抗TNF-α蛋白Fab抗体的制备和鉴定

Preparation and identification of the Fab antibodies against TNF-α

目的制备人源性抗肿瘤坏死因子(TNF)-α蛋白的可溶性Fab抗体。方法用固相化的TNF-α蛋白从人天然Fab噬菌体抗体库中筛选出表达抗TNF-α蛋白Fab抗体的阳性克隆,经酶切及连接反应构建可溶性表达噬菌粒并转化至大肠杆菌BL21(DE3)pLysS中,IPTG高效可溶性表达,SDS-PAGE及Western blot分析鉴定抗体表达情况,ELISA鉴定其抗原结合活性和特异性。结果筛选并表达了人源性抗TNF-α蛋白的Fab抗体,在非还原SDS-PAGE中形成相对分子质量47 kD的条带;Western blot分析证实表达的蛋白即Fab抗体;ELISA筛选出的2株抗体(TA-04、TA-13)具有良好的抗原特异性和抗原结合活性,与小牛血清白蛋白无交叉反应。结论成功制备并鉴定了人源性抗TNF-α蛋白的可溶性Fab抗体。

Objective To prepare human Fab （antigen-binding fragment） Abs against tumor necrosis factor -α（TNF-α）. Methods The specific anti-TNF-α clones were isolated from a ‘naive＇ human Fab antibody phage display library against the immobilized TNF-α antigens, then the clones were transformed to E. coli BI21 （DE3） pLysS cells to give Fab antibodies soluble expression by using IPTG as inducing agent. The expressed products were determined with SDS-PAGE, Westem blot and ELISA. Resuits The soluble products formed 47kD bands under non-inducing condition in SDS-PAGE. Western blot analysis showed the expression of human Fab antibodies. The ELISA demonstrated that Fab antibodies possessed good antigenic specificity as well as excellent combining activities with TNF-α antigens and had no cross-reactions with BSA. Conclusion The soluble human anti-TNF- α Fab antibodies were preparation and identified successfully.