Construction And Characterization Of ScFv Phage Display Library Against Fibrin

Thrombus imaging, a recently developed diagnostic method of thrombosis, has been taken as the most promising diagnostic technique for deep venous thrombosis for its non-invasive, accurate, high sensitive characterization. In this technique, a radionuclide-labeled agent that specifically binds the thrombotic components is injected into patients’ veins. The radionuclide-labeled agent accumulates in the area of thrombus and the exact blood clot localization can be detected by imaging method. Currently, the radioisotope binding agents are mainly monoclonal antibodies, including anti-fibrin, anti-platelet, anti-D-dimer etc. At present, radionuclide-labeled anti-fibrin monoclonal antibody imaging has shown excellent effect in animal experiments. However, severe HAMA response and low clearance rate in blood circulation seriously limit its clinical application. A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short flexible linker peptide of ten to about25amino acids. This protein retains the specificity of the original immunoglobulin and antigen affinity equivalent to Fab fragment, despite removal of the constant regions and the introduction of the linker. As the minimal functional unit of antigen binding, scFv shows many advantages, including low molecular weight, high penetrating capability, short half-life in vivo, low immunogenicity, applicability in prokaryotic expression system and easy operation in genetic engineering. Phage antibody library technology has solved this problem in scFvs construction perfectly, which skipping the step of monoclonal antibody construction. scFvs with much higher affinity and specificity than monoclonal antibodies can be gotten by several round screening. ScFv genes are expressed and the gene products displayed on the surface of filamentous bacteriophage as fusion proteins. ScFv genes are fused to phage genes, thus creating a linker between scFv phenotype and its encoded genotype.In this study, VH and VL gene segments were amplified from spleen of human fibrin immuned mouse. ScFv gene was assembled with a flex linker by splice overlap extension PCR(SOE-PCR). Then the scFv gene was cloned into the Sfi Ⅰ/Not Ⅰ site of pCANTAB5E phagemid and transformed into E coli TGI cells. Because there are just a few amino acids difference between fibrin and fibrinogen. To avoid getting cross reacting scFvs, the scFv library was negatively screened with human fibrinogen and positive screened with human fibrin. After4rounds of screening, antigen affinity and binding specificity of positive clones was determined by ELISA. The highest affinity clone was sequenced and solubly expressed in E. coli TOP10. The results suggested that the reservoir of anti-human fibrin scFv phage display library is8.7×106.40%clones showes high-binding affinity against human fibrin. Sequencing indicated the anti-human fibrin scFv gene has an open reading frame (ORF) of732bp encoding244amino acids. VBASE2database analysis showed there were obvious3complementarity determining regions and4framework regions in VH and VL genes respectively. The successful construction of anti-human fibrin scFv library provides basis for the research of novel thrombus imaging agent.