人白介素-38原核表达载体的构建及其蛋白表达、纯化  

Construction of human interleukin-38 prokaryotic expression vector and its protein expression,purification

作　　者：周密[1] 袁仙丽[1] 潘秀和 李燕[1] 李明才[1] 

机构地区：[1]宁波大学医学院免疫学研究室,浙江宁波315211

出　　处：《生物技术》2016年第2期140-145,共6页Biotechnology

基　　金：浙江省教育厅科研项目("重组IL-38对哮喘气道炎症的作用及应用研究";No.Y201327689);浙江省公益技术应用研究(实验动物)计划项目("IL-38转基因小鼠的建立及在高脂饮食诱导肥胖小鼠模型中的作用";No.2016C37139)

摘　　要：$目的%构建人白介素(interleukin,IL)-38原核表达载体,原核表达IL-38重组蛋白、纯化及活性分析。[方法]PCR扩增IL-38成熟蛋白编码区,构建IL-38原核表达载体p ET-44-h IL-38,转化BL21(DE3)菌株,用IPTG诱导,表达IL-38重组蛋白,并利用其C末端的组氨酸标签进行镍离子亲和层析纯化,将纯化的重组IL-38作用于脂多糖(LPS)诱导的THP-1细胞,研究纯化IL-38蛋白的生物学活性。[结果]构建的IL-38原核表达载体测序结果与Gen Bank中基因序列一致;IPTG诱导产生的IL-38蛋白主要以可溶性形式存在,纯化的目的蛋白经SDS-PAGE凝胶电泳分析和Western Blot鉴定发现,蛋白纯度可达98%,细胞实验证明纯化的目的蛋白具有较高的生物学活性。[结论]成功构建了IL-38的原核表达载体,制备了具有生物活性的IL-38重组蛋白。[Objective]To construct interleukin（ IL）- 38 prokaryotic expression vector,purify the recombinant protein,and test its biological activity. [Methods]Eukaryotic expression vector pc DNA3. 1- h IL- 38- V5- His was taken as template for PCR amplification to construct its prokaryotic expression vector p ET- 44- h IL- 38. The prokaryotic expression plasmids were transformed into host E. coli BL21（ DE3）. Then,IPTG was used to induce expression of target IL- 38 protein with 6 × His-tag in its C- terminal. The recombinant protein was purified through Ni2 ＋- c He Lating affinity chromatography by 6 × His-tag. The activity of purified protein was analyzed by stimulating LPS- induced THP- 1 cells. [Results]The IL- 38 protein induced by IPTG was mainly in the form of soluble pattern,and the purity was above 98% explained by SDS- PAGE gel electrophoresis and Western Blot analysis. The purified protein has a high biological activity through the cell stimulation experiments.[Conclusion]IL- 38 was cloned into the prokaryotic expression vector,bioactive IL- 38 protein was prepared.

关 键 词：IL-38 原核表达载体 重组蛋白 纯化 

分 类 号：R392.1[医药卫生—免疫学]