Gene clone of and expression Japan lamprey oral cavity gland KGD model protein possessing anti thrombotic action

An oral gland, lamprey technology, applied in genetic engineering, plant genetic improvement, peptide/protein composition, etc.

Inactive Publication Date: 2006-04-12
LIAONING NORMAL UNIVERSITY
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Problems solved by technology

[0004] At present, only two natural proteins with KGD motifs have been found in venomous snakes in nature: American rattlesnake venom barbourin (1) and Ussurista (2) , and the cDNA sequence of the Japanese lamprey oral gland KGD motif protein Lampetrin4 and its protein sequence and function have not been studied and reported at home and abroad, and the present invention is the first discovery in the world

Method used

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Embodiment Construction

[0034] The present invention will be further described below in conjunction with accompanying drawing.

[0035] 1. Extraction of total RNA: Trizol reagent from GIBCO BRL was used. details as follows:

[0036] (1) Take the oral gland of Japanese lamprey and quickly place it in liquid nitrogen for preservation;

[0037] (2) Weigh 0.2 g venom gland tissue, add 1 ml TRIzol reagent to prepare venom gland homogenate, and incubate at 4°C for 5 min;

[0038] (3) Add 0.2ml chloroform, close the lid tightly, shake vigorously for 15sec, and then place it on ice for 5min;

[0039] (4) 4°C, 12000xg, centrifuge for 15 minutes;

[0040](5) Transfer the upper aqueous phase to another centrifuge tube, add 0.5ml of isopropanol, and incubate on ice for 10 minutes;

[0041] (6) 4°C, 12000xg, centrifuge for 15 minutes;

[0042] (7) Discard the supernatant, add 1ml of 75% ethanol to the precipitate (containing RNA) to wash, and vortex to mix;

[0043] (8) Centrifuge at 10,000xg for 5 minutes ...

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Abstract

A gene clone and expression of the oral gland KGD pattern protein of Japanese lamprey with anti-thrombosis is disclosed. Its cDNA sequence and clone, its relative protein sequence, its expression in colibacillus and Piohia yeast, its combination with the glucoprotein gpó�b/IIIa on cell membrane of platelet to suppress the platelet coagulation, and its application in developing the anti-thrombosis medicines are disclosed.

Description

technical field [0001] The gene cloning and expression of the Japanese lamprey oral gland KGD motif protein with antithrombotic effect belongs to the field of biotechnology, involving a cDNA sequence and cloning of a KGD motif protein in the Japanese lamprey oral gland, and the corresponding The protein sequence and its expression in Escherichia coli or Pichia pastoris, and its specific combination with the glycoprotein gpIIb / IIIa on the platelet cell membrane competitively antagonize the combination of fibrinogen and platelets, preventing the activation of platelets and GPIIb / IIIa The conformational change of IIIa receptor blocks the binding of the receptor to a variety of ligands, thereby inhibiting the final common pathway of platelet aggregation and blocking the biological activity of thrombus formation. Background technique [0002] A class of drugs called "antiplatelet drugs" has been widely used clinically as an effective means for treating thrombotic diseases. Under...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435A61K38/17A61P7/02
Inventor 李庆伟
Owner LIAONING NORMAL UNIVERSITY
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