重组可溶性人IL-6的原核表达纯化和活性鉴定  

Prokaryotic expression purification and identification of recombinant human soluble IL-6

作　　者：刘彩艳[1] 赵自叶 郑娟[2] 段树燕 郭怀祖[3] 徐进[1] 王皓[3] 

机构地区：[1]上海交通大学药学院,上海200240 [2]聊城大学药学院,聊城252000 [3]第二军医大学肿瘤研究所,上海200433

出　　处：《现代免疫学》2013年第6期464-468,共5页Current Immunology

摘　　要：使用大肠杆菌BL21(DE3)重组表达可溶性人IL-6并对其进行纯化和活性鉴定。以人激活T细胞总RNA为模板,逆转录PCR扩增人IL-6的基因片段并将其克隆到原核表达载体pET32a(+)中,重组质粒转化大肠杆菌BL21(DE3),优化表达条件获得可溶性表达目的蛋白。细菌裂解上清经镍金属螯和层析纯化,SDS-PAGE鉴定其分子量大小,western blot鉴定其免疫原性,并通过IL-6依赖的细胞株T1165分析重组蛋白的活性。成功构建了pET32a(+)/IL-6表达载体,并获得了可溶性表达的重组人IL-6蛋白。SDS-PAGE显示,其分子量约为21kD,可被抗IL-6抗体特异性识别,并且该重组蛋白可刺激IL-6依赖的细胞株T1165增殖,比活性与标准品一致,约为1×106 U/mg。本实验获得了可溶性高表达的重组人IL-6蛋白,为进一步研究人IL-6奠定了基础。To express and purify of recombinant human IL-6 from E. coli, and evaluate its activity using IL-6 dependent cell line Tl165. eDNA was generated from total RNA of activated T cells by RT PCR and cloned into pET32a＋. The resulting plasmid pET32a（＋） /IL-6 was transferred to E. coli BL21（DE3）, and then overexpressed in E. coli cells by IPTG induction. Ni NTA affinity chromatography was used for the purification. The purified protein was characterized and activity was validated by SDS- PAGE and IL-6 dependent cell line Tl165. The recombinant human IL-6 was soluble expressed in prokaryotes with high effi- ciency. SDS-PAGE analysis showed the molecular weight of the protein was 21kD. It can be identified by mouse anti-human IL- 6 specific antibody and the activity was 1×10^6 U/mg, determined by stimulating the proliferation of Tl165 cell. The present study provides a novel method to get soluble active human IL-6 protein for further studying its biological activity.

关 键 词：IL-6 原核表达纯化 活性鉴定 

分 类 号：R329.8[医药卫生—人体解剖和组织胚胎学]