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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 screen recombinant anti-idiotypic antibodies against anti-gastric cancer monoclonal antibody (MGb1) by phage display technique and provide candidate molecules for the development of recombinant gastric cancer anti-Id vaccine. Methods Balb / c mice were immunized with monoclonal antibody MGb1 and splenocytes were isolated. Antibody VL and VH cDNA were amplified by RT-PCR respectively and linked to DNA by linker DNA. The ligation product of ScFv DNA and vector pCANTAB5E was transformed into E. coli TG1 and infected with M13KO7 to obtain a recombinant phage antibody ScFv library. After four rounds of panning with the monoclonal antibody MGb1, the clones were randomly selected to screen for anti-Id ScFv phage clones by ELISA, and their anti-Id types were preliminarily identified by competitive ELISA. Results The VL and VH cDNAs were about 320 and 340 bp, respectively, and the ScFv DNA was about 750 bp. After four rounds of panning of the antibody ScFv library, 18 phage clones exhibiting anti-Id ScFv were obtained in 50 randomly screened clones. Of the 18 clones, 4 showed beta or gamma anti-Id ScFv. Conclusion The recombinant bacteriophage antibody technique successfully screened the phage-displayed anti-IdScFv against the monoclonal antibody MGb1, which lays the foundation for further obtaining anti-Id ScFv that can induce anti-gastric cancer immunity.