关键词: 角蛋白;人源性抗体;Fab;制备;鉴定

    摘 要:目的 用基因工程技术制备人源性抗角蛋白抗体Fab片段，并对其纯度、抗原结合活性及特异性等进行鉴定. 方法 将从半合成噬菌体抗体库中成功地筛选到的特异表达抗角蛋白Fab片段的阳性克隆转化大肠杆菌，IPTG诱导表达，产物经金属鏊合层析纯化，并用ELISA鉴定抗原结合活性和特异性、SDS-PAGE鉴定纯度和蛋白印迹检测抗角蛋白抗体Fab片段识别的抗原组分. 结果 可溶性表达的抗角蛋白抗体Fab片段经金属鏊合层析可有效地纯化，蛋白印迹明确纯化产物为人Fab片段.所纯化的Fab片段在非还原SDS-PAGE中形成Mr 50×103 单一条带，在还原SDS-PAGE中可见Mr 23×103 ，Mr 25×103 两条带.ELISA和Western blot证实该片段具备良好的抗原结合活性和抗原特异性. 结论 成功表达并鉴定了人源性抗Mr 48×103 角蛋白的Fab可溶性片段，为人源性抗角蛋白抗体工程化、进一步研究该抗体的生物活性并提高其临床应用价值奠定了良好的基础.

    Preparation of human anti┐Mr 48×103 keratin Fab fragment by genetic engineering technology

    LI Cheng-Xin LIU Yu-Feng WANG Gang LI Chun-Ying WAN Ye-Hong

    1 Department of Dermatology，Xijing Hospital，Fourth Military Medical University，Xi'an710033，China，

    2 Department of Dermatology，Navy General Hospital，Beijing100036，China

    Keywords:keratin;human antibody;Fab;preparation;i-dentification

    Abstract:AIM To express human Fab fragment against Mr 48×103 keratin in E.coli，and identify its purity，combining activities with antigens and antigenic specificity.METHODS The specific anti-48kd keratin clone was selected from the established semisynthetic phage antibody library and trans-formed into E.coli xL1-blue.The specific Fab fragments were expressed by using IPTG as inducing agent.After being purified by immobilized metal ion affinity chromatography(IMAC) ......