Recombinant mouse beta-alexin 3 polypeptide, preparation and use thereof

A defensin, recombinant plasmid technology, applied in biochemical equipment and methods, recombinant DNA technology, microorganism-based methods, etc., to achieve high differential virulence, reduce production costs, and facilitate industrial production.

Inactive Publication Date: 2009-11-04
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although antifungal drugs with different mechanisms of action have played an important role in the treatment of fungal infections, with the gradual change of infectious strains, reports on drug-resistant strains have gradually increased, making the treatment of fungal infections face severe challenges

Method used

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  • Recombinant mouse beta-alexin 3 polypeptide, preparation and use thereof
  • Recombinant mouse beta-alexin 3 polypeptide, preparation and use thereof
  • Recombinant mouse beta-alexin 3 polypeptide, preparation and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1: Preparation of recombinant mouse β-defensin 3 polypeptide

[0030] 1. Obtain the mouse Mbd-3 gene

[0031] (1) Use Trizol reagent (purchased from Invitroge, USA) to extract the total RNA of the respiratory epithelium of mice injected with Escherichia coli LPS (purchased from Sigma, USA) for 12 hours;

[0032] 1) BALB / c mice were intraperitoneally injected with LPS at a dose of 2.5 mg / kg, and the mice were sacrificed by removing eyeballs and bleeding after 12 hours;

[0033] 2) Take 100 mg of mouse lung tissue under sterile conditions;

[0034] 3) Add 1ml Trizol;

[0035] 4) Fully grind in liquid nitrogen, mix by inverting for 10 times, and let stand at room temperature for 5 minutes;

[0036] 5) Add 0.2ml of chloroform, invert and mix for 10 times, and let stand at room temperature for 5 minutes;

[0037] 6) Centrifuge at 12000rpm for 15min at 4°C;

[0038] 7) Take the supernatant (about 0.4ml) and transfer it to another EP tube;

[0039]8) Add an eq...

Embodiment 2

[0090] Example 2: Determination of Anti-filamentous Fungal Activity of Recombinant Mouse β-Defensin 3 Polypeptide

[0091] 1. Preparation of culture medium

[0092] (1) 1 / 2 potato dextrose medium (1 / 2PDB: 10% potato, 1% glucose, natural pH), to which tetracycline (final concentration 10 μg / ml) and cefotaxime (final concentration 100 μg / ml) were added.

[0093] (2) Sabouraud dextrose agar medium (1% peptone, 2% glucose, natural pH)

[0094] 2. Preparation of filamentous fungal strains

[0095] Inoculate Aspergillus flavus (clinical isolate), Aspergillus niger (clinical isolate), Microsporum canis (clinical isolate), and Trichophyton rubrum (clinical isolate) on the Sabouraud dextrose agar medium, Grow at 28°C for 7-10 days.

[0096] 3. Preparation of experimental bacteria

[0097] Take about 5 mg each of Aspergillus flavus, Aspergillus niger, Microsporum canis and Trichophyton rubrum, place them in 5ml 1 / 2PDB medium, blow and blow 10 times with a sterile pipette, and take t...

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Abstract

The invention constructs procaryon recombinant plasmid of mouse beta-alexin 3 gene (Mbd-3) by gene engineering technology, comprising the steps of transferring the procaryon recombinant plasmid into engineering bacteria for induction efficient expression of mouse beta-alexin 3 polypeptide (MBD-3), extracting and purifying expression product and exonuclease to release active MBD-3 mature peptide. The invention detects the minimum inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of recombinant mouse beta-alexin 3 for resisting different mycelial fungus by mcroscale broth dilution method, proves that the recombinant mouse beta-alexin 3 polypeptide has function of resisting pathogenicity mycelial fungus so as to invent a novel medicament for curing mycotic infection.

Description

technical field [0001] The present invention relates to recombinant mouse β-defensin 3 polypeptide and its preparation method and use. Background technique [0002] β-defensins are widely distributed in epithelial cells of various organs of humans, mice, cattle, sheep, and pigs, including epithelial cells of the skin, respiratory tract, urinary tract, intestinal tract, and oral mucosa. Currently, about 30 human β-defensin genes and 45 mouse β-defensin genes have been discovered using bioinformatics methods (Michael et al., 2005). The upstream promoter of the mouse β-defensin 3 (MBD-3) gene has binding sites for pre-NF-KB, NF-IL-6 and STAT, etc., when the pathogen-associated pattern molecule (PAMP) and the pattern recognition After binding to receptors (including CD14, TLRs, etc.), with the participation of various pro-inflammatory factors (TNF-α, IL-1β), it activates transcription factors such as NF-κB, and then interacts with NF-κB and NF-κB in the promoter region. IL-6 b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/74C07K14/47A61K38/17A61P31/10C12P21/02C12R1/19
Inventor 李明远江滟李婉宜王月玲巩天祥王保宁杨远王欢蒋忠华
Owner SICHUAN UNIV
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