噬菌体抗体库技术制备胃癌单抗MGd1的抗独特型抗体

Generation of antiidiotypic antibody to monoclonal antibody MGd1 directed against gastric carcinoma by phage antibody library technique

目的获得胃癌单抗MGd1的噬菌体呈现型抗独特型抗体(抗-Id),为研制针对MGd1的抗-Id重组胃癌瘤苗创造条件。方法分离经单抗免疫鼠脾脏的mRNA,经RT-PCR分别扩增抗体VH和VLDNA,进而连接形成ScFvDNA。将ScFvDNA与载体pCANTAB5E的连接产物转化于大肠杆菌TG1,经M13KO7挽救后获得噬菌体呈现型ScFv文库。以MGd1对文库进行4轮筛选后,随机挑取克隆经ELISA筛选呈现抗-IdScFv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VH、VL和ScFvDNA分别约为340、320和750bp。抗体ScFv文库经4轮筛选后,在随机筛检的40个克隆中得到17个呈现抗-IdScFv的噬菌体单克隆。在17个克隆中,有3个呈现β或γ型抗-IdScFv。结论应用噬菌体抗体库技术成功制备了针对单抗MGd1的抗-IdScFv,从而为筛选新的胃癌重组抗-Id瘤苗奠定了基础。

Objective To pave the way for developing recombinant anti-idiotypic antibody （anti-Id）vaccine of gastric carcinoma by generating phage-displayed anti-Id to monoclonal antibody MGd1 directed against the cancer. Methods Balb/c mice were immunized i.p. with MGdl conjugated with KLH, and mRNA was isolated from the spleens of the immunized mice. VH and VL DNAs of the antibody were amplified separately by RT-PCR and assembled into ScFv DNAs with a linker DNA by PCR. 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 phage antibody ScFv library. After four rounds of panning to the library with MGd1, the MGd1 -positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminary identified by competition ELISA. Results The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After four rounds of panning to the antibody library, 17 MGd1-positive phage clones displayed anti-Id ScFv were selected from 40 enriched phage clones, among which 3 displayed β and γ type anti-Id ScFv. Conclusion The successful generation of anti-Id SeFv to monoclonal antibody MGd1 by recombinant phage antibody library technology might lay a foundation for screening of novel candidate molecules for developing recombinant anti-Id vaccine of gastric carcinoma.