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Chinese Polyphaga fibrinolytic enzyme gene and preparation thereof

A technology of ground beetle and plasmin, which is applied in the field of genetic engineering, can solve the problems of complicated separation and purification of plasmin, protein inactivation, and inability to obtain a large amount of plasmin, and achieve considerable medical value, inhibit thrombus formation and dissolve thrombus Effect

Inactive Publication Date: 2009-04-22
GUANGDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, most of the plasmin components in the ground beetle at home and abroad are obtained through separation and purification techniques. However, the process of separating and purifying plasmin from the organism is very cumbersome, and the protein from the organism is also easily inactivated during the purification process. plasmin

Method used

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  • Chinese Polyphaga fibrinolytic enzyme gene and preparation thereof
  • Chinese Polyphaga fibrinolytic enzyme gene and preparation thereof
  • Chinese Polyphaga fibrinolytic enzyme gene and preparation thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0022] Example 1 Extraction of total RNA from Wood Beetle

[0023] Take a live healthy female ground beetle, wash it with 0.1% by volume of diethylpyrophosphate (DEPC) solution, cut open the abdomen with scissors without RNase, cut about 30 mg of the digestive cavity and midgut muscle of the ground beetle, and add 1.2 ml The ice-cold Trizol solution (added twice) was thoroughly ground, and the homogenate was placed in a 1.5ml Eppendorf centrifuge tube, and placed in an ice bath for 5 minutes to fully lyse. Centrifuge at 12000rpm for 5min at 4°C, take the supernatant, add 0.2ml of chloroform, shake for 20sec, and place in ice bath for 10min. Centrifuge at 12000rpm for 15min at 4°C, carefully absorb the upper aqueous phase, and transfer it to another centrifuge tube. Add 0.5ml of isopropanol to each tube, mix well, and place in ice bath for 10min. Centrifuge at 12000rpm for 15min at 4°C and discard the supernatant. The precipitate was washed once with 1 ml of 75% ethanol, dri...

Embodiment 2

[0024] The reverse transcription of embodiment 2 mRNA

[0025] 20 μl reverse transcription reaction system, containing 1 μg total RNA prepared in Example 1, 2.0 μgoligo(dT) 18 , make up to 11 μl with RNase-free double distilled water, heat at 70°C for 5 minutes, then ice-bath for 10 minutes, then add 5×buffer (250mmol / LTris-HCl, 250mmol / LKCl, 20mmol several MgCl 2 , 50mmol / L dithiothreitol (DTT) 4μl, 10mmol / L dNTP mix 2μl, RNase-free double distilled water to make up to 19μl, 37°C for 5min, then add M-MuLV reverse transcriptase 1μl (200U), 42 Incubate at °C for 1 hour, heat at 70°C for 10 minutes to terminate the reverse transcription reaction, cool in an ice bath and store in a -20°C refrigerator.

Embodiment 3

[0026] Example 3 Obtaining of EFE-1 fragments

[0027] 1) Design of EFE-1 fragment primers

[0028] Referring to the published sequences of earthworm plasminase and centipede plasminase kinase on NCBI, the conserved segment between the sequences was analyzed with Clustal 1.8 software, and a pair of degenerate primers were designed with the help of Primer Premier 5 software:

[0029] Upstream primer: 5'-CTN ACY GCW GCY CAY TG-3', SEQ ID NO: 7, degeneracy 64, encoding amino acid sequence LTAAHC;

[0030] Downstream primer: 5'-CC NCC NGA RTC WCC CT-3', SEQ ID NO: 8, degeneracy 64, encoding amino acid sequence QGDSGG;

[0031] Where R=A+G Y=T+C W=A+T N=A+T+G+C.

[0032] 2) PCR amplification of EFE-1 fragment

[0033] PCR amplification uses a 25ul reaction system:

[0034] 1ul of the reverse transcription product obtained in Example 2;

[0035] 1×Ruffer (0.5mol / L KCl, 0.1mol / L Tris-HCl, pH8.4);

[0036] MgCl 2 2.0mmol / L;

[0037] dNTPs 0.2mmol / L;

[0038] Upstream primer:...

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Abstract

The invention discloses a eupolyphaga sinensis walker plasmin gene and a preparation method thereof. The gene consists of 811 ribonucleotides, a ribonucleotide sequence of the gene is shown in SEQ ID NO1, wherein 1st to 672nd ribonucleotides are a mature peptide sequence of the eupolyphaga sinensis walker plasmin gene, 224 amino acids are encoded, and an amino acid sequence of the amino acids is shown in SEQ ID NO3. The eupolyphaga sinensis walker plasmin gene is obtained through the following steps: firstly, cDNA is obtained through the reverse transcription of mRNA, a 5' end sequence of the gene is obtained through PCR augmentation, then a 3' RACE method is used to obtain a 3' end sequence of the gene, and finally the 5' end sequence and the 3' end sequence are spliced. The sequence of the gene expands a gene library of plasmin genes, obtains a large quantity of plasmin proteins through a method of genetic engineering according to the ribonucleotide sequence of the eupolyphaga sinensis walker plasmin gene at the same time, is applied to aspects of inhibiting the formation of thrombus and dissolving the thrombus, and has considerable medical value.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a fibrinolytic enzyme gene of the beetle beetle and a preparation method thereof. Background technique [0002] Eupolyphage sinensis Walker, also known as Tuyuan and Eupolyphage sinensis Walker, belongs to the family Arthropoda, Insecta, and is a traditional animal medicine for promoting blood circulation and removing blood stasis. Medical records and abundant clinical practice have already proved that it has important medicinal effects such as breaking blood and removing blood stasis, rejuvenating tendons and bones, reducing inflammation and dispelling stagnation, relaxing meridians, activating blood circulation, healing injuries and relieving pain. Modern pharmacological experiments have also fully proved that the ground beetle has medicinal effects such as dissolving thrombus, inhibiting platelet aggregation, and anticoagulation. [0003] Through the s...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N15/10
Inventor 韩雅莉李兴暖李张伟
Owner GUANGDONG UNIV OF TECH
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