重组 IL-33 融合表达载体的构建尧蛋白表达及纯化.pdfVIP

泸州医学院学报 2011年 第34卷 第5期 Journal of Luzhou MedicalCollege Vol.34 No.5 2011 471471 论著 重组IL-33融合表达载体的构建尧蛋白表达及纯化* 年四季,张金平,高 燕 ,杨 燕,向 丽,袁 青1 1 渊泸州医学院院病原生物学教研室曰 免疫学教研室袁四川泸州 646000冤1 摘 要 目的院构建人白介素-33渊IL-33冤硫氧还蛋白融合表达载体袁高效表达IL-33蛋白遥方法院采用RT-PCR从健康志愿 者外周血单核细胞渊PBMC冤mRNA中扩增IL-33 cDNA曰将扩增的IL-33 目的基因与融合表达质粒pET102/D-TOPO 连接袁 转化大肠杆菌BL21袁IPTG诱导表达并对表达产物进行纯化鉴定遥结果院扩增的IL-33cDNA大小为800bp左右遥表达的IL-33 融合蛋白大小为45kDa左右,Westernblotting鉴定显示表达的蛋白正确袁为IL-33 目的蛋白遥 但表达的大部分蛋白为无生物学 活性的包涵体袁在后续对表达蛋白进行纯化的同时对蛋白进行了复性袁得到了复性后具有生物学活性的IL-33蛋白遥结论院成功 制备了具有生物学活性的IL-33蛋白遥 关键词 IL-33曰融合表达曰纯化 中图分类号 R392.11 文献标识码 A 文章编号1000-2669渊2011冤5-0471-04 CONSTRUCTIONOFFUSIONEXPRESSION VECTORAND EXPRESSION, PURIFICATION OFIL-33 Nian Siji,etal Department ofPathogenBiology,LuzhouMedical College Abstract Objective:To construct interleukin-33 (IL-33)recombinant vector for expression of human IL-33 protein effectively through thioredoxin fusion expression system. Methods:cDNA of IL-33 was amplified with the mRNA ofperipheral blood mononuclear cells (PBMC)from healthy volunteer by RT-PCR.The amplified cDNA of IL-33 was ligated with the fusion expression vector pET102/D-TOPO, and then transformed into Escherichiacoli BL21 for expression. Results:The size of amplified cDNA of IL-33 was about 800bp. After Induced expression with IPTG, the size of expressed fusion protein was about 45kDa and the western blot results showed that it was IL-33 aim protein. As most of expressed protein was inclusion body which has no bioactivity, the protein was purified andrenaturated. Conclusion:the IL-33proteinwith bioactivitywas made successful