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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 IdScFv) ,为研制MG7的抗 IdScFv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连接形成ScFv基因片段。将ScFvDNA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M13KO7的作用下 ,获得重组噬菌体抗体ScFv。以单抗MG7对重组噬菌体抗体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分别约为 340、32 0和 75 0bp。经富集后 ,在随机筛检的 6 0个克隆中得到 2 4个噬菌体呈现型抗 IdScFv单克隆 ,阳性率为40 %。在 2 4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
Objective To obtain phage-displayed anti-idiotype single-chain antibody ScFv (anti-IdScFv) for gastric cancer monoclonal antibody (MAb) MG7, laying a foundation for the development of MG7 anti-IdScFv recombinant gastric cancer vaccine. Methods Balb/c mice were immunized with cross-linked MG7 monoclonal antibody and keyhole limpet hemocyanin (KLH). Splenocytes were used to isolate mRNA. The heavy and light chain variable region genes (VH and VL) of the antibody were amplified by reverse transcriptase polymerase chain reaction (PCR) and ligated with linker DNA to form the ScFv gene fragment. The ligation product of ScFvDNA and phagemid vector pCANTAB5E was transformed into E. coli TG1. Under the action of helper phage M13KO7, recombinant phage antibody ScFv was obtained. After four rounds of screening of the recombinant phage antibody ScFv with monoclonal antibody MG7, clones were picked randomly and screened by phage ELISA for monoclonal antibody against IdScFv, and the type of anti-idd Id was initially identified. Results The VH, VL and ScFvDNA were about 340, 390 and 750 bp, respectively. After enrichment, 24 phage-displayed anti-IdScFv clones were obtained from 60 randomly selected clones, with a positive rate of 40%. Of the 24 clones, 5 were β or γ anti-IdScFv. Conclusion The anti-IdScFv of monoclonal antibody MG7 can be successfully obtained by phage antibody technology, which has created the conditions for the development of anti-cancer research with anti-IdScFv.