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Cordyceps militaris fibrinoclase as well as preparation method and application thereof

A technology of fibrinolysis and Cordyceps militaris, applied in the field of bioengineering, can solve problems such as retention, and achieve the effects of improving production efficiency, shortening fermentation time, and good thrombolytic activity

Active Publication Date: 2019-04-26
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on the fibrinolytic enzyme of Cordyceps militaris still stays in the downstream stage of biological technology at present, and the gene research on the fibrinolytic enzyme has not been reported yet. The present invention utilizes molecular biological means to amplify the gene sequence encoding the fibrinolytic enzyme

Method used

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  • Cordyceps militaris fibrinoclase as well as preparation method and application thereof
  • Cordyceps militaris fibrinoclase as well as preparation method and application thereof
  • Cordyceps militaris fibrinoclase as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Screening of high-enzyme live Cordyceps militaris strains

[0033] Activation of 10 strains of Cordyceps militaris was carried out using solid plate medium, and a piece was cut from the slope and placed on a solid improved PDA plate (add 2% peptone, 0.3% KH on the basis of ordinary PDA solid medium) 2 PO 4 , 0.15% MgSO 4 , 0.002% VB 1 ) center, cultured at 24°C in the dark for 10 days, the mycelium covered the plate and did not turn color. Liquid culture fermentation was carried out on the activated 10 strains of Cordyceps militaris.

[0034] The culture conditions are: put 50mL culture medium in a 250mL Erlenmeyer flask, add a piece of bacteria block with a side length of about 1cm, and culture in a shaking flask at 24°C and 150r / min in the dark. From the 5th day, sample 1 mL every day, centrifuge at 4°C and 10,000 r / min for 5 minutes, take the supernatant as a crude enzyme solution for enzyme activity detection, and monitor for 20 days. Finally, consider...

Embodiment 2

[0035] Example 2: Identification of Cordyceps militaris Fibrinolytic Enzyme

[0036] (1) N-terminal sequencing of plasmin: take the high-enzyme active Cordyceps militaris strain obtained in Example 1 as the research object, carry out liquid fermentation on it and separate and purify the crude enzyme solution, and electrophoresis the protein samples according to Table 2 The method runs the gel, and transfers the protein to the PVDF membrane after the end. Before transfer, soak gel and filter paper of appropriate size in CAPS buffer for 5-10min. PVDF membranes were soaked in methanol for a few seconds and then also placed in CAPS electroblotting buffer. During installation, filter paper, PVDF membrane, glue, and filter paper are followed from bottom to top to drive out air bubbles.

[0037] The semi-dry method was adopted, and the transfer conditions were: 25V constant voltage, 1A, 60min. After the transfer was completed, the PVDF membrane was stained with Coomassie Brilliant...

Embodiment 3

[0045] Example 3: Induced expression and separation and purification of recombinant Cordyceps militaris fibrinolytic enzyme

[0046] E. coli cells containing the recombinant plasmid were cultured in 250 mL shake flasks containing 50 mL of LB broth supplemented with 100 μg / mL ampicillin and grown at 37 °C and 200 rpm until reaching an optical density of 0.6 at 600 nm. The culture was then added to 50 μg / mL IPTG to induce enzyme production, and culture was continued at 20 °C for 20 h. After incubation, the culture was centrifuged at 10000 rpm for 10 min at 4°C. Cells were then resuspended in cell culture medium. Lysis buffer (50mM NaH 2 PO 4 , 300mM NaCl, 10mM Tris, 20mM imidazole, pH 8.0) and disrupted by sonication. The crude extract was then centrifuged at 10000 rpm for 20 min at 4°C. Since the C-terminus of the pEASY Blunt E2 vector has a 6×His tag, nickel column affinity chromatography was used to purify the expression product. After the bacteria liquid was fermented ...

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Abstract

The invention provides cordyceps militaris fibrinoclase as well as a preparation method and application thereof, and belongs to the technical field of biological engineering. The invention provides the cordyceps militaris fibrinoclase which has the amino acid sequence shown as SEQ ID NO.1 and the gene sequence shown as SEQ ID NO.2. The cordyceps militaris fibrinoclase can be used for thrombolysis.Through a series of enzymatic property and in vitro and in vivo thrombolysis properties, the effect that the cordyceps militaris fibrinoclase has good thrombolysis activity is primarily studied and proved; through the building of other animal thrombus models, the in vivo thrombolysis mechanism is disclosed; by using the toxicological tests, the clinic application safety is mined; the scientific proving and data support are provided for the development of novel thrombus treatment medicine or functional food in future.

Description

technical field [0001] The invention relates to a cordyceps militaris fibrinolytic enzyme and its preparation method and application, belonging to the technical field of bioengineering. Background technique [0002] Cardiovascular and cerebrovascular diseases are one of the main causes of human death in the world, and intravascular thrombosis is a major cause of cardiovascular and cerebrovascular diseases. Intravascular thrombosis is caused by the conversion of fibrinogen to fibrin by thrombin, followed by the formation of insoluble fibrin clots (coagulation reaction), which are the main components of thrombus. To date, commercially used clinical thrombolytic agents such as tissue plasminogen activator (t-PA), urokinase, and streptokinase have some unavoidable side effects, such as bleeding and occlusive complications, Low specificity to fibrin degradation, allergic reactions and expensive. [0003] Cordyceps militaris, as a precious edible fungal resource with both medici...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/68C12N15/57C12N15/70A61K38/48A61P7/02
CPCA61K38/00A61P7/02C12N9/58C12N15/70C12Y304/21007
Inventor 刘英丽王静丁立万真孙宝国
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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