全人源抗Met双价抗体的制备与特性分析

Preparation and characterization of human anti-Met recombinant antibody diabody

目的以抗HGF受体Met特异性的全人抗体Fab基因为模板,合成并表达双价抗体Diabody。方法以天然抗体库中筛选出的Fab基因为模板,分别扩增抗体的重链可变区与轻链可变区基因,经重叠拼接合成Diabody基因,克隆于原核表达载体pBAD/gⅢA中表达。在不同的培养条件下,阳性克隆经IPTG诱导表达,ELISA、SDS-PAGE、Western-blot和细胞免疫荧光分析。结果培养基成分和诱导剂的浓度对目的蛋白的表达未见有明显的影响,低温诱导能够促进可溶性蛋白的产生;但电泳与印迹分析表明,在29kD出现预期大小蛋白条带。双价抗体经表达、变性与复性和IMAC亲和纯化后,用细胞免疫荧光分析表明,Diabody能够与S114细胞表达Met的胞外区特异性结合。结论本研究制备的双价抗体能够与其抗原特异性结合,该抗体有望与其他抗体分子结合,制备双特异性抗体,为肿瘤临床诊断或治疗提供候选分子。

Objective This project is to develop fully human antibody fragment diabody that specifically bind to cell receptor Met. Methods The variable region genes were amplified from VH and VL of anti-Met Fab selected from native antibody display library. The diabody genes were amplified by overlap PCR with VH and VL gene. Diabody gene was purified and digested and inserted into pBAD-g Ⅲ/A. Results The confirmed diabody gene was cloned into pBAD-gⅢ/A for expression. Diabody was verified by ELISA,SDS-PAGE,Western-blot and cell immuno-fluoresence detection. The results showed that one band at about 29 kD at the expected size, but most in inclusive body form. To analyze the immunological characters of diabody for Met binding,fluoresence assays were set up and carried out with S114 and NIH3T3 cell lines. The results demonstrate diabody could bind native Met specifically on the S114 cell surface. Conclusions The results showed that anti-Met diabody antibody fragment could recognize Met extracellular domain in native conformation. It could be a potential powerfully reagents bispecfic antibody and for clinical diagnostic and therapeutic application.