人源抗滋养层细胞表面抗原-2基因工程抗体Fab的制备及特性分析  

Preparation and Characterization of Human Anti-Trop-2 Engineering Antibody Fab

作　　者：林红[1] 梁洁[1] 张慧林[1] 唐奇[1] 苏亦平[1] Brian Cao 朱进[3] 管晓虹[1] 

机构地区：[1]南京医科大学卫生部抗体技术重点实验室 [2]Van Andel Research Institute,Antibody Lab,MI49503,USA [3]南京军区军事医学研究所

出　　处：《生物化学与生物物理进展》2010年第10期1101-1107,共7页Progress In Biochemistry and Biophysics

基　　金：南京市医学科技发展重点项目(ZKX09015);南京市科技发展项目(200901083)资助~~

摘　　要：应用噬菌体抗体库技术制备全人源抗滋养层细胞表面抗原-2(Trop-2)特异性Fab抗体片段.抗体库经细胞筛选和固相抗原筛选,获得特异性的阳性克隆.阳性载体经核酸序列分析后,构建工程菌,经IPTG诱导表达,SDS-PAGE和Western blot分析,呈现28ku和32ku大小的两条蛋白质条带.Fab分子经流式细胞术、细胞免疫荧光检测,结果表明,Fab能够与BxPc3细胞膜蛋白特异性结合,而与NIH3T3细胞不结合.免疫共沉淀与质谱分析结果表明,该Fab分子能够与Trop-2蛋白特异性结合.免疫组化显示,该抗体可结合胰腺癌细胞膜蛋白,在细胞培养液中加入Fab,能够抑制BxPc3细胞的生长.以上研究结果提示,该抗体有望成为胰腺癌临床影像诊断或治疗的候选分子.Fully human antibody fragment Fab that specifically binding to Trop-2 （trophoblast cell-surface antigens 2, Trop-2）, was selected from phage display antibody library. Positive phage-displayed antibody clones were selected on live cell lines and immobilized protein. The purified Fab was verified by SDS-PAGE and Western blot, which showed two bands at about 28 ku and 32 ku at the expected sizes. To analyze the immunological characters of Fab for Trop-2 binding, flow cytometry, immunoprecipitation assays and mass spectrometry were set up and carried out with BxPc3 and NIH3T3 cell lines. The results demonstrated Fab could bind native Trop-2 specifically on the BxPc3 cell surface. Peptide mapping fingerprint showed that the protein which bound is Trop-2 protein. Immunohistochemistry detection illuminated Fab could bind the membrance protein of pancreatic cancer tissue. In vitro cell growth inhibition assay showed that anti-Trop-2 Fab could inhibit the Trop-2 positive cell BxPc3 growth, it illuminated that anti-Trop-2 Fab bound the Trop-2 on Trop-2 positive cell surface. For Trop-2 negative cell line NIH3T3, no significant inhibition among the different dosages of Fab. The results showed that anti-Trop-2 Fab antibody fragments could recognize Trop-2 extracellular domain in native conformation, making them as potential powerful reagents for clinical therapeutic application.

关 键 词：噬菌体抗体库 胰腺癌 人源抗Trop-2抗体 

分 类 号：Q78[生物学—分子生物学]