Antimicrobial peptide Cm-CATH2, gene thereof, preparation method and application

An antimicrobial peptide and gene technology, applied in the field of biomedicine, to achieve the effects of good killing effect, strong broad-spectrum antibacterial activity and simple structure

Active Publication Date: 2016-12-07
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Antimicrobial peptide Cm-CATH2, gene thereof, preparation method and application
  • Antimicrobial peptide Cm-CATH2, gene thereof, preparation method and application
  • Antimicrobial peptide Cm-CATH2, gene thereof, preparation method and application

Examples

Experimental program
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Embodiment 1

[0021] Example 1 Separation and purification of antimicrobial peptide Cm-CATH2:

[0022](1) Fresh complete spleen tissue of a dying green sea turtle was obtained from the zoo, and the surface of the spleen tissue was washed with a little saline. After homogenization, dissolve with a small amount of normal saline, according to the ratio of PS solution and n-butanol 1:50 (V / V), stir PS and n-butanol at room temperature for 60min, centrifuge twice at 13000r / min for 20min, and then The pellet was lyophilized. Subsequently, the first step of SephadexG-50 gel filtration chromatography: 0.9g lyophilized powder was dissolved in 10ml 0.1M phosphate (Na2HPO4-NaH2PO4, pH 6.0) buffer solution, centrifuged at 12000rpm for 10min, and the supernatant was loaded on the equilibrated A good Sephadex G-50 gel size exclusion chromatography column (1.6cm x 90cm, Amersham Bioscience), eluted with the same buffer, flow rate 3mL / 10min, collected 3mL / tube with an automatic fraction collector, detecte...

Embodiment 2

[0025] Example 2 Cloning and gene sequencing of Cm-CATH2 precursor gene

[0026] Step 1: Extraction of total RNA from the liver of green sea turtles (the instruments and reagents used in the following experiments have been processed and are RNase-free):

[0027] A. Cut small pieces of about 1 g from various fresh tissues of freshly slaughtered green sea turtles, put them into liquid nitrogen pre-cooled cell cryopreservation tubes, and then quickly put them into liquid nitrogen for storage;

[0028] B. Take out the tissue material stored in liquid nitrogen, put it into a pre-cooled mortar, grind it quickly and fully, and add a little liquid nitrogen to the mortar during this period; transfer about 30mg of tissue powder to a pre-cooled 1.5ml Add 400 μl Buffer R-I (lysate, provided by RNA Miniprep Kit) to the centrifuge tube, repeatedly pump 8-10 times with a syringe with a 21-25 gauge needle, transfer to a 1.5ml centrifuge tube, add 150 μl Buffer R-I П, vortex for 15-30s, centr...

Embodiment 3

[0052] The chemical synthesis method of embodiment 3 Cm-CATH2:

[0053] (1) According to the deduced amino acid sequence of the mature peptide Cm-CATH2 of the gene encoding green sea turtle cathelicidin, its full sequence was synthesized with an automatic peptide synthesizer (Applied Biosystems).

[0054] (2) by HPLC reverse phase C 18 Column chromatography desalted and purified the synthetic polypeptide: the column used in the process was 4.6×250nm, Venusil XBP-C4; solvent A was 0.1% trifluoroacetic dissolved in 100% acetonitrile, solvent B was 0.1% trifluoroacetic dissolved in 100% water; gradient set For: 0.01min (A 15%, B 85%), 25min (A40%, B60%), 25.1min (A 100%, B 0%), 30min (stop); the flow rate is 1.0ml / min, the wavelength is 220nm , the volume is 5 μl, and the result shows that the purity of the synthesized polypeptide sequence is greater than 97%.

[0055](3) The molecular weight is determined by matrix-assisted laser desorption ionization time-of-flight mass spect...

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Abstract

The invention relates to an antimicrobial peptide Cm-CATH2, a preparation method thereof and an application. The Cm-CATH2 is a straight chain polypeptide containing 33 amino acid residues, the theoretical isoelectric point is 12.96, molecular weight is 4089.97Da, and net charge is +12. A gene of a precursor cathelicidin of the encoding antimicrobial peptide Cm-CATH2 comprises 486 nucleotides. The Cm-CATH2 has high antibacterial activity for Gram-negative bacteria, Gram-positive bacteria and fungi, comprises various clinical drug-resistant bacteria, is low in minimal inhibitory concentration and rapid and lasting in sterilizing effect, does not contain disulfide bonds and cyclic structures, has the advantages of low hemolysis, cell toxicity, difficulty in generating drug resistance and the like, can replace an antibiotic for preparing clinical drugs resisting pathogenic microorganism infection, diminishing inflammation and the like, is applied to additives in daily chemicals such as cosmetics, health care products, food and feed, and has an excellent application prospect. Chemical synthesis and gene engineering preparation are facilitated.

Description

technical field [0001] The invention provides a cathelicidin family broad-spectrum antimicrobial peptide Cm-CATH2 derived from green sea turtle (C.mydas) and its preparation method and its clinical application in anti-pathogenic microbial infection, anti-inflammatory and other clinical drugs, cosmetics, health products, food, feed The application in additives etc. belongs to the field of biomedical technology. Background technique [0002] Cathelicidins are a class of multifunctional antimicrobial peptides found in mammals, birds, reptiles, amphibians, and fish. Since the first discovery of Bac5 from bovine neutrophils, more and more cathelicidin antimicrobial peptides have been identified. Cathelicidin is usually synthesized in the form of a precursor, including a signal peptide of about 30 amino acid residues at the N-terminus, a conserved cathelin domain and a highly specific mature peptide region at the C-terminus, consisting of three parts. After the precursor of cath...

Claims

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Application Information

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IPC IPC(8): C07K14/46C12N15/12A61K38/17A61P31/04A61P31/10A61P29/00A61K8/64A61Q19/00A61Q17/00A23K20/147A23L3/3526
CPCA23L3/3526A23V2002/00A61K8/64A61K38/00A61Q17/005A61Q19/00C07K14/46A23V2200/10A23V2250/55
Inventor 于海宁王义鹏乔雪高久香
Owner DALIAN UNIV OF TECH
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