Tabanusyao Macquart antithrombotic enzyme tablysin and gene and application thereof

A gadfly protease and gene technology is applied in the field of biomedicine to achieve the effects of inhibiting the formation of thrombus in the tail vein of rats and inhibiting the hydrolysis of fibrinogen

Active Publication Date: 2011-05-25
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, many antithrombotic drugs are often limited by systemic side effects including hypotension and...

Method used

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  • Tabanusyao Macquart antithrombotic enzyme tablysin and gene and application thereof
  • Tabanusyao Macquart antithrombotic enzyme tablysin and gene and application thereof
  • Tabanusyao Macquart antithrombotic enzyme tablysin and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation and amino acid sequence determination of gadfly antithrombotic enzyme tablysin

[0030] 1. Dissection, homogenization and processing of gadfly salivary glands

[0031] Gadfly (Shaanxi Province, China, scientific name Tabanusyao Macquart) dissection method is as follows: use small scissors to cut the worm body along the midline of the back, put it into pre-cooled physiological saline (buffer solution containing 0.9% NaCl) for 3-5min, Pick out the salivary glands with tweezers, put them in Tris-HCl buffer solution with pH 7.2, and store them at -20°C. After dissecting all gadfly samples, they were fully homogenized on ice with a glass homogenizer, centrifuged at 10,000 rpm for 10 min, and the supernatant was freeze-dried at low temperature and stored at -20°C.

[0032] 2. Separation and purification of gadfly antithrombotic enzyme tablysin

[0033] Using the lyophilized powder collected above as a raw material, Tablysin was purified according to t...

Embodiment 2

[0038] Embodiment two: Cloning of gadfly antithrombotic enzyme tablysin gene

[0039] 1. Extraction of total RNA from gadfly salivary glands

[0040] A. Gadfly vivisection (from Shaanxi Province, China, scientific name T.yao Macquart), about 1 mg of salivary gland tissue was taken, added to pre-cooled 1 mL Trizol extraction buffer (product of Invitrogen, USA), and homogenized on ice for 15 minutes.

[0041]B. Add 1 / 5 volume of Trizol in chloroform, mix vigorously for about 15 seconds, leave at room temperature for 5 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, and take the supernatant.

[0042] C. Add an equal volume of isopropanol to the supernatant, leave it at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, wash the precipitate once with 75% ethanol, and dry it in the air. The precipitate at the bottom of the tube is the total RNA of the gadfly salivary gland.

[0043] 2. Purification of gadfly salivary gland mRNA

[0044] Gadf...

Embodiment 3

[0106] Example 3: In vitro recombinant expression of Tablysin

[0107] A. Construction of recombinant plasmid pET-Tablysin

[0108] Step 1: Gene amplification of the target gene using primers with restriction sites

[0109] Use primer NT3BD-F containing BamHI restriction site and protective base:

[0110] 5'-TAGGATCCGTT AACTACTGTAGATTACCCTGCCG-3' and primer NT3BD-R containing HindIII restriction site and protective base: 5'-GGAAGCTTAGTTAGATTTATTAGGGTCGATAG G-3' amplification, PCR reaction conditions: 95°C pre-denaturation for 4min, followed by the following The conditions were 30 cycles of 94°C for 30 sec, 57°C for 30 sec, and 72°C for 60 sec. Then 72°C for 10 minutes.

[0111] The PCR product was subjected to 1% agarose gel electrophoresis, the target band was cut out, and the target gene was recovered using a gel DNA recovery kit (Beijing Tiangen Biochemical Technology Co., Ltd.).

[0112] Step 2: Preparation of plasmid DNA

[0113] pick containing pET-32a + A single c...

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Abstract

The invention relates to Tabanusyao Macquart antithrombotic enzyme tablysin and a gene and application thereof, and belongs to the field of biomedicine. In the invention, a natural antithrombotic enzyme is separated by a biomechanical method and a coded gene of the enzyme is amplified; and a gene engineering product which has equivalent activity with natural protein is obtained by using an escherichia coli expression system. The Tabanusyao Macquart protease tablysin is active protein which is separated from a Tabanusyao Macquart salivary gland, wherein the molecular weight is 27KDa; and an amino acid sequence of the enzyme is shown as SEQ ID NO. 1. The gene of the Tabanusyao Macquart protease tablysin consists of a nucleotide sequence which is shown as SEQ ID NO. 2. Through the TabanusyaoMacquart protease tablysin, fibrinogen can be hydrolyzed, platelet aggregation can be suppressed, and carrageenin-induced rat caudal vein thrombosis can be suppressed; and the Tabanusyao Macquart protease tablysin does not have bleeding activity and can be applied to preparation of medicaments for treating thrombotic diseases.

Description

Technical field: [0001] The invention provides gadfly antithrombotic enzyme tablysin and its gene and application, belonging to the technical field of biomedicine. Background technique: [0002] Thromboembolic disease seriously affects human health and is one of the causes of the highest mortality. In the past few decades, the antithrombotic drugs that have been developed mainly include thrombolytic drugs, anticoagulant drugs and antiplatelet drugs. However, many antithrombotic drugs are often limited by systemic side effects including hypotension and bleeding. Therefore, it is necessary to develop new antithrombotic drugs with more potential and specificity. [0003] Research in traditional Chinese medicine has shown that many natural components derived from animals and plants have definite antithrombotic effects. Ancient medical books such as "Compendium of Materia Medica" and "Synopsis of the Golden Chamber" have recorded many prescriptions for promoting blood circulatio...

Claims

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Application Information

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IPC IPC(8): C12N9/64C12N15/57A61K38/48A61P7/02
Inventor 赖仞马冬莹
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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