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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pc DNA3.1-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p ET21a(+),构建成p ET21a-m IL-33质粒,转化至大肠埃希菌BL21,筛选出可表达m IL-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m IL-33蛋白,相对分子质量18 000。ELISA试验显示m IL-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m IL-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。
The aim is to express biologically active mouse IL-33 mature protein in Escherichia coli. Methods The mouse IL-33 mature protein gene was amplified from pcDNA3.1-IL-33 plasmid by PCR and inserted into the prokaryotic expression vector p ET21a (+) to construct p ET21a-m IL-33 plasmid. Transformed into Escherichia coli BL21 and screened an Escherichia coli engineering strain capable of expressing m IL-33. Induced by IPTG and purified by nickel column affinity chromatography. Results The purity of mIL-33 protein was up to 95% by SDS-PAGE. The relative molecular mass was 18,000. ELISA test showed that mIL-33 can effectively promote the production of Th2 type cytokines (IL-5) in mesenteric lymphocytes, which is significantly different from the untreated group. Conclusion The m IL-33 protein expressed in Escherichia coli has biological activity, which lays the foundation for the further study on the immunotherapy of IL-33 in inflammatory bowel disease.