全人源抗白介素21单链抗体的筛选与鉴定  

Selection and Characterization of Human Single Chain Fv (scFv) against Interleukin-21

作　　者：吴沁航[1] 王彤[1] 张弢[1] 张娟[1] 王旻[1] 

机构地区：[1]中国药科大学生命科学与技术学院,南京210009

出　　处：《药物生物技术》2012年第4期304-307,共4页Pharmaceutical Biotechnology

摘　　要：白介素21是具有免疫调节功能的I型细胞因子,在异常状态下可介导类风湿关节炎、系统性红斑狼疮和炎性肠病等多种自身免疫性疾病。为了拮抗白介素21的生物学活性,采用固相亲和筛选的方法,从Griffin1全人源噬菌体单链抗体库中筛选出抗白介素21的单链抗体。经过4轮亲和筛选,可溶性ELISA结果表明65%的噬菌体克隆可与IL-21发生特异性结合,经基因测序挑选出高亲和力的单链抗体FG8,转化至大肠杆菌HB2151中进行可溶性表达,采用镍柱亲和层析对表达的蛋白进行纯化后,SDS-PAGE和Western blot检测单链抗体相对分子质量为30 k。定量ELISA分析显示,单链抗体FG8与白介素21的结合呈浓度依赖性,可进一步研究用于炎症及自身免疫性疾病的诊断及靶向性治疗。Interleukin-21 （ IL-21 ） is a potent immunomodulatory four-a-helical-bundle type I cytokine, mainly produced by CD4^＋ T cells and has pleiotropic effects on both innate and adaptive immune responses. Abnormal expression of IL-21 is involved in the pathogenesis of inflammation and autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus and inflammatory bowel disease, suggesting that blocking the IL-21 signal might ameliorate autoimmune symptoms. Antibodies are some of the most powerful tools in therapy and diagnostics and are currently one of the fastest growing classes of therapeutic molecules. Single-chain variable fragments （scFvs） are becoming popular therapeutic alternatives to full length monoclonal antibodies since they are smaller, possessing different properties that are advantageous in certain medical applications. In this study,scFv antibodies against IL-2I were screened from Griffin 1 human phage display library. After four rounds of panning and selection on IL-21 absorbed immunotubes, individual clones were isolated, sequenced and characterized by Enzyme-Linked Immunosorbent Assay （ ELISA ）. 65% of the selected phage antibodies showed significant binding to IL-21, and a high affinity scFv FG8 was selected. The scFv FG8 was expressed in soluble form in Escherichia coli HB2151,purified by IMAC, the soluble 30 kDa FG8 scFv-antibody was verified in SDS-PAGE and Western-blot. The binding specificity of scFv FG8 was confirmed by quantitative ELISA, and a dose-dependent manner was obtained. The obtained scFv FG8 are fully human in nature, and have a higher therapeutic value compared with mouse and humanized antibodies, since it has a lower potential to cause adverse immunological effect. This study demonstrated that phage display libraries provide a valuable system for the rapid generation of specific antibody fragments, and the scFvs antibodies can be easily produced using bacterial protein synthesis systems. The scFv FG8 obtained can also be potentially served as drug

关 键 词：白介素21 噬菌体展示 单链抗体 酶联免疫法 自身免疫性疾病 FG8 

分 类 号：Q51[生物学—生物化学]