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Antithrombotic enzyme Eupolytinl from Eupolyphaga sinensis Walker and gene and application thereof

A technology for antithrombotic enzymes and terrestrial softshell worms, which is applied in the field of biomedicine

Active Publication Date: 2012-10-03
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, all antithrombotic drugs currently on the market are often accompanied by systemic side effects such as hypotension and bleeding. Therefore, it is urgent to develop new and safe antithrombotic drugs

Method used

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  • Antithrombotic enzyme Eupolytinl from Eupolyphaga sinensis Walker and gene and application thereof
  • Antithrombotic enzyme Eupolytinl from Eupolyphaga sinensis Walker and gene and application thereof
  • Antithrombotic enzyme Eupolytinl from Eupolyphaga sinensis Walker and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Preparation and Amino Acid Sequence Determination of Antithrombotic Enzyme Eupolytin1 from Ground Beetle

[0034] 1. Dissection, homogenization and processing of the midgut of the digestive tract of the ground beetle

[0035]The dissection method of Eupolyphaga sinensis is as follows: use small scissors to cut the insect body along the dorsal midline, pick out the digestive tract with tweezers, cut off the midgut, and put it into 0.05M acetic acid-sodium acetate buffer solution with pH5.0 In this method, after dissecting all ground beetle samples, they were centrifuged at 5000 g for 10 min, and the supernatant was stored at -80°C.

[0036] 2. Isolation and purification of the antithrombotic enzyme Eupolytin1 from the ground beetle

[0037] Using the supernatant collected above as a raw material, Eupolytin1 was purified according to the following procedures, and each step of separation and purification was tested for fibrinogen hydrolysis activity. The specif...

Embodiment 2

[0042] Example 2: Cloning of Eupolytin 1 Gene of Ground Beetle Antithrombotic Enzyme

[0043] 1. Extraction of total RNA from the midgut of the digestive tract of the ground beetle

[0044] A. Live dissection of the ground soft-shelled soft-shelled turtle, 60 ground soft-shelled soft-shelled soft-shelled turtle midgut tissues were taken, added to pre-cooled 1mL Trizol extract (Invitrogen, USA), and homogenized on ice for 15 minutes.

[0045] 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.

[0046] C. Add an equal volume of isopropanol to the supernatant, leave it at room temperature for 10 minutes, centrifuge at 12,000 rpm at 4°C for 10 minutes, wash the precipitate once with 75% ethanol, and dry it in the air.

[0047] 2. Purification of midgut mRNA in the digestive tract of the ground beetle

[0048] PolyATtract from PROMEGA, US...

Embodiment 3

[0086] Example 3: In vitro recombinant expression of Eupolytin1

[0087] A. Construction of recombinant plasmid pET-Eupolytin1

[0088] Step 1: Gene amplification of the target gene using primers with restriction sites

[0089] Using the primer 5′-BD1810-1:5′- ATTGTTGGTGGTT-3' and a primer containing a Hind III restriction site and a protective base 3'-BD1810-1: 5'-CCCCC CTAGTTGACTCCAG-3' was amplified using the midgut cDNA library of the digestive tract of the ground beetle as a template. The PCR reaction conditions were: pre-denaturation at 95°C for 4 minutes; then 30 cycles at 94°C for 30 sec, 57°C for 30 sec, and 72°C for 60 sec ; Finally, 72°C for 10 minutes. The PCR product will add an enterokinase restriction site at the 5' end of the target gene, and a Hind III restriction site and protective bases at the 3' end.

[0090] The product of the previous round of PCR was used as a template, and the primer 5’-BD1810-2: 5’-CGG containing KPN1 restriction site and protec...

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Abstract

The invention relates to antithrombotic enzyme Eupolytinl from Eupolyphaga sinensis Walker, a gene and application thereof, and belongs to the field of biomedicine. In the invention, the natural antithrombotic enzyme Eupolytinl is obtained from intestines in alimentary tracts of the Eupolyphaga sinensis Walker through separation and purification, an encoding gene of the natural antithrombotic enzyme Eupolytinl is cloned, and a genetic engineering product of which the activity is equivalent to that of a natural protein is obtained by utilizing an Escherichia coli expression system. A precursorof the antithrombotic enzyme Eupolytinl from the Eupolyphaga sinensis Walker consists of 254 amino acids, wherein 1th to 28th amino acids are a signal peptide part; 29th to 254th amino acids are mature Eupolytinl proteins; and a primary structure of an amino acid sequence of the precursor is shown as SEQ ID NO: 1. The encoding gene of the antithrombotic enzyme Eupolytinl consists of 888 nucleotide sequences, wherein 85th to 765th nucleotides encode the mature Eupolytinl proteins; and a nucleotide sequence of the gene is GenBank accession Gu187070. Through animal experiments, the recombinant expression antithrombotic enzyme Eupolytinl from the Eupolyphaga sinensis Walker which has a natural resource can be applied to the preparation of medicaments for treating thrombotic diseases.

Description

Technical field: [0001] The invention provides Eupolytin1, an antithrombotic enzyme of Eupolytin 1 and its gene and application, belonging to the technical field of biomedicine. Background technique: [0002] With the continuous improvement of people's living standards, the pace of life is getting faster and faster, busy work, unreasonable diet, irregular life schedule, smoking and drinking, and lack of physical exercise and many other incentives lead to more and more people suffering from Various cardiovascular diseases. According to the WHO, 18 million people die from heart disease and other cardiovascular diseases every year, accounting for about one-third of the global death toll. It is estimated that this number may exceed 20 million by 2020. 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/48C12N15/57A61K38/48A61P7/02
Inventor 赖仞杨海龙王义鹏肖瑶
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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