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利用巴斯德毕赤酵母(Pichia pastoris)表达系统对鼠源胰岛再生源蛋白(regenerating islet-derived protein,REG)REG3α二聚体进行表达,并对其体外抗肝癌细胞作用进行初步研究。通过DNA重组技术构建密码子优化的重组质粒pwPICZalpha-REG3α二聚体,用电转化的方法将线性化的重组质粒pwPICZalpha-REG3α二聚体导入毕赤酵母,经甲醇诱导表达重组蛋白,并用Ni-NTA Resin柱和阴离子交换柱对其进行纯化,用纯化后的蛋白进行体外抗肝癌细胞试验,通过MTT法测REG3α二聚体蛋白对肝癌细胞的半数有效浓度。SDS-PAGE和Western blot鉴定结果显示在29.6 ku处有特异性条带,并且以剂量和时间依赖的方式抑制人肝癌细胞SMMC-7721的活性。研究表明获得了具有生物活性的鼠源重组REG3α二聚体蛋白,为进一步研究REG3α二聚体蛋白功能提供理论依据。
The REG3α dimer of rat regenerating islet-derived protein (REG) was expressed by the Pichia pastoris expression system and its anti-hepatocarcinoma cell activity in vitro was studied. The codon-optimized recombinant plasmid pwPICZalpha-REG3α dimer was constructed by DNA recombination technology. The linearized recombinant plasmid pwPICZalpha-REG3α dimer was introduced into Pichia pastoris by electrotransformation and the recombinant protein was induced by methanol. NTA Resin column and anion exchange column to purify the purified protein with anti-liver cancer cells in vitro test by MTT assay REG3α dimer protein half effective concentration of liver cancer cells. The results of SDS-PAGE and Western blot showed that there was a specific band at 29.6 ku, and the activity of SMMC-7721 was inhibited in a dose-and time-dependent manner. Studies have shown that the biologically active mouse recombinant REG3α dimer protein obtained for the further study of REG3α dimer protein function provides a theoretical basis.