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Snake venom thrombin-like enzyme and its encoding gene and application

A thrombin-like, gene-encoding technology, applied in applications, genetic engineering, plant genetic improvement, etc., can solve problems such as limited yield and achieve broad application prospects.

Inactive Publication Date: 2007-07-04
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no thrombin-like enzymes have been found in the sea snake family, and the snake-like thrombin used clinically is purified from snake venom, and the yield is limited, and the impure snake-like miscellaneous proteins or enzymes contained in it It is one of the reasons for the relatively large toxic side effects

Method used

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  • Snake venom thrombin-like enzyme and its encoding gene and application
  • Snake venom thrombin-like enzyme and its encoding gene and application
  • Snake venom thrombin-like enzyme and its encoding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Obtaining of TLE and expression of snake venom thrombin-like mature protein

[0052] 1. Obtaining TLE

[0053] Include the following steps:

[0054] 1) The venom gland tissue was isolated from the flat-chin sea snake (Lapemis curtus) and the blue-gray sea snake (Hydrophiscaerulescens) captured in the Beibu Gulf of China, and the total RNA of the venom of the two sea snakes was extracted;

[0055] 2) extracting mRNA from the total RNA of the two kinds of sea snake venoms, and using the mRNA as a template to reverse transcribe and synthesize their respective cDNA sequences;

[0056] 3) Ligate the two synthetic cDNAs with the T7 phage vector (Novagen) respectively, and obtain the sea snake phage display polypeptide library through in vitro packaging;

[0057] 4) ELISA method was used to screen the phage display peptide library constructed in step 3) with fibrinogen (fibrinogen, sigma company) as the target molecule, and phage clones with specific binding abili...

Embodiment 2

[0066] Embodiment 2, detection of enzyme activity of snake venom thrombin-like mature protein

[0067] 1. Determination of amidolysis activity

[0068] At 37°C, add a chromogenic substrate (N-α-p-tosyl-Gly-Pro-Arg-p-nitronilide, Sigma Company) to a concentration of 2mM in 50mM phosphate buffer (pH8.0) and Example 1 The obtained snake venom thrombin-like mature protein to a concentration of 1mg / mL, after reacting for 10min, by detecting OD 405 The changes of the snake venom-like thrombin mature protein to measure the amidolysis activity, the experimental results show that the snake venom-like thrombin mature protein can degrade the chromogenic substrate, with amidolysis activity (1062.5 enzyme activity U / min / mg enzyme), It shows that the thrombin-like mature protein of the snake venom is similar to the thrombin-like enzymes of other land snakes, and both have amidolysis activity, which can degrade the amide bond in the protein polypeptide.

[0069] 2. Determination of arginin...

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Abstract

The present invention discloses one kind of reptilase and its coding gene and application, and aims at provides one kind of reptilase and reptilase mature protein and their coding genes as well as medicine with the reptilase and / or reptilase mature protein as active component and with thrombolysis and thrombosis preventing effects. The reptilase of the present invention has one of the amino acid residue sequences: 1. SEQ I in the sequence list; and 2. the amino acid residue sequence of SEQ I through substitution, deletion and / or addition of 1-10 amino acid residues and coding protein with thrombolysis and thrombosis preventing effects. The recombinant expressed reptilase mature protein amidase activity an arginine esterase activity similar to those of reptilase, can degrade specifically the beta chain of fibrinogen and fibrinogen caused thrombosis, and has the functions of lowering viscosity and thrombolysis.

Description

technical field [0001] The present invention relates to snake venom-like thrombin and its coding gene and application, in particular to snake venom-like thrombin, snake venom-like thrombin mature protein and their coding genes and the snake venom-like thrombin or snake venom-like thrombin mature protein as active ingredients Drugs with thrombolytic and antithrombotic effects. Background technique [0002] Thrombosis is a common complication of surgical operations, an important factor for reocclusion after modern interventional angioplasty, and a cause of morbidity and death in various cardiovascular and cerebrovascular diseases. Physiological thrombosis is a means of hemostasis, while pathological thrombosis can lead to dysfunction of related organs. At present, thrombotic diseases in the heart and brain have become one of the diseases with the highest morbidity and mortality rates, which seriously threaten human health. According to statistics, there are about 15 million ...

Claims

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Application Information

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IPC IPC(8): C12N9/74C12N15/57C12N15/63A61K38/48
Inventor 顾军陈师勇
Owner PEKING UNIV
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