全人源性肝癌单链抗体的表达、纯化及功能鉴定  

Expression,purification and activity analysis of human single-chain antibody against hepatocellular cancer

作　　者：赵清涛[1] 马龙洋[1] 薛国柱[1] 赵爱志[1] 窦科峰[1] 

机构地区：[1]第四军医大学西京医院肝胆外科,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2007年第7期649-651,656,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(59637050)

摘　　要：目的:在大肠杆菌中表达人源性肝癌单链抗体(scFv),并分析他的结合活性。方法:应用噬菌体表面呈现技术获得人源性肝癌scFv,利用重叠延伸PCR将VL和VH基因以(Gly4ser)3linker连接成单链,插入表达载体pET28a(+),诱导目的蛋白表达,对包涵体进行溶解、复性、纯化,得到可溶性目的蛋白,应用非竞争细胞ELISA检测与肝癌细胞结合的特异性及结合能力。结果:在A600为0.8时开始诱导,持续6h,目的蛋白表达量占菌体总蛋白的26%,包涵体经过复性纯化后,得到纯度达到95%的重组scFv,其亲和常数为:3.6×107mol/L。结论:实现了人源性肝癌scFv的蛋白表达,抗体蛋白与肝癌细胞具有较强的特异性结合能力,为今后进行免疫学检测和开发肿瘤靶向治疗提供了研究手段。AIM： To express and characterize an active form of a single-chain antibody （scFv） from the gene of human phage antibodies which is specific for hepatocellular cancer. METHODS： The complementary DNAs encoding the variable regions of the light chain （VL） and heavy chain （VH） were connected by a （Gly4Ser）3 linker using a splicing by overlap extension polymerase chain reaction. The resultant scFv gene was cloned to the pET28a（ ＋ ） vector and expressed in E. coil as inclusion bodies. Then the inclusion bodies were solubUized, denatured and renatured. Finally, the affinity constant of scFv was determined by noncompetitive enzyme immunoassay. RESULTS： The target protein amounted 26% of the total protein in the condition of A600 at 0.8 for 6 hours. After renatured, the purity of target protein was 95% and the affinity constant of the scFv was 3.6×10^7 mol/L. CONCLUSION： An active form of scFv which is specific for hepatocellular cancer can bind selectively with hepatocellular cancer cells, which provides a theoretical basis for immunological detection and clinical use of scFv.

关 键 词：肝癌 单链抗体 表达 生物活性 

分 类 号：R392.11[医药卫生—免疫学]