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目的应用噬菌体展示技术构建抗人纤维蛋白单链抗体(scFv)文库,筛选高亲和力抗人纤维蛋白scFv并进行鉴定。方法利用人纤维蛋白免疫小鼠,分别扩增小鼠VH和VL基因,经重叠延伸聚合酶链反应(PCR)将VH和VL基因拼接成scFv基因,SfiⅠ/NotⅠ双酶切克隆入pCANTAB 5E噬菌粒载体,转化E.coli TG1构建成库,采用人纤维蛋白原对抗体库进行负筛选,人纤维蛋白进行正筛选,酶联免疫吸附分析(ELISA)检测阳性克隆的抗原特异性并进行测序分析。结果构建了库容为8.7×106的抗人纤维蛋白scFv库,ELISA测定显示scFv具有较高的抗原特异性;抗人纤维蛋白scFv基因序列长732 bp,编码244个氨基酸,VH和VL基因均有明确的3个互补决定区和4个骨架区。结论成功构建了抗人纤维蛋白scFv文库,并筛选到高亲和力的抗人纤维蛋白scFv,为新型血栓显像剂的开发奠定了实验基础。
Objective To construct a scFv library by phage display technique and screen high-affinity anti-human fibrin scFv for identification. Methods Mice were immunized with human fibrin to amplify the mouse VH and VL genes. The VH and VL genes were spliced into scFv by overlap extension polymerase chain reaction (PCR). The scFv gene was digested with Sfi I / Not I and cloned into pCANTAB 5E The plasmid vector was transformed into E. coli TG1 into a library, negative screening of the antibody library with human fibrinogen, positive screening of human fibrin, and antigenic specificity of positive clones were detected by enzyme-linked immunosorbent assay (ELISA) and sequenced analysis. Results An anti-human fibronectin scFv library with a capacity of 8.7 × 106 was constructed. The result of ELISA showed that scFv was highly antigen-specific. The sequence of anti-human fibrin scFv was 732 bp in length and encoded 244 amino acids. Both VH and VL genes Clear three complementary decision area and four skeleton area. Conclusion The anti-human fibrin scFv library was successfully constructed and high-affinity anti-human fibrin scFv was screened, which laid the experimental foundation for the development of novel thrombotic imaging agents.