针对单克隆抗体MGb1的重组抗独特型抗体的筛选与鉴定

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VH cDNA，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VH cDNA分别约为320和340bp，ScFv DNA约为750bp。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-Id ScFv，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。

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

Objective T9 provide candidate molecules for developing recombinant anti-idiotypic antibody (anti-Id) vaccine of gastric carcinoma by selection of recombinant anti-Id to monoclonal antibody ( McAb) MGb1 directed against the cancer with phage display technique.Methods Balb/c mice were immunized with MGb1 and the mRNA was isolated from the spleens of the immunized mice. The VL and VH 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 MGb1, the MGb1-positive clones were selected from the enriched phages by ELISA. The types of the anti-Id 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 MGb1, 18 MGb1-positive phage clones displayed anti-Id ScFv were selected from 50 pre-selected phage clones, among which 4 clones displayed β or γ type anti-Id ScFv. Conclusion The phagedisplayed anti-Id ScFvs to McAb MGb1 are successfully selected by recombinant phage antibody technique, which might lay a foundation for screening the anti-Id ScFv possessing the characteristics of inducing anti-gastric carcinoma immunity.