cDNA sequence and amino acid sequence of lugworm fibrinolytic enzyme with thrombolysis activity

A sea earthworm and fibrinolytic enzyme technology, applied in the field of medical bioengineering

Inactive Publication Date: 2012-10-31
WEIFANG MEDICAL UNIV
View PDF2 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no research or report about the cDNA sequence, amino acid sequence and function of sea earthworm fibrinolytic enzyme at home and abroad

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • cDNA sequence and amino acid sequence of lugworm fibrinolytic enzyme with thrombolysis activity
  • cDNA sequence and amino acid sequence of lugworm fibrinolytic enzyme with thrombolysis activity
  • cDNA sequence and amino acid sequence of lugworm fibrinolytic enzyme with thrombolysis activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Cloning and sequencing of full-length cDNA of sea earthworm fibrinolytic enzyme.

[0050] Experimental Materials:

[0051] 1. Sea earthworms, collected from the offshore of Yantai City;

[0052] 2. Trizol reagent, 3′RACE kit, 5′RACE kit (Beijing Gibbett), reverse transcription kit (fermentas company), T-A Easy kit, Wizard purified DNA kit, DNase, RNase H (Promega company), high-fidelity Taq enzyme (Dalian Baobio).

[0053] Experimental method: such as figure 1 shown, including the following steps:

[0054] 1. Design primer sequences:

[0055] Primer name sequence 3R 5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3′ 3F1 5′-GGATCTTGCAATGGTGACAGCGGTG-3′ 3R1 5′-GGCCACGCGTCGACTAGTAC-3′ 5R 5′-TTTTTTTTTTTTTTTTTT-3′ 5F1 5′-GGCCACGCGTCGACTAGTACGGGGGGGGGGGGG-3′ 5R1 5′-CACCCTCTCTGATCCAGTCACGGAAG-3′ 5F2 5′-CCACGCGTCGACTAGTACG-3′ 5R2 5′-GGAGTAGGCAGATGGGTAGGAGGTC-3′

[0056] 3R is a reverse transcription primer containi...

Embodiment 2

[0110] Construction of recombinant expression vector and expression in Escherichia coli.

[0111]Experimental materials: Endonucleases BamHI and XhoI (Dalian Bao Biology), expression vectors pET-21a, E.coliBL21 are preserved in our laboratory, IPTG (Beijing Dongsheng Taibo), ultrasonic cracker (Shanghai Bilang).

[0112] Experimental method: such as figure 2 shown, including the following steps:

[0113] 1. According to the obtained cDNA sequence, design a pair of primers:

[0114] F 5′-TTAT GGATCC ATTGTTGGTGGCGATGAG-3′ R 5′-AATA CTCGAG GATGTCAGACACCTCTCTGATC-3′

[0115] upstream primer F contains BamHI Restriction site, contained in the downstream primer R wxya Restriction sites.

[0116] Using the cDNA of sea earthworm fibrinolytic enzyme as a template, pfu DNA polymerase was used to amplify the cDNA fragment without the coding region of the signal peptide, that is, the active peptide.

[0117] 2. The PCR product was connected with the pro...

Embodiment 3

[0121] Isolation and purification of recombinant sea worm fibrinolytic enzyme.

[0122] Experimental materials: nickel column packing, ultrafiltration tube (Beijing Dongsheng Taibo).

[0123] Experimental method: such as figure 2 shown, including the following steps:

[0124] 1. Pick a single colony of engineered bacteria, inoculate it in 5ml LB medium containing 100μg / ml ampicillin, and cultivate overnight at 37°C with shaking. Transfer to 500ml of the same medium and continue to culture at 37°C for 4h. Add IPTG (isopropyl-β-D-thiogalactoside) to 1.0 mM, continue to culture at 30°C for 4 hours, and collect the bacteria by centrifugation at 8000×g for 5 minutes.

[0125] 2. Resuspend the bacteria in 25ml Tris-CL buffer (20mM, pH7.4, 10mM imidazole, 0.5M NaCl), sonicate the cells, centrifuge at 12000×g for 30min at 4°C, and take the supernatant.

[0126] 3. Flow the supernatant over Ni 2+ Resin column, washed with 50ml washing solution (20mM Tris-CL, pH7.4, 10mM imidazole...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a cDNA sequence and an amino acid sequence of coding lugworm fibrinolytic enzyme which are respectively shown in SEQIDNo.1 and SEQIDNo.2 in a sequence table, and belongs to the field of medical biological engineering. The cDNA sequence has the beneficial effects that according to the cDNA sequence of the lugworm fibrinolytic enzyme, the coding gene of the fibrinolytic enzyme can be cloned from digestive tract tissue of lugworm, and can be expressed in a prokaryotic expression system or a eukaryotic expression system through a gene recombination technology. The recombinant lugworm fibrinolytic enzyme has plasminogen activation activity, and can be used as a new thrombolytic medicament in genetic engineering.

Description

[0001] technical field [0002] The invention relates to a sea earthworm fibrinolytic enzyme, specifically a cDNA sequence and an amino acid sequence of the sea earthworm fibrinolytic enzyme with thrombolytic activity, belonging to the field of medical bioengineering. Background technique [0003] Thrombotic disease is a serious threat to human health, and it is urgent to find a new type of thrombolytic agent that is safe, efficient and low-toxic. Fibrinolytic enzymes mostly belong to the serine protease family. The protease catalytic active center of this family contains a highly conserved sequence GDSGGP near Ser, which mainly cleaves the peptide bond at the carboxyl end of Lys or Arg in fibrin and fibrinogen to degrade it into Many soluble small peptides have anticoagulant effects; some plasmin also acts as a plasminogen activator and indirectly exerts a thrombolytic effect. The clinically approved fibrinolytic enzymes include urokinase, streptokinase, staphylokinase, an...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/68
Inventor 赵春玲鞠吉雨于文静初金鑫陈丽梅连波
Owner WEIFANG MEDICAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap