Antimicrobial peptide Pc-CATH1 and gene thereof, chemical synthesis method and application thereof

An antimicrobial peptide, pc-cath1 technology, applied in the field of biomedicine, achieves the effect of super strong serum stability, small molecular weight, and rapid bactericidal action time

Inactive Publication Date: 2011-07-06
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the molecular biological and molecular pharmacological basis behind its medicinal value has not yet been reported

Method used

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  • Antimicrobial peptide Pc-CATH1 and gene thereof, chemical synthesis method and application thereof
  • Antimicrobial peptide Pc-CATH1 and gene thereof, chemical synthesis method and application thereof
  • Antimicrobial peptide Pc-CATH1 and gene thereof, chemical synthesis method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Cloning and gene sequencing of antimicrobial peptide Pc-CATH1 precursor gene, including:

[0036] The RNeasy Mini Kit was used to extract the total RNA from the bone marrow of the ring-necked pheasant, and the Creator TM SMART TM The cDNA library kit was used to construct the cDNA library of ring-necked pheasant bone marrow. Reverse Transcriptase reverse transcription synthesizes the first strand of cDNA, the primers are:

[0037] Forward Oligo dT primer: 5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3′

[0038] Reverse CDSIII primer: 5′-ATTCTAGAGGCCGAGGCGGCCGACATGT(30)N -1 N-3' (N=A, G, C, or T; N -1 = A, G, or C).

[0039] The second strand was synthesized by Advantage DNA Polymerase, the primer was: forward 5′-AAGCAGTGGTATCAACGCAGAGT-3′, and the reverse primer was CDSIII.

[0040] Two specific forward primers (P1, P2) and one reverse non-specific universal primer (CDSIII) were designed, and the cDNA of cathelicidin was amplified by semi-nested PCR using the cDN...

Embodiment 2

[0104] The chemical synthesis method of Pc-CATH1:

[0105] 1. According to the amino acid sequence of the mature peptide Pc-CATH1 deduced from the gene encoding ring-necked pheasant cathelicidin, its full sequence was synthesized with an automatic polypeptide synthesizer (Applied Biosystems), and purified by desalting and desalting by HPLC reverse-phase column chromatography.

[0106] II. Molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).

Embodiment 3

[0108] Pharmacological experiments of antimicrobial peptide Pc-CATH1:

[0109] 1. Detection of antibacterial activity of Pc-CATH1:

[0110] Dissolve chemically synthesized Pc-CATH1 in sterile ultrapure water at a concentration of 2 mg / ml; pick up newly activated microorganisms with an inoculation loop, and spread them evenly on a new LB agar plate; place a circle with a diameter of 0.5 cm Place a piece of sterile filter paper on the above-mentioned agar plate, and then drop 10 μl of Pc-CATH1 sample solution onto the piece of paper; put it in a constant temperature incubator at 37°C for 12-24 hours; CATH1-sensitive strains were recorded.

[0111] 2. Determination of Pc-CATH1 on the minimum inhibitory concentration (Minimum Inhibitory Concentration, MIC) of sensitive strains. In this experiment, human cathelicidin LL-37, traditional antibiotics ampicillin and kanamycin were used as positive controls, and sterile liquid LB was used as negative controls; the minimum inhibitory c...

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Abstract

The invention relates to an antimicrobial peptide Pc-CATH1 from Phasianus colchicus and a gene thereof, a chemical synthesis method and application in the field of biopharmaceuticals and belongs to the technical field of biomedicine. The antimicrobial peptide Pc-CATH1 is a straight-chain polypeptide encoded by (Phasianus colchicus) Cathelicidin genes, and the complete sequence of the antimicrobial peptide Pc-CATH1 is as follows: arginine-isoleucine-lysine-arginine-phenylalanine-tryptophan-proline-valine-valine-isoleucine-arginine-threonine-valine-valine-alanine-glycine-tyrosine-asparaginate-leucine-tyrosine-arginine-alanine-isoleucine-lysine-lysine-lysine; and a gene for encoding a precursor of the antimicrobial peptide Pc-CATH1 consists of 607 nucleotides, wherein the 367th to 445th nucleotides are used for coding mature peptide part. The antimicrobial peptide Pc-CATH1 is smaller in molecular weight, stronger in bactericidal action and broader in spectrum and has strong effect of killing various clinical drug-resistant bacteria; the antimicrobial peptide Pc-CATH1 has a simple structure without a disulfide bond or a ring structure and is convenient for chemical synthesis and the preparation of gene engineering; and in addition, the antimicrobial peptide Pc-CATH1 has the beneficial characteristics of no hemolysis, no cytotoxicity, super-strong serum stability and the like.

Description

technical field [0001] The invention provides a cathelicidin family broad-spectrum antimicrobial peptide Pa-CATH1 derived from Phasianus colchicus and its gene, a chemical synthesis method and application, belonging to the technical field of biomedicine. Background technique [0002] Cathelicidin is a family of multifunctional antimicrobial peptides composed of an N-terminal signal peptide region, a middle conserved cathelin domain and a C-terminal highly specific mature peptide region. species and fish) have been found. Like defensin, cathelicidin is a host defense peptide unique to most vertebrates including humans, which constitutes a key component of the natural immune response of vertebrates and serves as a bridge connecting natural immunity and specific immunity. Cathelicidin has broad-spectrum antibacterial activity, not only has very strong bactericidal activity against Gram-positive bacteria, Gram-negative bacteria, some fungi and viruses, but also has effects on m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/465C07K1/00C07K1/20C12N15/12C12Q1/18
Inventor 于海宁王义鹏
Owner DALIAN UNIV OF TECH
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