Recombinant lamprey lysin as well as preparation method and application thereof in preparing antithrombotic medicament

A technology of lamprey and lysin, which is applied in the field of medicine and medical engineering, to achieve enhanced antithrombotic effect, large safety range, and mild toxicity

Inactive Publication Date: 2011-10-12
LIAONING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far there has been no report on the preparation of antithrombotic drugs from the oral gland of Japanese lamprey

Method used

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  • Recombinant lamprey lysin as well as preparation method and application thereof in preparing antithrombotic medicament
  • Recombinant lamprey lysin as well as preparation method and application thereof in preparing antithrombotic medicament
  • Recombinant lamprey lysin as well as preparation method and application thereof in preparing antithrombotic medicament

Examples

Experimental program
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Embodiment Construction

[0061] a. Preparation of eellysin gene:

[0062] (1) The oral glands of Japanese lamprey were taken and quickly stored in liquid nitrogen;

[0063] (2) Weigh 0.2g lamprey oral gland, add 1ml TRIzol reagent to prepare venom gland homogenate, incubate at 4°C for 5min;

[0064] (3) Add 0.2ml chloroform, close the cap tightly, shake for 15sec, and place on ice for 5min;

[0065] (4) 4°C, 12000xg, centrifuge for 15 minutes;

[0066] (5) Transfer the upper aqueous phase to another centrifuge tube, add 0.5ml of isopropanol, and incubate on ice for 10 minutes;

[0067] (6) 4°C, 12000xg, centrifuge for 15 minutes;

[0068] (7) Discard the supernatant, add 1ml 75% ethanol to the precipitate to wash, and vortex to mix;

[0069] (8) Centrifuge at 10,000xg for 5 minutes at 4°C to obtain the RNA precipitate of eellysin;

[0070] (9) After air drying, dissolve RNA with TE or RNase-free water for later use;

[0071] b. Perform RT-PCR amplification to obtain the target cDNA;

[0072] Th...

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Abstract

The invention discloses a recombinant lamprey lysin protein as well as a preparation method and application thereof in preparing an antithrombotic medicament. The recombinant lamprey lysin protein is a product which is obtained through the following steps: constructing a lamprey protein lysin gene on a pET28a (+) vector; and carrying out efficient induction expression on the gene in Escherichia coli. The lamprey lysin cDNA (complementary deoxyribonucleic acid) sequence 351bp is long, and the protein consists of 117 amino acids, can perform specific binding with glycoprotein GpIIb/IIIa on a blood platelet cell membrane, competitively antagonizes the binding of fibrinogen and blood platelets, prevents the activation of the blood platelets and variation of GpIIb/IIIa receptor conformation and blocks the binding of the receptor and various ligand, so as to inhibit the final common passageway for blood platelet aggregation so as to block thrombosis. The recombinant lamprey lysin has an obvious antithrombotic effect.

Description

technical field [0001] The invention relates to a preparation of recombinant lamprey lysin, which belongs to the field of medicine and medical engineering, and can block thrombus formation with low effective dosage, large safety range, high action intensity, mild adverse reaction and low toxicity Method and application in preparation of antithrombotic drugs. Background technique [0002] Under normal circumstances, the hemostasis, coagulation system and fibrinolytic system in the body are in a physiological balance to maintain normal blood flow. However, in some pathological conditions, such as vascular intimal injury or venous blood flow blockage, platelets are activated and adhere to the subendothelial tissue, which triggers a series of hemostasis and coagulation processes, and finally forms a thrombus. The main components of thrombus are activated platelets and fibrin. Therefore, specific anti-activated platelet and anti-fibrin drugs can be used as thrombus-directing ag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C12N15/12C12N15/10C12N15/70A61K38/17A61P7/02C12R1/19
Inventor 李庆伟王继红吕莉张艳
Owner LIAONING NORMAL UNIVERSITY
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