Construction Anti-activated Human Platelet Phage Display ScFv Library And Screening Specific ScFv Antibody Against Platelet Membrane Receptors

The variable region of heavy-chain and light-chain (VH and VL) of immunoglobulin gene were amplified from the spleen cells of Bal b/c mouse immunized with actived human platelets, VH and VL are joined to construct single-chain Fv fragment (ScFv) gene in vitro. The recombinant ScFv gene was ligated into phagemid vector, then the ScFv were displayed in Filamentous phage. Thus, the anti-actived human platelet phage display ScFv library was constructed. From the library, the specific ScFv antibody against several platelet membrane receptors such as GPIIb, GPIIIa, GPIIb/IIIa and recombinant thrombin receptor PAR-1 (rPARl) have been screened. Target antigens, GPII b, GPIIIa and GP II b/IIIa were purified from solubilized platelet solution using affinity chromatography. The rPARl was expressed in E.coli. The function of recombinant peptide is similar to native PAR-1.The gene of the selected clones were sequenced, their sequence were conformed to mouse Ig gene. The gene of high affinity anti-rPARl specific phage ScFv clone APAR31 was expressed and the expressed ScFv fragment retain binding activity to target antigen. These results also demonstrated preliminarily the availability of the library. It is useful for application of phage display technique in thrombosis and hemastasis.