针对单克隆抗体MGb1的重组抗独特型抗体的筛选与鉴定(英文)  

Screening and identification of recombinant anti-idiotypic antibody against monoclonal antibody MGb1

作　　者：郑英如[1] 李蓉芬[1] 李力[2] 黄刚[1] 张立[1] 胡大强[1] 

机构地区：[1]第三军医大学基础医学部生物化学与分子生物学教研室,重庆400038 [2]第三军医大学大坪医院野战外科研究所妇产科,重庆400042

出　　处：《免疫学杂志》2006年第1期86-89,共4页Immunological Journal

基　　金：ThisworkwassupportedbytheNationalNaturalScienceFoundationofChina(30200338)

摘　　要：目的利用噬菌体呈现技术筛选针对抗胃癌单克隆抗体(简称单抗)MGb1的重组抗独特型抗体(抗-Id),为研制胃癌重组抗-Id瘤苗提供候选分子。方法以单克隆抗体MGb1免疫Balb/c小鼠,取脾分离mRNA。RT-PCR分别扩增抗体VL和VHcDNA,经linker DNA连接形成ScFv DNA。将ScFv DNA与载体pCANTAB5E的连接产物转化于大肠杆菌TG1,经M13KO7感染后,获得重组噬菌体抗体ScFv文库。以单抗MGb1对文库进行4轮淘选后,随机挑取克隆经ELISA筛选呈现抗-Id ScFv的噬菌体单克隆,进而经竞争ELISA对其所属抗-Id类型进行初步鉴定。结果VL和VHcDNA分别约为320和340 bp,ScFv DNA约为750 bp。抗体ScFv文库经四轮淘选后,在随机筛检的50个克隆中得到18个呈现抗-Id ScFv的噬菌体单克隆。在18个克隆中,有4个呈现β或γ型抗-Id ScFv。结论经重组噬菌体抗体技术成功地筛选到了针对单抗MGb1的噬菌体呈现型抗-IdScFv,从而为进一步获得能诱导抗胃癌免疫的抗-Id ScFv奠定了基础。Objective To provide candidate molecules for developing recombinant anti-idiotypic antibody （anti-ld） vaccine of gastric carcinoma by selection of recombinant anti-ld to monoclonal antibody （McAb） MGbl directed against the cancer with phage display technique. Methods Balb/c mice were immunized with MGbl and the mRNA was isolated from the spleens of the immunized mice. The VL and V, cDNAs 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 competent E. coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage antibody ScFv library. After four rounds of biopanning to the library with MGbl, the MGbl-positive clones were selected from the enriched phages by ELISA. The types of the anti-ld ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA. Results The VL and VH cDNAs was about 320 bp and 340 bp, respectively. The ScFv DNA were about 750 bp. After four rounds panning to the phage antibody ScFv library with MGbl, 18 MGbl-positive phage clones displayed anti-ld SeFv were selected from 50 pre-selected phage clones, among which 4 clones displayed 13 or T type anti-ld ScFv. Conclusion The phage- displayed anti-ld ScFvs to McAb MGbl are successfully selected by recombinant phage antibody technique, which might lay a foundation for screening the anti-ld ScFv possessing the characteristics of inducing anti-gastric carcinoma immunity.

关 键 词：胃癌 抗独特型抗体 噬菌体呈现技术 肿瘤疫苗 

分 类 号：R392[医药卫生—免疫学]