鼠源REG3α二聚体蛋白的体外表达及抗肝癌细胞功能  

Expression of mouse REG3α dimer in vitro and the anti-hepatoma cells efficacy

作　　者：陶佳丽[1] 郭夏斐 范阔海[1] 张亮[1] 段马魁 王志瑞[1] 姜俊兵[1] 

机构地区：[1]山西农业大学动物科技学院,山西太谷030801

出　　处：《畜牧与兽医》2017年第5期112-117,共6页Animal Husbandry & Veterinary Medicine

基　　金：山西省科技攻关项目(20100312016)

摘　　要：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 codon- optimized murine REG3α dimer was expressed in Pichia pastoris and its anti- hepatoma cells efficacy was investigated in vitro. According to the codon usage preference of P. pastoris, the recombinant plasmid pwPICZalpha-REG3α was constructed. Then the recombinant plasmid was linearized and transformed into P. pastoris by electroporation. The expression of recombinant P. pastoris was induced by methanol. The murine REG3α dimer was captured using ProteinIsoTM Ni-NTA resin and further purified using a strong anion exchange resin Poros 50 HQ. The anti-hepatoma ceils efficacy of the recombinant REG3α dimer was assessed using MTI＂ test to determine 50% effective concentration of REG3α dimer on SMMC-7721 cells. The SDS-PAGE and Western blotting results showed a specific band with the molecular weight of 29. 6 ku. The recombinant murine REG3α dimer had a potent anti-hepatoma activity in a time-dependent and dose-dependent manner. It indicates that the murine recombinant REG3α dimmer of good bioactivity was successfully obtained. This study provides a theoretical foundation for the research on the function of REG3α dimer.

关 键 词：巴斯德毕赤酵母 REG3α二聚体 表达 纯化 抗肝癌细胞 

分 类 号：Q786[生物学—分子生物学]