抗人纤维蛋白噬菌体单链抗体库的构建和鉴定

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。

Construction and characterization of single chain Fv phage display library against human fibrin

Objective To construct single chain Fv(scFv) library against human fibrin by phage display technique,and characterize the anti-fibrin scFv clones selected from the library.Methods Total RNA was extracted from the splenocytes of the BALB/c mice immunized with human fibrin.Complementary DNA fragments of variable heavy(VH) and variable light(VL) chains of antibodies were obtained by polymerase chain reaction(PCR) and assembled into scFv by overlap extension PCR.ScFv fragment was cut by NotⅠand SfiⅠ,and then ligated into a pCANTAB 5E phagemid vector.The phagemids containing scFv cDNA were transformed into E.coli TG1 cells to generate scFv antibody library.Four rounds of enrichment and panning were performed to select specific anti-fibrin scFv from the library.The antigen binding activity was evaluated via enzyme-linked immunosorbent assay(ELISA).The high affinity scFv clone was analyzed by sequencing.Results A scFv antibody library against human fibrin with a diversity of approximately 8.7 × 106 was constructed by phage display.Sequencing analysis of one positive clone showed that the anti-fibrin scFv was 732 bp,and encoded 244 amino acids.VH and VL genes each contained three complementarity determining regions(CDR) and four framework regions(FWR).Conclusion It is demonstrated that the successful construction of a phage scFv library against human fibrin is lay the foundation for the development of a new thrombus imaging agent.