Preparation Of Human Thrombin And Fibrinogen

This paper has recited the properties and application of human fibrinogen concentrate and thrombin ,and summarized the progress in research and preparation in the world of human f ibrinogen concentrate and thrombin. It introduced the function mechanism of human fibrinogen concentrate and thrombin, and effective factor of content or activity. Having done a great deal of experiments, we get the optimum processing conditions of that human fibrinogen concentrate preparated from cohn fraction I by cryoprecipitation method and human thrombin was preparated from cohn fraction III by Ion Exchange Chromatography. S/D treatment was incorporated into the scheme to inactivate lipid-enveloped viruses.In the research, the monofactor method was used to determine the proper range of each factor , then the mult if actor orthogonalizing design method was used to find the optimum processing conditions of preparation of human fibrinogen concentrate and thrombin. Through the experiments, the optimum processing conditions like these were got.(1) Preparation of human fibrinogen concentrate : The fibrinogen-rich cohn fraction I was solubilized in a tris/citrate/lysine buffer, pH6.8, and concentrated to 10-20 protein g/l. The solution was clarified using a 1um filter, and treated with a mixture of 0. 3% and 1% Tween 80 at 25C for 8h. The solution was subject to two-step 10% ethanol precipitation .The precipitation was solubilized in a tris/NaCl buffer, pH7.0, at 30C.(2)Preparation of human thrombin:The PCC-rich cohn fraction III precipitation was solubilized in a 0.06mol/l tris-HCl buffer, pH7. 5. The solution was subjected to 3% PEG6000, and soya bean lecithoid was used to activate prothrombin. Then S/D treatmnt was incorporated into inactivate virus. Thrombin was further purified with CM ion-exchange liquid chromatographic column. The gel was washed with a buffer containing 0. 06mol/l tris-HCl, pH7. 5 , and thrombin was eluted with a buffer containing 0.06mol/l tris-HCl, 1% NaCl, pH7. 5 at 3. 8ml/minProtein determinations showed that clottability of f ibrinogen was over 85% and thrombin specific activity was over 70 U/ml.The creativeness of this research is: Soya bean lecithoid was used to activate prothrombin that was preparated from cohn fraction III precipitation. Thrombin was further purified with CM biosep FF (made in china) using one-step ion-exchange liquid chromatography. This process has never been reported at home, it is initiate in our country.The significance of this research is: cohn fraction I and fraction III precipitation are discarded by most of blood product companies in our country at present. Fibrinogen and thrombin preparated in these processes have applied widely in surgery now. These process can raise the economic consideration of these businesses and fit in with our national condition.CHENG Wen-kang (chemical engineering) Directed by ZHANG Xiao-yan , GUO Li-an...