Venom Separation And Purification Of Recombinant Thrombin-like And The Preparation Of Affinity Ligands

Thrombin-like enzymes belong to the snake venom serine protease. It can convert fibrinogen into fibrin. The hydrolyzed product-fibrin can form a fibrin microclot, which isn't cross-linked and is susceptible to degration by plasmin, leading to a consequent decrease in blood viscosity. So the thrombin-like enzymes have been used extensively in clinical medicine to cure and prevent thrombotic and myocardial infraction diease.At the base of the study of the thrombin-like enzyme gloshedobin, the work of this thesis has been expanded from three ways.(1) The gene of gloshedobin was cloned into the vector pET 32a (+), strain E.coli BL21 (DE3), and the expression products were distributed in cytoplasm. The recombinant gloshedobin in the supernant was separated and purified by followed two combinations of chromatographic methods. One is a combination of immobilized Ni2+ affinity chromatography, hydrophobic interaction chromatography on Phenyl Sepharose FF, anion exchange chromatography on Source 15Q, which achieved the purity of 88%, the product recovery of 37.9% and the purification fold of 26. The other is a combination of immobilized Ni2+ affinity chromatography, weak anion exchange chromatography on DEAE Sepharose FF, strong anion exchange chromatography on Source 15Q, which achieved the purity of 75%, the product recovery of 7.3% and the purification fold of 15.(2) The egg yolk antibodies was collected by the precipitation of PEG-6000, which got the total IgY of 4mg/ml. And the specific IgY was purified by immunoaffinity chromatography column, resulting in the purification fold of 200. These recombinant gloshedobin were purified by a protocol involving Octyl Sepharose FF, egg yolk antibody (IgY)-immunoaffinity chromatography and Source Q, resulting in the purity of 80%, 34.8% activity yield and the purification fold of 29.5.(3) The purification of thrombin-like enzyme from crude snake venom was accomplished by a combined chromatography including cation exchange chromatography on SP Sepharose FF, gel filtration chromatography on Superdex G-75 and anion exchange chromatography on Source 15Q. And finally two single fraction, enzyme I and enzyme II, which contained the fibrinogenolytic activity and arginine esterase activity. The enzymatic characteristics were assayed. The molecular mass of enzyme I was 34kDa, and the value of Km and Vmax were3.4 10-4M and6.26 10-4mM/s respectively. The molecular mass of enzyme II was 38kDa, andthe value of Km and Vmax were 5.7 10-4M and 7.36 10-4mM/s respectively. By Comparative estimation of the inhibitory efficiency of five different inhibitor to two kinds of enzyme, for enzyme I , 4-aminobenzamidine was the most strong inhibitor, and guanidine hydrochloric was the most weak inhibitor, which were the same to enzyme II.