具有天然N端的小鼠高活性IL-18的高效表达与纯化  

The high efficiency expression of the authentic mouse interleukin-18 with na-tural N-terminus and high biological activities

作　　者：杨永杰[1] 陈松华[1] 葛锡锐[1] 

机构地区：[1]中国科学院上海生命科学研究院生物化学与细胞生物学研究所,中国科学院研究生院,上海200031

出　　处：《现代免疫学》2005年第6期445-449,共5页Current Immunology

基　　金：中国科学院知识创新工程项目(KSCX2-3-04-03);科技部2001年度科技基础性工作专项基金资助项目(2001DEA10007)

摘　　要：IL-18是IL-1家族成员,与IL-1β相似,IL-18也是以前体形式合成,其成熟和分泌需要Caspase-1的作用。我们正确克隆了小鼠IL-18全长cDNA,并成功构建了真核表达载体pcDNA3-proIL-18,能在真核细胞COS7中得到正确表达;为了得到具有天然N端的成熟IL-18(mIL-18),IL-18cDNA上Caspase-1的识别位点被转换成FactorXa的作用位点,并克隆到原核表达载体pGEX-4T-1中,IPTG能大量诱导GST-IL-18融合蛋白的可溶性表达,经一步亲和纯化后,能得到30～50mg融合蛋白/升菌;经过FactorXa作用融合蛋白,能得到大量(5～8mg/升菌)、高纯度(>95%)的mIL-18。与商品化的IL-18相比,所得mIL-18能诱导小鼠脾细胞表达更高水平的IFN-γ,表现出更强的生物学活性,这为进一步在小鼠系统中研究IL-18的功能、人成熟IL-18蛋白的高效表达、纯化以及其后可能的临床应用奠定了基础。Interleukin-18（IL-18） is an inducer of IFN-γ and a co-inducer of the Thl cytokines, sharing structural features with the IL-1 family of proteins. Like IL-1β, IL-18 is synthesized as biologically inactive precursor （proIL-18） which can be cleaved by Caspase-1 （IL-1β-converting enzyme, ICE） to form the biologically active and mature cytokine. We had correctly cloned the full length of IL-18 cDNA, and two expression vectors were successfully constructed, one of which was the eukaryotic expression vector pcDNA3-IL-18, in which IL-18 could be correctly expressed as a 24 Mr×10^3 protein and a 18 Mr×10^3 band could be detected with the co-transfection of Caspase-1, and the other was prokaryotic expression vector pGEX-IL-18（Xa）, in which the Caspase-1 cleavage site was mutated into a Factor Xa site. With the induction of IPTG, the fusion protein GST-IL-18（Xa） was highly expressed in E.coli and could be purified with Glutathione Sepharose 4B to obtain 30-50 mg of the fusion protein in one liter of cultures. After the cleavage with Factor Xa, the mature IL-18 with authentic N-terminus was obtained （5-8 mg/L of culture） and it showed high purity over 95% and exhibited much higher biological activities in comparison with the commercial IL-18. These results provide a practical way to produce highly active and mature IL-18 with high efficiency.

关 键 词：IL-18 RT-PCR 克隆 表达 纯化 

分 类 号：R392.1[医药卫生—免疫学]