羊白细胞介素-18成熟蛋白的酵母表达和生物学活性研究

为了研究重组羊白细胞介素(gIL)-18在酵母系统中的高效表达,进一步阐明该重组蛋白的生物学活性,以含有gIL-18基因的重组质粒为模板进行PCR扩增,构建重组表达质粒pPICZ-gIL-18,转化于毕赤酵母GSll5,以甲醇诱导表达,经sDS-PAGE和western blot分析证实了重组蛋白的表达,分泌的重组gIL-18表达量为100 mg/L.经纯化后用MTT法和MDBK-VSV法检测表达的重组IL-18体外生物活性,利用免疫试验检测了重组蛋白对羊痘疫苗的免疫增强作用.实验结果表明,该蛋白具有诱导MDBK细胞分泌IFN-γ和刺激PBMC增殖的生物学活性,比活性为1.5×105 u/mg.体外具有增强羊痘疫苗的活性.毕赤酵母分泌表达的重组gIL-18具有良好的生物学活性,为gIL-18作为免疫佐剂和免疫治疗剂的大规模应用奠定了基础.

Expression of goat IL-18 mature protein in Pichia mammalian expression system

For expression of goat interleukin-18 (gIL-18) in pichia pastoris system, the IL-18 gene was amplified by PCR and cloned into pPICZa A vectors, then transformed into pichia. pastoris system GS115. High level expression of recombinant protein up to 100 mg/L was achieved following inducing with methanol. Bioactivity of recombinant protein was analyzed by MTT and MDBK-VSV methods. The enhanced immunity was measured by goat pox vaccine test. The recombinant protein could stimulate PBMCs proliferation and induce IFN- γ production in MDBK cells. It also showed immune enhancement to the antigen of goat pox vaccine. The recombinant gIL-18 expressing in yeast system retained nature bioactivity and could be a valuable resource for the large-scale clinical use as an immune adjuvant or therapeutic cytokines.