肝癌细胞HepG2特异性结合双价抗体的构建、表达及鉴定

发布时间：2018-11-27 18:00

【摘要】： 目的从噬菌体抗体库中筛选获得的肝癌细胞HepG2特异性结合单链抗体(single chain Fv, scFv),为进一步提高其亲合力(avidity),构建和表达与肝癌细胞HepG2特异结合的双价抗体(Bivalent single chain Fv, BsFv),鉴定其生物学活性,并和亲本单链抗体进行比较。 方法①以与肝癌细胞特异结合的单链抗体SLH10基因为模板,设计引物引入中间连接肽(G4S)及酶切位点AscI,通过PCR方法扩增出两个scFv片段,将其连接,并克隆至原核表达载体pAB1;②将表达载体转化大肠杆菌TG1,挑取单克隆细菌进行扩增,提取质粒酶切、测序鉴定;③阳性菌于23℃经IPTG诱导表达,在不同时间点收集菌液进行SDS-PAGE分析,并进行亚细胞定位分析,确定最适表达条件后进行大量表达,Ni2+-NTA层析柱纯化抗体,对纯化产物进行SDS-PAGE分析和Western blot鉴定;④通过FCM测定纯化产物与L02细胞和HepG2细胞结合活性,并进一步测定与其他几种肿瘤细胞株结合特性,比较BsFv、亲本scFv亲合力。 结果①提取重组菌质粒,酶切经凝胶电泳可见约1500bp大小片段条带,与BsFv基因大小相符;测序结果提示两个扩增基因片段以及中间连接肽序列正确无误,表明BsFv构建成功;②scFv及BsFv表达菌于23℃经IPTG诱导随时间的延长，蛋白产量上升;收集处理诱导12h菌体，亚细胞定位分析提示细菌外周质中存在目的蛋白表达，分子量大小分别为30kD、60kD左右，与理论值相符;③经IPTG诱导大量表达，收集上清超滤浓缩，收集菌体经处理得到外周质产物，所有产物经NiZ+一NTA层析柱纯化，并通过SDS一PAGE分析、W己stem blot鉴定表明表达蛋白的分子量与理论值一致;④FCM分析纯化抗体分别与几种细胞株的结合活性，，结果表明BsFv与亲本scFv比较，与HePGZ细胞结合率增加，与L02细胞及其他肿瘤细胞株结合率下降，表明BsFv与HePGZ细胞株亲合力有所增加。 结论成功构建与表达双价抗体BsFv;与亲本抗体scFv比较，BsFv与细胞结合的亲合力增加，本实验为进一步将其作为肿瘤靶向分子研究奠定了基础。
[Abstract]:Objective to construct and express bivalent antibody (Bivalent single chain Fv, a bivalent antibody to hepatoma cell line HepG2, which was selected from phage antibody library to construct and express bivalent antibody (Bivalent single chain Fv, which is specific for HepG2 binding to single chain antibody (single chain Fv, scFv),) in order to further enhance its affinity (avidity),. BsFv), identified its biological activity and compared it with parent scFv. Methods 1 using the SLH10 base of single chain antibody (scFV) specifically binding to hepatoma cells as template, two scFv fragments were amplified by PCR and cloned into prokaryotic expression vector pAB1; by introducing intermediate junction peptide (G4S) and restriction enzyme site (AscI,) into the primer. (2) the expression vector was transformed into Escherichia coli TG1, and the monoclonal bacteria were amplified, the plasmids were digested and sequenced. (3) the positive bacteria were induced by IPTG at 23 鈩

本文编号：2361612