Department of Basic Medicine, Luzhou Medical College, Luzhou, Sichuan Province 646000, China.
Appl Biochem Biotechnol. 2012 Jun;167(3):498-509. doi: 10.1007/s12010-012-9676-x. Epub 2012 May 6.
Interleukin (IL) 33 plays very important roles in inflammatory and allergic diseases. To select human single-chain Fv fragments (scFvs) against IL-33, a nonimmune phage library system was constructed. The full-length cDNA library was synthesized for amplification of the variable heavy chain (V(H)) and variable light chain (V(L)). By overlapping extension PCR for splicing V(H) and V(L), the full-length scFv library DNA were amplified and then transformed into Escherichia coli TG1. The scFv library was constructed successfully which contained 2.5×10(8) independent clones with full-length scFv inserts. The results of fingerprint maps of the scFvs by BstN I and DNA sequencing from the library at random proved that the library was diverse. The human IL-33 was amplified, expressed, and purified. The purified IL-33 with bioactivity was biotinylated and used as antigen for selection of scFv library by phage display. After three rounds of affinity selection, about 30 % of clones have specific binding activity with IL-33. Five of those with good binding activity were transformed into E. coli strain HB2151 for soluble expression. The selected scFvs were further identified by western blot and sequencing. Those selected scFvs could be used for further research of their effect on inflammatory and allergic diseases such as asthma by blockade of IL-33.
白细胞介素 (IL) 33 在炎症和过敏疾病中起着非常重要的作用。为了筛选针对 IL-33 的人源单链 Fv 片段 (scFv)，构建了一个非免疫噬菌体文库系统。合成全长 cDNA 文库以扩增可变重链 (V(H)) 和可变轻链 (V(L))。通过重叠延伸 PCR 拼接 V(H)和 V(L)，扩增全长 scFv 文库 DNA，然后转化入大肠杆菌 TG1。成功构建了包含 2.5×10(8)个独立克隆且全长 scFv 插入物的 scFv 文库。通过 BstN I 对 scFv 的指纹图谱和随机从文库中进行 DNA 测序的结果证明了文库的多样性。扩增、表达和纯化了人源 IL-33。具有生物活性的纯化 IL-33 被生物素化并用作噬菌体展示筛选 scFv 文库的抗原。经过三轮亲和选择，约 30%的克隆与 IL-33 具有特异性结合活性。其中 5 个具有良好结合活性的克隆被转化入大肠杆菌菌株 HB2151 进行可溶性表达。通过 Western blot 和测序进一步鉴定了所选 scFvs。这些筛选出的 scFvs 可用于进一步研究其通过阻断 IL-33 对哮喘等炎症和过敏疾病的影响。
