Prokaryotic Expression And Activity Research Of Vasotab Ty,a Antithrombosis Bioactive Proteins From Salivary Gland Of The Horsefly, Tabanus Yao Macquart

Thrombotic disease (TD) imperil human health seriously as a usual clinical disease.It is the leading cause of human death and disability in the world.In recent years, various therapeutic treatment of thromboembolism complication such as anti-clotting, anti-platelet, and other anti-thrombosis agents emerged with the continuous increase of TD incidence.However, more or less side effects are present in each of them.Therefore, it’s essential to hunt for a high quality thrombolytic agent. Under the long-term environmental pressure, haematophagous arthropods have evolved perfect molecular mechanisms to get a successful blood meal.These mechanisms of interaction between blood-sucking arthropods and vertebrate host are hot spots of international research, which can find lots of potential medical leading molecules.By proteomics or peptidomics and coupling transcriptome analysis with pharmacological testing, the whole length sequence of Vasotab TY, a inhibitor of platelet aggregation,was found by our lab. Then Vsotab TY was expressed through pET prokaryotic expression system in Escherichia coli BL21(DE3). Separated and purified by nickel affinity chromatography, formic acid digestion, Sephadex G-50gel chromatography and reversed-phase highperformance liquid chromatography (RP-HPLC) and so forth, tracked by protein SDS-PAGE and platelet aggregation inhibitory activity analysis, pure Vasotab TY with platelet aggregation inhibitory activity was produced.We studied the anti-thrombosis activity in vivo, such as bleeding time which assessed by a tail transection method, carrageenan-induced mouse-tail thrombosis model, rat arteriovenous shunt thrombosis model, and confirmed that Vsotab TY including the KGD domain is a novel receptor antagonist of platelet glycoprotein Ⅱb/IIIa (GPⅡb/Ⅲa) and displays potent antithrombotic properties. In addition, we studied the bleeding side effect of Vasotab TY and found that Vasotab TY didn’t cause bleeding under the concentration of150μg/kg. This progress relieve the restriction of the resources poverty. More important, our research provide a substantial foundation to the excavation for anti-thombosis leading molecular, the industrial exploitation and the clinical application of the traditional Eastern medicine insect material.