| Thrombotic diseases are diseases caused by two pathological changes such as thrombosis and embolism.In recent years,the incidence of thrombotic diseases has increased rapidly worldwide,and the disability rate and mortality rate are high,which seriously endange the healthy of humans.In the past ten years,thrombolytic drugs have been widely used in the treatment of thrombotic diseases.Based on the thrombolytic mechanism of these drugs,they can be divided into two types: one is plasminogen activator,and the other is plasminogen activator.The species is a plasmin-like protein.However,although these thrombolytic drugs play a role in the prevention and treatment of thrombosis,and are widely used,there are disadvantages that cannot be ignored: excessive fibrinolysis causes body bleeding,and fibrin substrate specificity is low.And expensive and so on.Therefore,finding safer,more effective and cheaper thrombolytic drugs from various sources remains an urgent problem to be solved.In the past decade of research,new thrombolytic agents of microbial origin have shown good characteristics and have gradually attracted the interest of researchers.Such as nattokinase.In recent years,there have been reports of higher fibrinolytic active proteins in metabolites from fungi,such as Aspergillus ochraceus,Fusarium oxysporum etal.Endophytic fungi are a special and important group,and there are few reports on endophytic fungi and thrombolytic drugs.In this study,a strain of high-yield fibrinolytic enzyme was screened from endophytic fungi,and its enzymatic properties were explored.New strain resources were provided for the discovery of novel plasmin for the development of thrombolytic drugs.A total of 486 plant endophytes were isolated and purified from the collected plant samples.The skim milk powder plate was used as the primary screening model.The protease-producing strains were first screened.The fibrin plate was used as the rescreening model to further screen for the production of fibrinolytic enzyme.The strain was finally screened from 486 strains of endophytes to obtain a strain with high production of fibrinolytic enzyme.The combination of morphological and molecular biological identification methods was used to identify the strain producing as Myrothecium roridum.The method of purifying various proteins is combined,and the fibrinolytic enzyme is separated and purified from the crude fermentation extract of the strain,and finally the purification process is: fermentation crude extract→ammonium sulfate primary precipitation→dialysis→anion exchange chromatography→cation exchange layer Analysis → Native-PAGE.The conditions of each of the purification steps were optimized.The final purification conditions were as follows: the optimum interval for primary precipitation of ammonium sulfate was 50%~70%;the dialysis desalting buffer was 0.1M,pH(7.2~7.4)Tris-HCl;the anion exchange chromatography: the filler was DEAE-Sepharose FF,The pH of the buffer system is 6.5;cation exchange chromatography: the packing is Sepharose-6FF,the pH of the buffer system is 4.5;the purity of plasmin is further improved by Native-PAGE electrophoresis,and the protein spectrum is identified by gel extraction.As a result,it was analyzed that the enzyme of interest belongs to the peptidase M36 family.Explore the enzymatic properties of this plasmin.The molecular weight of plasmin was estimated to be about 30 kDa by SDS-PAGE protein electrophoresis.The effect of temperature,pH,metal ions and protease inhibitors on the activity of the enzyme was determined by the method of detecting the activity of fibrin plate.The optimum temperature of the enzyme is about 40°C,Moreover,it can maintain high enzyme activity after incubation at 37°C for more than 4h.It is speculated that the enzyme can adapt to the body temperature environment of the human body;the enzyme can maintain a certain activity between pH 3 and 11,and has a wide range of activities pH adaptability;metal ions have no obvious effect on the activity of the enzyme.It is speculated that the enzyme can adapt to the complex ion environment of the human body;the activity of the enzyme and the SDS-PAGE protein electrophoresis method,the kinase activity and substrate of the enzyme The nature is explored,and it is finally determined that the enzyme can directly hydrolyze fibrin to play a thrombolytic effect;the enzyme has a hydrolysis effect on fibrinogen,which is presumed to prevent thrombus formation.By simulating the experiment of gastric juice environment in vitro,it was further confirmed that the plasmin could maintain certain activity under the environment of lower pH(3~5);blood clots were prepared by collecting venous blood from rats,and the thrombolytic test was simulated in vitro to further verify The plasmin has the ability to dissolve thrombus.At the same time,the effect of enzyme on the morphology of vascular endothelial cells(HUVEC)was observed.The results showed that the cell morphology of HUVEC was significantly affected by the dose of plasmin.Variety.The above experiment provides a data reference for further investigation of the plasmin.In summary,this experiment investigated the screening of a strain of high-yield fibrinolytic enzyme from the endophytic fungi of plants.The strain was identified as Myrothecium roridum.The suitable conditions for the separation and purification of the plasmin has been explored;the enzymatic properties were explored,and the enzyme was able to adapt to the human body temperature environment(37 ° C tolerance)and complex ion environment(metal ions have no significant effect on the activity of the enzyme)The plasmin can exert a thrombolytic effect by directly degrading fibrin,having the activity of hydrolyzing fibrinogen,and the enzyme has a wide pH adaptability(pH 3 to 11),and it is speculated that the plasmin has the potential developed into oral and intravenous prevention of thrombolytic drugs;In addition,in vitro simulated thrombolytic assays and cytotoxicity experiments provide some basic data for further pharmacodynamic studies of plasmin. |
