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Fusarium plasmin and preparation method thereof
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A Fusarium and fibrinolytic enzyme technology, applied in the field of biopharmaceuticals, to achieve the effect of small molecular weight, good thrombolysis and low cost
Inactive Publication Date: 2012-09-05
SHANGHAI LAIYI BIOMEDICAL RES & DEV CENT +1
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Problems solved by technology
In recent years, many microorganisms capable of producing plasmin have been continuously screened, but there is no report on the use of Fusarium chrysanthemum stem to isolate and prepare plasmin
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Embodiment 1
[0031] Example 1 , Fusarium plasmin preparation
[0032] Follow the steps below to prepare Fusarium plasmin:
[0033] 1. Put the chrysanthemum stem endophytic fungus Fusarium strain (preservation number CGMCC No.2347) on the PDA slant medium, cultivate it at 28°C for 4-5 days, then inoculate it on the seed medium, cultivate it at 28°C for 2 days, then press 10% of the inoculum is inoculated in the fermentation medium, cultured at 28°C for 6 days, and the culture is centrifuged or filtered to obtain the fermentation broth.
[0034] 2. Add ammonium sulfate to the obtained fermentation broth to 40% saturation, stand at 4°C for 4 hours, centrifuge to obtain a supernatant, continue to add ammonium sulfate to 60% saturation, stand at 4°C for 4 hours, and centrifuge to obtain protein precipitation.
[0035] 3. Dissolve the obtained precipitate with a small amount of Tris-HCl buffer solution (pH 7.4), collect the permeate through a 0.1micron microfiltration membrane, and then ultra...
Embodiment 2
[0039] Example 2 , Fusarium plasmin identification
[0040] Get the obtained Fusarium plasmin HCCB00699S1 in embodiment 1 and carry out following detection:
[0041] 2.1. In the study of the activity characteristics of Fusarium plasminase, the plate method is mainly used, specifically: in a 9cm-diameter dish, first add 5ml of 0.5% fibrinogen solution, and then add 100μl (5U / ml) thrombin 5ml of 1% agarose solution, shake quickly, and let stand at room temperature for 2h. Put 10 μl of Fusarium plasmin or control solution (blank fermentation medium and Tris-HCl buffer) on the fibrin plate with a micro-injector, keep it warm at 37°C for 18 hours, take it out, and measure the diameter of the lytic ring with a caliper . Take the square of the diameter of the dissolved circle as the ordinate, and the concentration of the standard urokinase as the abscissa to draw a graph, and the unit of activity of the sample can be found on the graph. The result is as figure 1 shown.
[0042...
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Abstract
The invention discloses fusarium plasmin and a preparation method thereof. The fusarium plasmin is prepared by fermenting fusarium sp. An N-terminal amino acid residue sequence of the fusarium plasmin is QASSGTPATIRVLVV, the molecular weight is 28, 000 Da, and the isoelectric point is 8.1. The fusarium plasmin has the fibrinolytic activity which can directly degrade fibrin, has the effects of good thromboclasis and remarkable inhibition on the formation of venous thrombus, and has smaller molecular weight; at the same time, the fusarium plasmin has the advantages of rich production raw materials, simple and convenient production technology, and low cost, and can remedy defects of the prior clinical medicaments such as urokinase on the production and treatment, so the fusarium plasmin has development value.
Description
technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a Fusarium plasminase and a preparation method thereof. Background technique [0002] Thrombosis is the narrowing and occlusion of the vascular lumen due to thrombus, and is a disease in which ischemia and infarction of major organs cause dysfunction. Thrombosis belongs to cardiovascular and cerebrovascular diseases. According to statistics, cerebral thrombosis, cerebral infarction, myocardial infarction, coronary heart disease, arteriosclerosis and other cardiovascular and cerebrovascular diseases take the lives of 12 million people every year, which is close to 1 / 4 of the total death toll in the world. Become the number one enemy of human health. In my country, more than 2.6 million people die from cardiovascular and cerebrovascular diseases every year, and 75% of the surviving patients are disabled. Although thrombosis is related to blood vessels and m...
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