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Antimicrobial peptide Ea-CATH1 of Equus asinus and genes and application thereof

An antimicrobial peptide, ea-cath1 technology, applied in the field of biomedicine, can solve the problems that the research on active protein peptides has not yet been reported, and achieve the effect of strong killing effect, small molecular weight, and rapid bactericidal action time

Inactive Publication Date: 2010-09-29
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on its active ingredients and pharmacological properties mainly focuses on small organic molecules, and the research on its active protein peptides has not yet been reported.

Method used

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  • Antimicrobial peptide Ea-CATH1 of Equus asinus and genes and application thereof
  • Antimicrobial peptide Ea-CATH1 of Equus asinus and genes and application thereof
  • Antimicrobial peptide Ea-CATH1 of Equus asinus and genes and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Cloning and gene sequencing of donkey antimicrobial peptide Ea-CATH1 precursor gene, including:

[0030] The total RNA of donkey spleen was extracted with RNeasy Mini Kit, and the Reverse Transcriptase reverse transcription synthesis cDNA first strand. Two specific forward primers (P1, P2) and one reverse non-specific universal primer (CDSIII) were designed, and the cDNA of donkey cathelicidin gene was amplified by semi-nested PCR using single-stranded cDNA as a template.

[0031] Forward P1: 5′-GGACCATGGAGACCCAGAGG-3′;

[0032] Forward P2: 5′-ATGGAGACCCAGAGGGACAGTT-3′;

[0033] Reverse Non-specific Universal Primer for CLONTECH Creator TM SMART TM 3'PCR Primer CDSIII in the cDNA LibraryConstruction Kit, its sequence is: 5'-ATTCTAGAGGCCGAGGCGGCCGACATGT(30)N -1 N-3' (N=A, G, C, or T; N -1 = A, G, or C).

[0034] The obtained positive single clones were subjected to gene nucleotide sequence determination, and the general primers for pMD19-T vector sequencing:

...

Embodiment 2

[0084] The chemical synthesis method of Ea-CATH1:

[0085] 1. According to the amino acid sequence of mature peptide Ea-CATH1 deduced from the gene encoding domestic animal donkey cathelicidin antimicrobial peptide, its full sequence was synthesized with an automatic peptide synthesizer (433A, Applied Biosystems), and purified by HPLC reverse-phase column chromatography desalting.

[0086] II. Molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).

Embodiment 3

[0088] Pharmacological experiment of donkey antimicrobial peptide Ea-CATH1 from livestock:

[0089] 1. Detection of antibacterial activity of Ea-CATH1:

[0090] Dissolve chemically synthesized Ea-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 Ea-CATH 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.

[0091] 2. Determination of Ea-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 ...

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Abstract

The invention relates to an antimicrobial peptide Ea-CATH1 of Equus asinus and genes and applications thereof, belonging to the technical field of biological medicine. Ea-CATH1 is one genetic coding straight-chain polypeptide of Cathelicidin of Equus asinus, and the complete sequence of Ea-CATH1 is as follows: lysine-arginine-arginine-glycin-serine-valine-threonine-threonine-arginine-tyrosine-glutamine-phenylalanine-leucine-methionine-isoleucine-histidine-leucine-leucine-arginine-proline-lysine-lysine-leucine-phenylalanine-alanine. The gene coding Ea-CATH1 precursor is composed of 550 nucleotides and the part coding mature peptide is the 391-465th nucleotides. Ea-CATH1 has smaller molecular weight, stronger bactericidal effect and broader spectrum and extremely strong killing effect on various clinical drug-resistance bacteria; Ea-CATH1 has simple structure, does not contain disulfide bond and ring structure and is suitable to be prepared by chemical synthesis and gene engineering; and Ea-CATH1 also has useful characteristics such as no hemolysis and ultrastrong serum stability.

Description

technical field [0001] The invention provides a cathelicidin family broad-spectrum antimicrobial peptide Ea-CATH1 of domestic animal donkey (Equus asinus) and its gene 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 many clinical drug-res...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12C12Q1/18G01N21/31
Inventor 于海宁王义鹏迟连利孙文敬冯菲菲
Owner DALIAN UNIV OF TECH
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