Anti-inflammatory effect of fusion protein anticoagulant dual function of TAP-SSL5
|School||Third Military Medical University|
|Keywords||Atherosclerosis Inflammation Anticoagulant Tick ??anticoagulant peptide Staphylococcal superantigen -like protein -5 P-selectin glycoprotein ligand -1 Clotting factor Xa|
Background and Objective: atherosclerosis (atherosclerosis, AS) is a chronic inflammatory process, inflammatory reaction in atherosclerotic lesions occur, plays a key role in the development process; coagulation system and platelet activation were also involved, further promotes atherosclerosis and thrombotic complications and development. In this study, one kind with the granulocyte cell surface glycoprotein P-selectin ligand -1 (P-selectin glycoprotein ligand-1, PSGL-1) binding and inhibit \staphylococcal superantigen-like protein-5, Staphylococcus aureus superantigens like protein -5), and functions as a guide molecule and inhibit the coagulation factor Xa (factor Xa, FXa) active molecules - tick anticoagulant peptide (tick anticoagulant peptide, TAP) fusion, prepared by the recombinant fusion protein TAP-SSL5. By combining the anti-inflammatory effect and TAP SSL5 anticoagulant effect combined with a period of atherosclerosis and its prevention and treatment of thromboembolic complications provide a new method. Methods 1, obtained by PCR technique SSL5, TAP gene was cloned into the pET32a expression vector, and synthesized by Gene Synthesis TAP-SSL5 recombinant fusion protein gene was cloned into pET22b expression vector and, in E. coli BL21 transfect (DE3) for expression, the final application of ion exchange chromatography, or metal ion affinity chromatography for separation and purification. 2, in vitro experiments parts: the fusion protein was detected by flow cytometry in human TAP-SSL5 surface binding of granulocytes, and the mouse anti-human PSGL-1 antibody (KPL-1-PE) and granulocyte binding competition inhibition; with calcein-AM labeled HL60 cells or neutrophils, TAP-SSL5 detect the marked cells to P-selectin (P-selectin) adhesion of coated surface; FXa inhibition activity using the chromogenic substrate color method, in S2765 as substrate, TAP-SSL5 detect the inhibition of FXa activity; both viper venom factor X activator (RVV-X) activates human or mouse plasma FX, further testing of its FXa activity in plasma inhibition. 3, part animal experiments: the use of ferric chloride-induced rat model of inferior vena cava thrombosis, research TAP-SSL5 antithrombotic effect; using rat carotid artery balloon injury model, giving TAP-SSL5 (3 mg / kg / d, ip) three weeks, the control group was given normal saline, to understand TAP-SSL5 on neointimal formation after vascular injury effects; for the study of TAP-SSL5 on atherosclerotic plaque formation, selecting 12 weeks old male apoE knockout (apoE-/ -) mice, using high-fat diet, and while giving TAP-SSL5 (3mg/kg/d, ip), given normal saline control group, n = 10, 12 weeks later, mice aorta, detect AS plaque formation, and through the vessel wall protein microarray expression levels of inflammatory cytokines. Results: 1, TAP-SSL5 with granulocytes and HL60 cell binding, and competitive inhibition of the binding KPL-1; 30μg/ml TAP-SSL5 inhibit human neutrophils and HL60 cells in the surface of the P-selectin adhesion, inhibition rates were 67.9% and 81.6% (p lt; 0.001); TAP-SSL5 on FXa concentration-dependent inhibition of the activity of a final concentration of 3μM of TAP-SSL5 can produce significant for FXa inhibition, using purified FXa, mouse plasma, human plasma detected inhibition rates were 93.7%, 75.5%, and 94.7% (p lt; 0.001). 2, a single intravenous injection of 3mg/kg of TAP-SSL5, can significantly inhibit the FeCl3-induced inferior vena cava thrombosis, inhibition rate of 52.7% (p lt; 0.001); TAP-SSL5 can significantly inhibit rat carotid artery neointimal formation after injury, compared with the control group, neointimal area can reduce 32.0% (p lt; 0.05), the intima / media thickness ratio (intima-media thickness ratio) decreased by 41.9% ( p lt; 0.001); TAP-SSL5 can effectively inhibit the apoE-/ - mice AS plaque formation and inhibit murine aortic wall in a variety of inflammatory cytokine expression. CONCLUSION: The recombinant fusion protein TAP-SSL5 inhibit neutrophil surface of the P-selectin adhesion, and inhibition of FXa activity; experiments further confirmed TAP-SSL5 inhibit thrombus formation, inhibition of arterial neointimal formation after vascular injury and hyperlipidemia Feeding apoE-/ - mice AS plaque formation, these effects were associated with TAP-SSL5 possess anticoagulant and anti-inflammatory activity of the recombinant fusion protein TAP-SSL5 is expected to become the treatment of vascular injury and thrombotic diseases biological products class of drugs.