羊白细胞介素-18成熟蛋白的酵母表达和生物学活性研究  

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

作　　者：刘文强[1] 刘桂芹[1] 路建彪[1] 赵宏坤[2] 许传田[3] 

机构地区：[1]聊城大学农学院,山东聊城252059 [2]山东农业大学动物科技学院,山东泰安271018 [3]山东省农科院畜牧所,山东济南250100

出　　处：《中国预防兽医学报》2009年第12期969-973,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(30800130);山东省中青年科学家奖励基金(2006BS02012)

摘　　要：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GS115,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100mg/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1.5×105u/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。For expression of goat interleukin-18 （gIL-18） in pichia pastons system, the IL-18 gene was amplified by PCR and cloned into pPICZ α A vectors, then transformed into pichia pastotis 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.

关 键 词：山羊 白细胞介素-18 酵母表达系统 活性 

分 类 号：S852.4[农业科学—基础兽医学]