Establishment Of Technical Platform For Thrombolytic Drugs Of Enzymic Nature And Quality Analysis Of Product

With the rapid expansion of thrombolytic drugs market, the natural thrombolytic agent has occupied nearly 70% market share, most of which are enzymes. Thrombolytic agent of enzymic nature is playing a more and more important role. For development and utilization better, it is urgent to need a platform of technical methods and ideas provided for acquiring such active ingredients, so that we can extract valuable ingredients of thrombolysis from natural organisms efficiently and rapidly. The main purpose of this study is to build a technical platform, from pretreatment of raw materials, choice of methods and technologies, optimization of condition, to the identify and quality analysis of active components ,which forms a series of technical methods, in order to provide reference for purification and detection of the other thrombolytic drugs from the similar source, thus it will be more beneficial to large-scale,efficient and rapid isolation and extraction.The complexity of marine environment makes marine life with a variety of bioactive substances, which have the advantages of novel structure, unique,function, low molecular weight, strong activity and have great potential to be explored. Nereis Virens protease found from the marine life-Nereis Virens, has a strong effect of fibrinolysis and the potential role of decomposing thrombus. The thrombolytic components in Nereis Virens had been researched, and the result of research showed that Nereis Virens protease has strong and specific thrombolytic activity, small molecular weight, short time of role, no bleeding and is easy to be controlled. Nereis Virens protease is a kind of promising and novel thrombolytic enzymes.Nereis Virens protease as a new active substance, is crucial to have low-cost, high yield technology.of extraction and purification.Therefore, this study took isolation and extraction of a single ingredient of Nereis Virens protease for example, which elaborates the construction of the technical platform for thrombolytic drugs of enzymic nature. We hope that Nereis Virens protease could be developed into drug for injection by using reasonable and effective purification technology ,so that we would carry out large-scale industrial production.Before carrying out purification work ,we first used the method of non-denaturing polyacrylamide gel electrophoresis for preparing and separating Nereis Virens coelomic fluid ,then the activity of isolated bands were detected separately by fibrin assay plate. Nereis Virens protease having three active components, in order to further purify the isozyme, acquire a single component, then develop into high pure agent for injection, We chose the second component with relatively high content among the three active components as target protein, then we researched on the basic characterization of target component . The results showed that the molecular weight of the component was about 30KD and the isoelectric point was around 4.5.We apply the method of ammonium sulfate precipitation to extract the stock suspension of Nereis Virens crudely, trying to precipitate by different concentrations of ammonium sulfate, Lastly we select 40% saturation ammonium sulfate to prepare crude extract of Nereis Virens. Then, according to the characterization of target protein, we used different principle chromatography for the refined separation. First of all, according to different hydrophobicity, We selected hydrophobic interaction chromatography to isolate the sample after salting-out, and determined that 1.0mol / L (NH4)2SO4—phosphate buffer solution is the best salt concentration for bonding. Secondly, We used DEAE anion exchange chromatography to purify the sample according to different protein molecules having different charge. In order to obtain better results, we groped the optimal pH of buffer according to the isoelectric point of target protein, then adopted gradient elution and stage elution to identify salt concentration of elution and determined to choose 100mMNaCl-phosphate buffer (0.02M, pH = 7.5) to elute. Finally, we obtained a single component. of Nereis Virens protease by using gel filtration chromatography according to different molecular weight. After the whole process, the recovery of the single component of Nereis Virens protease was 35% and the activity increased by 246 times.By a variety of combination of purification technologies, we isolated a single active component from coelomic fluid of Nereis Virens. The purity of Nereis Virens protease had been identified with UV - visible absorption spectroscopy, Native-PAGE, SDS-PAGE, HPLC and a kind of improved cellulose acetate electrophoresis according to different principles. The results show that the prepared single component product of Nereis Virens protease , A280 / A260 = 2.091 > 1.8, didn't be contaminated by nucleic acid. Native-PAGE, SDA-PAGE, and the improved cellulose acetate electrophoresis displayed a single band and no other impurity; HPLC- gel column is a peak and peak area 97.55%. So the purity meet the requirements of Pharmacopoeia.In the methods of purity testing, we adopted an innovative electrophoresis and improved the traditional cellulose acetate electrophoresis creatively, using neutral buffer system of pH = 7.0, and loading sample in the middle of film. So we can observe the acidic protein and basic protein in the sample simultaneously by this kind of electrophoresis, which is direct-viewing and easy, simple equipment, and lower cost, especially for real-time detecting separation of sample , removal of impurity during the purification process , evaluating and guiding us to choose better methods and conditionsThe concentration of Nereis Virens protease product was measured by UV absorption difference method, Brad-ford and Kjeldahl Nitrogen Determination method. The result was 0.434167mg/ml, 0.41753 mg / ml, 0.4206mg/ml respectively, and the results were nearly, so we could determine the concentration of our product by the three methods above. The titer of Nereis Virens protease product is equivalent to 27 241 U(urokinase unit) per mg.so the product with higher activity have the potentiality of being developed into a kind of novel thrombolytic drug for treating and prevention of thrombotic disease. In order to make products of Nereis Virens protease easy to be preserved and transported, we groped the best amount of protective agent for freeze-drying process through experimental comparison, then we concluded that the rate of dextran and protein was 1:100 (W / W).The content of this research is rich ,having innovation in some degree and having great value of potential applications. The construction of technical platform combinated different methods of isolation and purification, and solved the problems successfully in the process of separation and extraction. The target ingredient was isolated precisely with high purity, which not only provides theoretical and technical support for separating the similar thrombolytic agents of enzymic nature, but also establishes solid foundation for Nereis Virens protease injection transforming into industrial production, with good prospects.