [ Methods ] The rSalmosin - Mel gene was linked to pPIC9K to construct pPIC9K/rD - M expression vector. Then transformed it into Pichia pastoris GS115. The fermentation time, methanol concentration and medium pH were optimized, rD - M was purified by QSepharoseHP and Sephadex G - 75 chroma- tography. The inhibitory effect of rD - M on the proliferation of breast cancer ceils were detected by MTF assays. [ Results ] Yeast engineering strain with high secretion expression level of rD - M fusion protein was obtained. The optimal expression con- dition of rD- M was： the temperature for induction was 30℃ ,the pH value was 6.0,methanol concentration was 1.5% ,the time for induction was 96 hours. The purity of the recombinant protein was higher than 95% by the method of chromatography. MTF results showed that 0. 25 ,0. 5,1,2 and 4 nmol/L of rD - M could inhibit the proliferation of MCF -7 cells in dose - de- pendent. [ Conclusion] rSalmosin - Mel was expressed to be secreted in Pichia pastoris successful and lay the foundation for the genetic engineering, production of anti - tumour drug and its medical research.