小鼠IL-4重组质粒的构建及目的蛋白的表达和纯化  

The Construction of Recombinant Mouse Interleukin 4 Prokaryotic Expressing Plasmid and the Expression and Purification of Target Protein

作　　者：罗晓蕾[1] 任潇潇[1] 黄浓郁[1] 邱吉[1] 李倩[1] 周西坤[1] 李炯[1] 

机构地区：[1]四川大学华西医院生物治疗国家重点实验室,成都610041

出　　处：《四川大学学报（医学版）》2009年第4期579-583,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家重点基础研究发展规划项目(2004cb518800)资助

摘　　要：目的构建重组小鼠白细胞介素-4(rmIL-4)原核表达载体,在大肠杆菌BL21(DE3)中诱导表达,纯化目的蛋白并鉴定。方法将优化后的小鼠IL-4cDNA片段克隆到原核表达载体pET-32a(+)上,获得重组质粒pET32/rmIL-4,转入BL21(DE3)中经IPTG诱导表达得到包涵体。经复性处理后,依次用阴离子交换柱和分子筛纯化蛋白。对纯化得到的蛋白用Western blot检验免疫学特异性,用mIL-4生长依赖性细胞株CTLL-2增殖实验和动物实验测定其生物学活性。结果经酶切和测序证实重组质粒pET32/rmIL-4构建成功。诱导得到的包涵体用3mol/L盐酸胍溶液洗涤后溶于加有β-巯基乙醇的7mol/L盐酸胍溶液中变性,在pH9.5的条件下梯度透析复性。纯化出的蛋白经Western blot鉴定,能与抗小鼠IL-4抗体特异性结合;经细胞和动物实验鉴定,rmIL-4蛋白具有生物学活性,能刺激mIL-4生长依赖性细胞株CTLL-2增殖,使小鼠体内细胞因子发生从TH1到TH2的漂移。结论成功制备了高纯度、具有生物学活性的小鼠IL-4,为进一步研究其生物学功能提供了条件。Objective To construct Recombinant Mouse Interleukin 4 prokaryotic expressing plasmid, express it in E. coli strain BL21 （DE3）, purify and identify the expressed cytokine. Methods The optimized raiL- 4 cDNA fragment was cloned into the prokaryotie expressing vector pET-32a （＋） to generate pET32/rmlL-4 and transformed into BL21 （DE3） cells. After induction, the expressed protein was found to be in the inclusion of E. coli cells. The induced product was purified through Q Sepharose Fast Flow Column and Gel Filtration Column under renaturing condition. The purified protein was identified by Western blot analysis, and the biologic activity was identified by the generation of raiL-4 dependence cell CTLL-2 and in vivo experiment of mouse psoriasis model. Results The recombinant plasmid pET32/rmlL-4 has been constructed correctly. The inclusion body was washed with 3 mol/L guanidine hydrochloride and denaturized in 7 mol/L guanidine hydrochloride. Then, the denaturized protein was gradient dialysis in the condition of pH 9. 5. The protein we purified has the right immunology specificity and biologic activity. Conclusion The recombinant mouse interleukin-4 with high purity and biologic activity was prepared in this study,which will become the basis for the further study of the biologic activity of IL-4.

关 键 词：重组小鼠白细胞介素-4蛋白纯化生物学活性 

分 类 号：Q786[生物学—分子生物学]