| The thrombotic diseases are the leading death cause of human being and extend to human health threatening effects. Formation of blood clot (thrombus) can cause embolism in the blood circulatory system of brain, heart, lung and so forth, which subsequently results in coronary heart disease, cerebral thrombosis, arteriosclerosis and other diseases. The thrombotic diseases have always been the difficulty of the medicine area because of the high rate of disability and death. Also, as thrombotic diseases are age-related, the prevention and treatment of it are becoming more and more urgent with the increasingly serious aging problem in China.Thrombolysis is one of the most effective and widely used methods in treating and preventing the thrombotic diseases, through using thrombolytic drugs to achieve blood clot lysis. The main thrombolytic drugs in clinical application so far are urokinase (UK), streptokinase (SK), and tissue-type plasminogen activator (t-PA), etc. Although those drugs have obvious thrombolytic effect, there are shortcomings like short half-time, high price and so on. It is urgent to discover an efficient, safe, economic thrombolytic drug. Fibrinolytic enzymes occur ubiquitously in microbes, plants and animals. Microbes as the main source of thromolytic agents, have lots of advantages including culturing readily, fermentation products of high yield and requiring simple equipments. It has always been the focus of the research and development of thrombolytic drugs.Based on the preliminary work of our laboratory, isolation and purification of fibrinolytic enzymes were carried out from Bacillus subtilis fermentation broth. To obtain the pure fibrinolytic enzymes from Bacillus subtilis, a two-step purification procedure was established including ammonium sulfate precipitation, ion exchange chromatography. The stability and substrate-specificity properties were investiagted toward the purified fibrinolytic enzyme. And then the preliminary fibrinolytic enzyme activity in vivo was evaluated as well. The followings are the main results:1. Pure enzyme preparation:The enzymes were salted-out from fermentation broth with 60% saturated ammonium sulfate.Protein precipitate was collected by centrifugation. After dissolving, the resulting sample was dialyzed agaisnt the acetate buffer solution (0.02 M pH 5.6), and then the sample was further purified by cation exchange chromatography to obtain the pure enzyme products. In the purification process, purity and activity of thrombolytic enzyme was evaluated by SDS-PAGE and enzymatic activity assay.2. Investigation of stability and substrate-specificity:The fibrinolytic enzyme stability in the solution state at different temperature(4℃、37℃) was examined. It turned out that enzyme retained 90% activity in the 9-day’s degradation experiment under the condition of 4℃. While the enzymatic activity decreased rapidly in the first 3 days and the enzymatic activity could not be detected 5 days later under the condition of 37℃. It showed that it has well stability under the condition of 4℃. In addition, it is found that fibrinolytic enzyme posseses the direct fibrinolytic activity based on the "heating" fibrin plates experiment.3. Preliminary study on the fibrinolytic activity in vivo:The results demonstrated that the enzyme is high-performance in blood clot lysis. After intravenous injection, the animal blood was taken forty-minutes later. It is observed that after the blood coagulation, drug injection group was still capable of dissolving thrombus. Erythrocytes were morphology integrity under the microscope. Also, the fibrinolytic activity and enzyme concentration were correlated well. |
PDF Full Text Request