Fengtian He, Yongzhan Nie, Baojun Chen, Taidong Qiao, Zheyi Han, Daiming Fan
Department of Biochemistry and Molecular Biology, Third Military Medical University, Chongqing 400038. hefengtian@.163.net
Chin Med Sci J. 2002 Dec;17(4):215-9.
To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti-Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.
Balb/c mice were immunized i.p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.
The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed beta or gamma type anti-Id ScFv.
The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.
制备针对胃癌相关MG7单克隆抗体（McAb）的噬菌体展示抗独特型抗体单链可变片段（抗Id ScFv），为研制该肿瘤的抗Id ScFv疫苗奠定基础。
用钥孔戚血蓝蛋白（KLH）偶联的MG7 McAb经腹腔免疫Balb/c小鼠，取免疫小鼠脾脏提取mRNA。分别扩增抗体的重链和轻链（VH和VL）基因，通过PCR用连接子DNA组装成ScFv基因。将ScFv基因连接到噬菌粒载体pCANTAB5E，连接产物转化感受态大肠杆菌TG1。用M13K07辅助噬菌体感染转化菌，产生在M13噬菌体顶端展示ScFv的重组噬菌体。经4轮用MG7进行淘选后，用ELISA从富集的噬菌体中筛选出MG7阳性克隆。通过竞争ELISA初步鉴定所选噬菌体克隆上展示的抗Id ScFv类型。
VH、VL和ScFv DNA片段大小分别约为340 bp、320 bp和750 bp。从60个富集的噬菌体克隆中筛选出24个MG7阳性克隆，其中5个展示β或γ型抗Id ScFv。
利用重组噬菌体抗体技术可成功筛选到针对MG7 McAb的抗Id ScFv，为利用抗Id ScFv研究胃癌的防治开辟了道路。
