胃癌单克隆抗体MG7的噬菌体呈现型抗独特型单链抗体ScFv的制备

目的 获得胃癌单克隆抗体（简称单抗）MG7的噬菌体呈现型抗独特型单链抗体ScFv（抗－Id ScFv），为研制MG7的抗－Id ScFv重组胃癌瘤苗奠定基础。方法 以MG7单抗与匙孔血蓝蛋白（KLH）的交联物免疫Balb/c小鼠，取脾分离mRNA。以逆转录聚合酶链反应技术分别扩增抗体重、轻链可变区基因（VH和VL），经linkerDNA连接ScFv基因片段。将ScFv DNA与噬粒载体pCANTAB5E的连接产物转化于大肠杆菌TG1。在辅助噬菌体M13K07的作用下，获得重组噬菌体抗体ScFv。以单抗MG7对重组噬菌体抗ScFv进行四轮筛选后，随机挑取克隆，经噬菌体ELISA筛选抗－IdScFv单克隆，并对其所属抗－Id类型进行初步鉴定。结果 VH、VL和ScFvDNA分析约为340、320和750bp。经富集后，在随机筛检的60个克隆中得到24个噬菌体呈现型抗－IdScFv单克隆，阳性率为40％。在24个克隆中，有5个为β或γ型抗－IdScFv。结论 用噬菌体抗体技术能够成功地获得单抗MG7的抗－IdScFv，为开展用抗－IdScFv防治胃癌的研究创造了条件。

Production of phage-displayed anti-idiotypic antibody single chain variable fragments to MG7 monoclonal antibody directed against gastric carcinoma

OBJECTIVE: To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti-Id ScFv) MG7 to monoclonal antibody directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer. METHODS: Balb/c mice were immunized i.p. with purified MG7 monoclonal antibody conjugated with keyhole limpet hemocyanin. mRNA was isolated from the spleens of immunized mice. Heavy and light chain genes (VH and VL) of antibody were amplified separately and assembled into ScFv genes with a specially constructed linker DNA by RT-PCR. The ScFv genes 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, which displayed ScFv fragments as a fusion with gene 3 protein on the tips of M13 phage. After four rounds of panning with monoclonal antibody MG7, the MG7-positive clones were selected with the enzyme-linked immunosorbent assay (ELISA) from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were primarily identified by competition ELISA. RESULTS: The VH, VL and ScFv DNAs were about 340, 320 and 750 bp respectively. Twenty-four MG7-positive clones were selected from 40 enriched phage clones. Five of the 24 clones displayed beta or gamma type anti-Id ScFv. CONCLUSION: The anti-Id ScFv frangments to MG7 monoclonal antibody can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma using anti-Id ScFv.