结肠癌单抗MC5的噬菌体呈现型单链可变区片段的制备  

Preparation of Phage Displayed Single Chain Variable Fragments of Monoclonal Antibody MC5 Recognizing Colorectal Carcinoma

作　　者：何凤田[1] 李蓉芬[1] 张艳[1] 吉清[1] 陈宝军[2] 乔太东[2] 樊代明[2] 

机构地区：[1]第三军医大学生物化学与分子生物学教研室,重庆400038 [2]第四军医大学西京医院消化病研究所,陕西西安710032

出　　处：《癌症》2002年第6期636-639,共4页Chinese Journal of Cancer

摘　　要：背景与目的:MC5是一种特异性良好的针对人结肠癌的鼠源性单克隆抗体,而将鼠源性抗体小型化可使其用于在体研究时引起人抗鼠抗体反应的可能性大大降低。本研究的目的是制备MC5的噬菌体呈现型单链可变区片段(ScFv)。方法:从分泌MC5的杂交瘤细胞分离mRNA,RT-PCR分别扩增抗体的重、轻链可变区DNA(VH和VLDNA),两者经linkerDNA连接形成ScFvDNA。将ScFvDNA与噬粒载体pCANTAB5E的连接产物转化于大肠杆菌TG1,经M13KO7辅助噬菌体感染后,获得重组噬菌体抗体ScFv。以高表达MC5结合抗原的细胞株SW480对重组噬菌体抗体ScFv进行两轮筛选后,随机挑取克隆经ELISA筛选呈现MC5ScFv的噬菌体单克隆,经竞争ELISA对阳性克隆结合抗原的能力进行鉴定。结果:VH、VL和ScFvDNA分别约为340bp、320bp和750bp。在随机筛检的25个克隆中得到10个呈现MC5ScFv的噬菌体单克隆,其中结合抗原能力强的克隆有3个。结论:用噬菌体呈现技术成功地制备了单抗MC5的ScFv,为拓展该抗体的应用范围奠定了基础。Background &Objective:MC5 is a murine monoclonal antibody with a good specificity to human colorectal carcinoma and smaller murine antibody can significantly decrease the possibility of developing human antimouse antibody response in vivo study. The aim of this study was to prepare single chain variable fragments (ScFv) of MC5. Methods: mRNA was isolated from the hybridoma cell line producing MC5, and the DNAs encoding variable domains of heavy and light chains(VH and VL DNAs)of the antibody were amplified separately by RT PCR and assembled into ScFv DNAs with a linker DNA. The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield recombinant phage antibody ScFvs. After two rounds of panning with cell line SW480 highly expressing MC5 binding antigen, the phage clones displayed ScFv fragments of the antibody were selected by ELISA, and the affinity of the positive phage clones was assayed by competition ELISA. Results:The VH,VL, and ScFv DNAs were about 340bp, 320bp, and 750bp, respectively. Ten phage clones displayed ScFv of MC5 were selected from 25 enriched phage clones, and 3 of the 10 phage clones had higher affinity of binding to the antigen. Conclusion:The phage displayed ScFv fragments of monoclonal antibody MC5 are successfully produced by phage display technique, which may provide a way for broadening the application range of the antibody.

关 键 词：结肠癌 单克隆抗体 单链可变区片段 噬菌体呈现技术 

分 类 号：R735.3[医药卫生—肿瘤]