Cloning Of Lectin-EGF Domain Of Human P-selectin And Screening Its Specific ScFv Antibody

P-selectin(CD62P. PADGEM. GMP140)is a member of selectin's family of cell adhesion molecules, mainly involves in the process of white cell-endothelium cell initial adhesion, closely related to immune injury, inflamation, thrombus formation and lymphocyte recycling.Totol RNA was extracted from human platelets, P-selectin cDNA was synthesized by RT-PCR. Lectin-EGF domain gene was amplified with PCR, The genetically engineered bacterium for human P-selectin's Lectin-EGF domain was successfully constructed which is verified by restriction enzyme cutting , PCR and sequence analyzing. And then cloned into E.coli DH5 a . human P-selectin's Lectin-EGF protein fragments were then produced as inclusion bodies. After denaturalization and renaturalization, it was bound to a 3 cm dish in a series of concentrations, and anti-actived human platlet phage display ScFv library was used to a four round panning for specific ScFv antibody against human P-selectin's Lectin-EGF domain. Three positive clone was panned out, which is verified by PCR and sequence analyzing. And ScFv was produced by pET20b(+) system which provided a basia for further work.