靶向bFGF与FGFR1Ⅲc结合位点的单克隆抗体制备及其生物学活性研究  

Production and biological activity study of mouse monoclonal antibody against the binding site of human b FGF with FGFR1Ⅲc

作　　者：罗镇明[1] 龚隆财 杨雁青 李雨蒙[1] 黄建芳[1] 宋其芳[1] 王宏[1] 

机构地区：[1]暨南大学生命科学与技术学院分子免疫与抗体工程重点实验室,广州510632

出　　处：《免疫学杂志》2015年第2期151-155,共5页Immunological Journal

基　　金：国家自然科学基金(81202449);广东省科技计划项目(2012B010300016);国家大学生创新创业训练计划(201410559069)

摘　　要：目的针对碱性成纤维细胞生长因子(b FGF)与成纤维生长因子受体1c(FGFR1Ⅲc)的结合位点,制备鼠抗人b FGF单克隆抗体并研究其生物学活性。方法用重组表达的人b FGF免疫Balb/c小鼠,取小鼠脾脏常规融合制备杂交瘤细胞,通过间接和竞争ELISA筛选出与FGFR1Ⅲc有共同结合位点的抗b FGF单抗。制备小鼠腹水并利用Protein G亲和柱纯化腹水单抗,SDSPAGE鉴定纯化产物;间接ELISA测定单抗亚类、亲和常数,竞争ELISA测定抗体IC50;Western blot和免疫荧光对单抗的生物学活性初步鉴定;CCK-8法检测单抗对肺癌细胞株LL/2的增殖抑制活性。结果筛选出了2株稳定表达b FGF抗体的细胞株Mab E12和Mab D9,竞争ELISA测得其IC50分别为1.564和1.96μg/ml,免疫球蛋白分类两者均为Ig G1,间接ELISA测得Mab E12的亲和常数为5.66×108L/mol;Western blot和免疫荧光表明该抗体能与LL/2细胞内天然合成的b FGF相结合,并对其增殖产生了较明显的抑制作用。结论成功建立了1株能特异性识别b FGF-FGFR1Ⅲc合位点的单抗,能有效的中和b FGF对肺癌细胞株LL/2促增殖作用,为研究肿瘤治疗途径奠定了基础。This study designed to produce mouse monoclonal antibody（Mc Ab） against b FGF of human directly at the binding site of b FGF with FGFR1 Ⅲ c and study its biological activity. Balb/c mice were immunized with recombinant b FGF of human,and then the spleen lymphocytes of the mice were fused with SP2/0 mouse myeloma cells. Hybridoma cell lines secreting Mc Ab against the binding site of b FGF with FGFR1 Ⅲc were screened out by indirect and competitive ELISA. The Mc Ab in ascites was purified by using affinity chromatography with protein G and then identified by SDS-PAGE. ELISA was further carried out to determine the antibody isotype,affinity constant and IC50. The biological activity of Mc Ab was investigated by using western blot,immunofluorescence assays,and CCK-8 assay. Data showed that two hybridoma cell lines,named Mab E12 and Mab D9 and stably expressed anti-b FGF monoclonal antibody,were obtained. ELISA showed the IC50 of Mab E12 and Mab D9 were1.564 μg/ml and 1.96 μg/ml respectively,and the affinity constant of the Mab E12 was 5.66×10^8L/mol,furthermore,their isotypes were both belonged to Ig G1. Mab E12 could specifically react with nature b FGF expressed in LL/2cells and obviously inhibited the proliferation of LL/2 cells. In conclusion,a hybridoma cell line secreting Mc Ab that can specifically recognize the binding site of b FGF with FGFR1Ⅲ c was developed successfully,and the Mc Ab can effectively neutralize b FGF to block its effect of promoting LL/2 proliferation,which will facilitate the study of cancer therapy.

关 键 词：b FGF FGFR1Ⅲc 单克隆抗体 肺癌 

分 类 号：R392[医药卫生—免疫学]