噬菌体呈现技术制备结肠癌抗独特型抗体

 以纯化的鼠抗人结肠癌细胞单克隆抗体 (简称单抗 )MC5与钥孔血蓝素 (KLH)的交联物经腹腔免疫Balb c小鼠 ,取脾分离mRNA .RT PCR分别扩增抗体重、轻链可变区基因片段 (VH 和VLcD NA ,大小分别约为 340bp和 32 0bp) ,二者经linkerDNA连接形成ScFv(singlechainvariablefragment)DNA(约 75 0bp) .将ScFvDNA与噬菌粒载体pCANTAB5E的连接产物转化于大肠杆菌TG1,经辅助噬菌体M13KO7感染后 ,获得重组噬菌体抗体ScFv文库 .以单抗MC5对ScFv文库进行 4轮亲和筛选后 ,随机挑取 80个克隆经酶联免疫吸附实验 (ELISA)筛选出 2 2个呈现ScFv形式抗独特型抗体(抗 IdScFv)的噬菌体单克隆 .竞争抑制实验表明 ,在 2 2个阳性克隆中有 4个克隆所呈现的抗 IdScFv属β或γ型 .针对单抗MC5的噬菌体呈现型抗 IdScFv的制备 ,为筛选新的结肠癌重组抗 Id瘤苗候选分子奠定了基础

Generation of Anti-idiotypic Antibody of Colorectal Carcinoma by Phage Display

MC5 is a mouse monoclonal antibody directed against human colorectal carcinoma cells. Balb/c mice were immunized i.p. with purified MC5 conjugated with keyhole limpet hemocyanin. mRNA was isolated from the spleens of the immunized mice. The cDNAs of heavy and light chain variable domains of the antibody (V H and V L cDNAs, about 340 bp and 320 bp respectively) were amplified separately by RT PCR and assembled into ScFv (single chain variable fragment) DNAs (about 750 bp) with a specially constructed 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 recombinant phage antibody ScFv library. After four rounds of panning to the library with MC5, 22 MC5 positive clones were selected by ELISA from 80 preselected phage clones. Competitive inhibition assay indicated that 4 out of the 22 positive clones could display β or γ type anti idiotypic antibody(anti Id)ScFv. The successful generation of the phage displayed anti Id ScFv to MC5 might lay a foundation for screening and identification of new candidate molecules for developing recombinant anti Id vaccine of colorectal carcinoma.