胃癌单克隆抗体MG7的噬菌体呈现型抗独特型单链抗体ScFv的制备  

Production of phage displayed anti idiotypic antibody single chain variable fragments to MG7 monoclonal antibody directed against gastric carcinoma

作　　者：何凤田[1] 聂勇战[2] 韩者艺[2] 陈宝军[2] 乔太东[2] 樊代明[2] 

机构地区：[1]第三军医大学基础部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,西安710032

出　　处：《中华医学杂志》2001年第1期33-36,共4页National Medical Journal of China

基　　金：国家杰出青年基金资助项目 (395 2 5 0 2 0 ) ;国家 86 3高科技计划基金资助项目! (10 2 10 0 1 0 6 )

摘　　要：目的　获得胃癌单克隆抗体 (简称单抗 )MG7的噬菌体呈现型抗独特型单链抗体ScFv(抗 IdScFv) ,为研制MG7的抗 IdScFv重组胃癌瘤苗奠定基础。方法　以MG7单抗与匙孔血蓝蛋白(KLH)的交联物免疫Balb/c小鼠 ,取脾分离mRNA。以逆转录聚合酶链反应技术分别扩增抗体重、轻链可变区基因 (VH和VL) ,经linkerDNA连接形成ScFv基因片段。将ScFvDNA与噬粒载体pCANTAB5E的连接产物转化于大肠杆菌TG1。在辅助噬菌体M13KO7的作用下 ,获得重组噬菌体抗体ScFv。以单抗MG7对重组噬菌体抗体ScFv进行四轮筛选后 ,随机挑取克隆 ,经噬菌体ELISA筛选抗 IdScFv单克隆 ,并对其所属抗 Id类型进行初步鉴定。结果　VH、VL和ScFvDNA分别约为 340、32 0和 75 0bp。经富集后 ,在随机筛检的 6 0个克隆中得到 2 4个噬菌体呈现型抗 IdScFv单克隆 ,阳性率为40 %。在 2 4个克隆中 ,有 5个为 β或γ型抗 IdScFv。 结论　用噬菌体抗体技术能够成功地获得单抗MG7的抗 IdScFv ,为开展用抗Objective To generate phage displayed anti idiotypic antibody single chain variable fragments (anti Id ScFv) MG7 to monoclonal antibody directed against gastric carcinoma so as to lay a foundation for developing anti Id ScFv vaccine of the cancer. Methods Balb/c mice were immunized i.p. with purified MG7 monoclonal antibody conjugated with keyhole limpet hemocyanin. mRNA was isolated from the spleens of immunized mice. Heavy and light chain genes (VH and VL) of antibody were amplified separately and assembled into ScFv genes with a specially constructed linker DNA by RT PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E.coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage, which displayed ScFv fragments as a fusion with gene 3 protein on the tips of M13 phage. After four rounds of panning with monoclonal antibody MG7, the MG7 positive clones were selected with the enzyme linked immunosorbent assay (ELISA) from the enriched phages. The types of the anti Id ScFv displayed on the selected phage clones were primarily identified by competition ELISA. Results The VH, VL and ScFv DNAs were about 340、320 and 750 bp respectively. Twenty four MG7 positive clones were selected from 40 enriched phage clones. Five of the 24 clones displayed β or γ type anti Id ScFv. Conclusion The anti Id ScFv frangments to MG7 monoclonal antibody can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma using anti Id ScFv.

关 键 词：胃肿瘤 噬菌体 胃癌 单克隆抗体 MG7 单链抗体 制备 

分 类 号：R735.2[医药卫生—肿瘤] R392－33[医药卫生—临床医学]