基因工程技术制备的人源性抗Mr48×10^3角蛋白抗体Fab片段的纯化与活性鉴定

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

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 EL ISA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 Mr5 0× 10 3单一条带 ,在还原 SDS- PAGE中可见 Mr 2 3× 10 3,Mr 2 5× 10 3两条带 .EL ISA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 4 8× 10 3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 .

AIM To express human Fab fragment against M r 48×10 3 keratin in E.coli , and identify its purity, combining activities with antigens and antigenic specificity. METHODS The specific anti 48 kd keratin clone was selected from the established semisynthetic phage antibody library and transformed 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) , the expressed products were verified by ELISA, SDS PAGE and Western blot. RESULTS Soluble anti keratin Fab fragment could be efficiently purified by IMAC, and the purified products were identified to be human Fab fragment by Western blot. The purified Fab fragment formed a single band under non reducing conditions and two bands under reducing conditions in SDS PAGE. The purified Fab fragment possessed good antigenic specificity as well as the excellent combining activities with antigens as verified by ELISA and Western blot. CONCLUSION The high level expression and identification of soluble human anti keratin Fab fragment may lay a potentially good foundation of engineering human anti keratin Fab, of further researching its biological activities and of facilitating its potential clinical application.