重组小鼠 IL－33成熟蛋白的原核表达、纯化及活性检测

摘要

Objective To express mouse IL-33 mature protein with biological activity.Methods The gene coding IL-33 mature protein was amplified from pcDNA3.1-IL-33 by PCR and then inserted into the vector pET21a(+) to construct the recombinant expression plasmid pET21a-mIL-33.The expression construct was then transformed into Escherichia coli BL21 ( DE3) to express mIL-33.The mIL-33 protein was induced to express by IPTG and then purified by nickel affinity chroma-tography.Results The purified mIL-33 had a molecular weight of 18 000 with a purity of 95%as shown by SDS-PAGE. As demonstrated by ELISA, mIL-33 could effectively promote the production of Th2 type cytokines ( IL-5) by the lympho-cytes from mesenteric lymph nodes(MLN).IL-5 production by the MLN lymphocytes was significantly different between those with and without mIL-33 treatment.Conclusion The bioactive mIL-33 protein was successfully obtained in this study.It would be potentially used for the immunotherapy of inflammatory bowel disease.%目的：为了在大肠埃希菌中表达具有生物学活性的小鼠IL－33成熟蛋白。方法利用PCR技术从pcDNA3．1－IL－33质粒中扩增小鼠IL－33成熟蛋白的基因，将其插入原核表达载体pET21a（＋），构建成pET21a－mIL－33质粒，转化至大肠埃希菌BL21，筛选出可表达mIL－33的大肠埃希菌工程菌株。经IPTG诱导表达，用镍柱亲和层析法纯化。结果经SDS－PAGE分析获得纯度高达95％的mIL－33蛋白，相对分子质量18000。 ELISA试验显示mIL－33可以有效的促进肠系膜淋巴细胞产生Th2型细胞因子（ IL－5），与未处理组的差异具有显著的统计学意义。结论利用大肠埃希菌表达系统表达了具有生物活性的mIL－33蛋白，为继续开展IL－33在炎症性肠病的免疫治疗研究奠定了基础。