抗粘蛋白1单链抗体的构建及其功能初步分析  

Construction and characterization of anti-mucin1 single chain Fv antibody fragment

作　　者：陈庆峰[1] 马中良[1] 王笑峰[2] 李福年[1] 郑玲莉[1] 

机构地区：[1]青岛大学医学院附属医院乳腺外科,山东青岛266003 [2]青岛大学医学院微生物教研室,山东青岛266003

出　　处：《第三军医大学学报》2010年第16期1770-1772,共3页Journal of Third Military Medical University

摘　　要：目的分离鼠源抗粘蛋白1(mucin1,MUC1)单克隆抗体可变区基因,构建有结合功能的单链抗体,并初步检测结合活性。方法提取杂交瘤细胞总mRNA,合成cDNA后,PCR扩增鼠特异性抗体可变区编码基因,通过9个氨基酸的连接子构建表达单链抗体的原核重组表达载体,体外诱导表达纯化后,采用Westernblot和流式细胞术方法分析单链抗体与变性或天然状态下抗原的结合活性。结果在杂交瘤细胞中成功分离到抗体轻重链可变区基因,重组链接后形成单链抗体能够在大肠杆菌中分泌表达,Westernblot和流式细胞术分析表明该单链抗体能够与变性或天然状态下的T47D肿瘤细胞表面MUC1特异性结合。结论成功制备具有生物活性的抗肿瘤相关MUC1单链抗体,该单链抗体具有与MUC1特异性结合的生物学活性。Objective To construct single chain variable fragment antibody （scFv） from a mouse monoclonal anti-mucin1 （MUC1） antibody secreting cell lines.Methods Isolation of mRNA of hybridoma cells,synthesis of cDNA,and antibody genotyping was performed using mouse antibody specific primers.Related variable genes were fused with a 9-amino-acid linker to obtain a scFv,which was characterized on denatured and natural antigen by Western blotting and flow cytometry.Results Variable regions of antibodies heavy and light chain coding genes were isolated form a monoclonal anti-MUC1 hybridoma cell line.ScFv was obtained by the combination of the 2 variable genes,which could be produced in E.coli.Western blotting and FACS analysis showed that the obtained scFv could bind to nature and denatured MUC1 of T47D tumor cells specifically.Conclusion A functional anti-MUC1 scFv is obtained successfully,which can bind to tumour associated MUC1 specifically.

关 键 词：乳腺肿瘤 粘蛋白类 单链抗体 

分 类 号：R392-33[医药卫生—免疫学] R392.11[医药卫生—基础医学]