具有中和活性的抗人IL-1β单链抗体的构建  

Construction of anti-hIL-1β scFv with neutralizing activity

作　　者：李宁[1] 徐黎明[1] 李天鹤[1] 任桂萍[1] 陈睿[1] 张宇[1] 阚方明[1] 叶贤龙[1] 谷学佳[1] 丁良君[1] 李德山[1] 

机构地区：[1]东北农业大学生命科学学院生物制药教研室,哈尔滨150030

出　　处：《免疫学杂志》2011年第7期594-598,共5页Immunological Journal

基　　金：哈尔滨市科技创新人才发展计划资助(2006RFXXS002);东北农业大学创新团队发展计划资助(CXZ001-1);哈尔滨科技创新人才研究专项资金项目(青年科技创新人才)(2010RFQXN109);国家自然科学基金青年科学基金项目(31001084)

摘　　要：目的构建抗人IL-1β单链抗体(anti-hIL-1βscFv)的原核表达载体,并对其表达的蛋白进行生物学活性检测。方法使用PCR的方法从含有抗人IL-1β抗体基因的质粒中获得抗人IL-1β的单链抗体基因,将其插入原核表达载体pET-27b中,构建重组表达载体pET-27b-hIL-1βscFv。将该载体转化大肠杆菌Rosetta(DE3)后诱导表达,经凝胶过滤色谱柱上复性得到可溶的抗人IL-1β的scFv蛋白。使用ELISA方法鉴定scFv抗体的特异性结合活性,使用Real time-PCR的方法检测scFv抗体的中和活性。结果成功地对抗人IL-1β单链抗体进行诱导、表达和复性,蛋白相对分子质量约为28 000。ELISA结果证实scFv蛋白对hIL-1β具有特异性结合能力。Real time-PCR实验结果证实该scFv抗体可以有效阻断人IL-1β刺激人T细胞表达细胞因子IL-18及IL-1β的作用。结论通过原核表达系统得到的抗人IL-1β单链抗体经复性后,与人IL-1β蛋白有特异性结合能力,并且表现出中和活性,为进一步研究人IL-1β相关疾病及其治疗性抗体奠定了基础。We aimed to prepare scFv antibody against human IL-1β and identify its bioactivity for further research on diseases associated with IL-1β.Firstly,anti-hIL-1β scFv gene was subcloned into pET-27b.Then the recombinant vector pET-27b-hIL-1β scFv was constructed and transformed into the host bacteria Rosetta.The scFv protein was expressed by IPTG induction,and characterized by SDS-PAGE and ELISA after on-column refolding.The neutralizing activity was detected by Real time-PCR.The anti-hIL-1β scFv was expressed and refolded successfully,and showed specific binding activity to hIL-1β.The result of Real time-PCR demonstrated that anti-hIL-1β scFv can block IL-1β to stimulate human T cell to express IL-18 and IL-1β.In conclusion,anti-hIL-1β scFv antibody with neutralizing activity was successfully constructed and expressed in prokaryotic system.

关 键 词：IL-1Β 单链抗体 包涵体复性 中和活性 Realtime-PCR 

分 类 号：R392-33[医药卫生—免疫学]