重组IL-33融合表达载体的构建、蛋白表达及纯化  

CONSTRUCTION OF FUSION EXPRESSION VECTOR AND EXPRESSION,PURIFICATION OF IL-33

作　　者：年四季[1] 张金平[1] 高燕[2] 杨燕[1] 向丽[1] 袁青[2] 

机构地区：[1]泸州医学院病原生物学教研室,四川泸州646000 [2]泸州医学院免疫学教研室,四川泸州646000

出　　处：《泸州医学院学报》2011年第5期471-474,共4页Journal of Luzhou Medical College

基　　金：国家自然科学基金项目(31000415);四川省科技厅项目(2009JY0124);四川省卫生厅项目资助(80164;100229)

摘　　要：目的:构建人白介素-33(IL-33)硫氧还蛋白融合表达载体,高效表达IL-33蛋白。方法:采用RT-PCR从健康志愿者外周血单核细胞(PBMC)mRNA中扩增IL-33 cDNA;将扩增的IL-33目的基因与融合表达质粒pET102/D-TOPO连接,转化大肠杆菌BL21,IPTG诱导表达并对表达产物进行纯化鉴定。结果:扩增的IL-33 cDNA大小为800bp左右。表达的IL-33融合蛋白大小为45kDa左右,Western blotting鉴定显示表达的蛋白正确,为IL-33目的蛋白。但表达的大部分蛋白为无生物学活性的包涵体,在后续对表达蛋白进行纯化的同时对蛋白进行了复性,得到了复性后具有生物学活性的IL-33蛋白。结论:成功制备了具有生物学活性的IL-33蛋白。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 of peripheral 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 Escherichia coli 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 and renaturated.Conclusion： the IL-33 protein with bioactivity was made successfully.

关 键 词：IL-33 融合表达 纯化 

分 类 号：R392.11[医药卫生—免疫学]