应用噬菌体展示技术制备抗HGF受体Met特异性、高亲和力的全人抗体scFv片段。方法: 以从天然抗体库中筛选出的 Fab 基因可变区VH和VL为模板，分别在CDR区引入有限突变和随机突变，扩增出scFv基因库，克隆于pComb3XSS中，构建scFv次级突变抗体库，筛选高亲和力克隆。结果:经5轮细胞筛选和2轮抗原固相筛选，选取60个克隆进行ELISA检测，选择较高亲和力的噬菌体抗体，克隆于原核表达载体pBAD-gIII/A中，经诱导表达、SDS-PAGE和Western blotting分析，在相对分子质量30 000处出现预期大小的蛋白条带。单链抗体经表达、变性与复性和IMAC亲和纯化后，用于流式细胞术、免疫沉淀分析，结果表明，scFv能够与S114细胞表达Met的胞外区特异性结合，其亲和常数Kd 值为4.76×10 -8 mol/L，与突变前抗体的亲和力（ Kd值为5.24×10 -6 mol/L）相比，抗体的亲和力提高约100倍。竞争ELISA结果显示，亲和力成熟后抗体的抗原结合表位未变。 结论: 经过CDR突变和抗体库筛选，有效提高了抗体的亲和力，该抗体有望成为临床肿瘤诊断或治疗的候选分子。

To prepare human high affinity antibody fragment scFv that specifically binds to cell receptor Met by phage display technique. Methods: The variable regions were amplified from VH and VL of anti-Met Fab genes screened from the natural immune Fab antibody phage display library. The scFv genes were amplified by overlap PCR with different VH and VL library genes assembled by pairs of different CDR mutation primers. scFv DNA was purified and digested with SfiI and was then inserted into pComb3XSS. Positive phage-displayed antibodies with high affinity were selected on live cell lines. Results: After 5 cycles of cell screening and 2 cycles of antigen screening, 60 positive clones were subjected to ELISA. The selected high affinity scFv gene was cloned into pBAD/gIII for expression and was then studied by SDS-PAGE and Western blotting; a band was showed at about 30 000 as expected. To analyze the immunological characters of scFv for Met binding, flow cytometry and immunoprecipitation assays were carried out with S114, MKN45 and NIH3T3 cell lines. The results of flow cytometry and immunoprecipitation demonstrated scFv could bind to natural Met specifically on the surface of S114 and MKN45 cells (Kd=4.763×10 -8 mol/L), and the affinity was about 100 times higher than before mutation (Kd= 5.24×10 -6 mol/L). The results of ELISA showed that there was no change in the binding site of antigen after maturation of the affinity. Conclusion: It is indicated that anti-Met scFv antibody fragment can recognize Met extracellular domain in natural conformation with relatively high affinity, and it may be a potential candidate for clinical diagnosis and therapy.

江苏省卫生厅重大科技项目（No. wkj2004-2-12)