山羊IL-18成熟蛋白在昆虫细胞／杆状病毒中的表达及其活性分析  

Expression of goat IL-18 mature protein in insect/baculovirus and determination of bioactivity of the recombinant protein

作　　者：王婷婷[1,2] 王希辉[2] 范忠玲[2] 陈金龙[2] 曹丙蕾[2] 孔娜[2] 胡敬东[2] 赵宏坤[1,2] 

机构地区：[1]青岛农业大学食品科学与工程学院山东省肉类食品质量控制工程技术研究中心,山东青岛266109 [2]山东农业大学动物科技学院,山东泰安271018

出　　处：《细胞与分子免疫学杂志》2011年第2期173-176,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：山东省科技攻关重大专项(SDSP2005041005)

摘　　要：目的:在昆虫细胞/杆状病毒中表达山羊白介素18(gIL-18)成熟蛋白,并对其生物学活性进行分析。方法:将编码山羊白介素18(gIL-18)成熟蛋白的基因克隆到杆状病毒转移载体pFastBac Dual上,构建重组质粒pFastBac Dual-gIL18,将该重组质粒转化至DH10Bac感受态细胞,经三重抗性与蓝白斑筛选,获得杆状病毒重组质粒Bacmid-gIL18,在Cellfectin作用下,将Bacmid-gIL18转染昆虫细胞sf9,收获感染后不同时间段的细胞,进行SDS-PAGE、Western blot检测和生物学活性分析。结果:重组蛋白获得正确表达具有良好的免疫原性;生物学活性分析表明,重组蛋白能够促进淋巴细胞增殖、诱导淋巴细胞分泌IFN-γ。结论:用昆虫细胞/杆状病毒系统成功表达了gIL-18成熟蛋白,且具有良好的免疫原性和生物学活性,从而为进一步研究gIL-18作为免疫调节剂和免疫佐剂在生产上的应用奠定了基础。AIM： To express goat IL-18 in insect/baculovirus and detect the bioactivity of the recombinant protein. METHODS： The mature goat interleukin-18 （glL-18） gene was cloned into the baculovirus transfer vector pFastBac Dual, and then the resulting eukaryotic expression plasmid pFastBac Dual-giLl8 was transformed into DHI0Bac, followed by the identification of Bacmid-glL18 recombinat plosmid by three antibiotics and blue-white patch. Finally, the recombinant bacmid was transfected into sf9 insect cells by Cellfectin and the transfected cells were harvested at different times. Then the expressed protein was identified by SDS-PAGE, Western blot and bioactivity assay. RESULTS： The recombinant protein recognized and bound to its specific antibody. Bioactivity assay showed that the recombinant protein stimulated the proliferation of lymphocytes and induced IFN-γ production in spleen lymphocytes. CONCLUSION： The mature glL-18 protein has been expressed successfully in insect/baculovirus expression system, and have good immunogenicity and bioactivity. The study paves a way for application of glL-18 as an immunomodulator or immune adjuvant.

关 键 词：山羊IL-18 杆状病毒 昆虫细胞 生物学活性 

分 类 号：S852[农业科学—基础兽医学] R392.11[农业科学—兽医学]