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Fibrinolytic active protein, preparation method and application thereof, pharmaceutical composition and nucleic acid encoding protein

A fibrinolytic and active protein technology, which is applied in the field of fibrinolytic active protein and its preparation method and application, as well as in the field of pharmaceutical compositions, can solve the problem of large individual differences in dosage, poor specificity, easy to cause bleeding, easy occurrence of gastric mucosal damage and Gastrointestinal bleeding and other problems, to achieve the effect of strong fibrinolytic activity and high specific activity

Pending Publication Date: 2021-08-10
BEIJING UNIV OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aspirin is often used as an antiplatelet drug, and its side effects are prone to gastric mucosal damage and gastrointestinal bleeding; warfarin is often used as an anticoagulant drug, but its use is limited due to the large individual differences in dose and the need for real-time monitoring; thrombolysis Drug streptokinase and urokinase can easily lead to serious adverse reactions such as bleeding due to poor specificity

Method used

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  • Fibrinolytic active protein, preparation method and application thereof, pharmaceutical composition and nucleic acid encoding protein
  • Fibrinolytic active protein, preparation method and application thereof, pharmaceutical composition and nucleic acid encoding protein
  • Fibrinolytic active protein, preparation method and application thereof, pharmaceutical composition and nucleic acid encoding protein

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preparation example Construction

[0040] According to the second aspect of the present invention, the fibrinolytic active protein provided by the present invention can be obtained by separation, purification and artificial synthesis, or its coding gene can be synthesized first, and then obtained by biological expression. The present invention provides a fibrinolytic active protein A preparation method for dissolving active protein, the method comprising the following steps:

[0041] (1) Add earthworm homogenate to deionized water, adjust the pH to 6-9, separate to obtain the first solution, and separate the first solution to obtain the second solution by at least one ethanol precipitation; the second solution is separated by at least one ethanol precipitation and separation obtain the first extract;

[0042] (2) Separating and purifying the first extract by first column chromatography, replacing the solvent in the first eluent with deionized water after collecting the first eluent, desalting and concentrating ...

Embodiment 1

[0076] (1) Homogenize fresh Earthworm (frozen) to obtain Earthworm slurry, weigh the Earthworm slurry, add 2 times the weight of deionized water to extract, adjust the pH value to 9, stir for 15 minutes, and centrifuge to collect the supernatant; Add absolute ethanol to the supernatant until the ethanol concentration is 70%, and refrigerate in a refrigerator at 4°C for 12 hours; collect the supernatant by centrifugation, add absolute ethanol to the supernatant until the ethanol concentration is 78%, and refrigerate in a refrigerator at 4°C for 12 hours Collect the precipitate by centrifugation, add an appropriate amount of deionized water to dissolve the precipitate, and freeze-dry the solution to obtain the initial extract of earthworm fibrinolytic active protein;

[0077] (2) Ion exchange method was used to separate and purify the fibrinolytic activity protein primary extract of Dilonga: the NGC protein chromatography system was used as the separation and purification device,...

Embodiment 2

[0080] (1) Homogenize fresh Earthworm (frozen) to obtain Earthworm slurry, weigh the Earthworm slurry, add 6 times the weight of deionized water to extract, adjust the pH value to 6, stir for 15 minutes, and centrifuge to collect the supernatant; Add absolute ethanol to the supernatant until the ethanol concentration is 75%, and refrigerate in a refrigerator at 4°C for 12 hours; collect the supernatant by centrifugation, add absolute ethanol to the supernatant until the ethanol concentration is 85%, and refrigerate in a refrigerator at 4°C for 12 hours Collect the precipitate by centrifugation, add an appropriate amount of deionized water to dissolve the precipitate, and freeze-dry the solution to obtain the initial extract of earthworm fibrinolytic active protein;

[0081] (2) Ion exchange method was used to separate and purify the initial extract of Dilong fibrinolytic active protein; the NGC protein chromatography system was used as the separation and purification device, an...

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Abstract

The invention relates to the technical field of biology, in particular to a fibrinolytic active protein which has any one of amino acid sequences shown as (I), (II) and (III): (I) having an amino acid sequence shown as SEQ ID NO.1, (II) having an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO.1; (III) an amino acid sequence having at least 90% consistency with the amino acid sequence as shown in SEQ ID NO.1; the fibrinolytic active protein has direct fibrinolytic activity, can activate plasminogen, further enhances the fibrinolytic activity of the fibrinolytic active protein, has a strong dissolving effect on in-vitro thrombus, and can be applied to preparation of drugs for treating thrombotic diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fibrinolytic active protein, a preparation method and application thereof, and a pharmaceutical composition. Background technique [0002] Many diseases that seriously endanger human health, such as myocardial infarction, ischemic stroke, and disseminated intravascular coagulation, are all related to the formation of thrombus. Prevention and treatment of thrombosis usually takes measures from the steps of promoting fibrinolytic activity, reducing platelet function, protecting vascular endothelium, and inhibiting blood coagulation. There are certain deficiencies in the clinical use of corresponding drugs. Aspirin is often used as an antiplatelet drug, and its side effects are prone to gastric mucosal damage and gastrointestinal bleeding; warfarin is often used as an anticoagulant drug, but its use is limited due to the large individual differences in dose and the need for real-time ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/68A61K38/48A61P7/02
CPCC12N9/6408A61P7/02C12Y304/21007A61K38/00
Inventor 杜守颖刘海杨万青吴娅丽马韫楠汪文杰钟宛凌李鹏跃
Owner BEIJING UNIV OF CHINESE MEDICINE
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