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Bloody noun antibacterial peptide temporin-La, genes thereof and use in pharmacy

An antimicrobial peptide and bullfrog technology, which is applied in the field of biomedicine, can solve the problems of limited research on skin active peptides, achieve the effects of inhibiting the growth of bacteria and fungi, convenient artificial synthesis, and a wide antibacterial spectrum

Inactive Publication Date: 2009-07-08
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The application of amphibian drugs has a long history in my country, but the research on their active ingredients and pharmacological properties mainly focuses on small organic molecules such as alkaloids, and there are not many studies on their skin active peptides

Method used

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  • Bloody noun antibacterial peptide temporin-La, genes thereof and use in pharmacy
  • Bloody noun antibacterial peptide temporin-La, genes thereof and use in pharmacy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Bullfrog antimicrobial peptide gene cloning:

[0040] I. Extraction of Total RNA from Bullfrog Skin

[0041] A. The living bullfrog was cleaned with double distilled water, and the sterilized needle was paralyzed from the medullary cavity of the bullfrog. Take 50-100 mg bullfrog skin tissue and put it into a dry-baked mortar, add 1 mL TRIZOL Reagent (product of Invitrogen Company) Grind in an ice water bath.

[0042] B. After fully mixing, transfer to a 1.5mL centrifuge tube (DEPC tube) and place at 15-30°C for 5min, add an equal volume of chloroform, vortex mix for 15s, and centrifuge at 12000×g for 15min at 4°C.

[0043] C. Add 500 μL of isopropanol to the supernatant, place at 15-30°C for 5 minutes, vortex for 15 seconds, and centrifuge at 12,000×g for 10 minutes at 4°C. Discard the supernatant, add 1 mL of 75% ethanol to rinse the precipitate, centrifuge at 7500×g for 5 min, and repeat once. Dry it in an ultra-clean workbench for 90 seconds, and dissolve it in 30...

Embodiment 2

[0109] Preparation of bullfrog antimicrobial peptide

[0110] I. Sample preparation method: deduce the amino acid sequence of the bullfrog antimicrobial peptide according to the gene encoding the bullfrog antimicrobial peptide, and synthesize its full sequence with an automatic peptide synthesizer APEX396. Reversed phase C by HPLC 18 Desalted and purified by column chromatography.

[0111] II. Molecular weight was determined by electrospray ionization (ESI) with an emission voltage of 4.5Kv.

[0112] III, the bullfrog antimicrobial peptide of purification is identified its purity (flow velocity 1.0ml / min with high performance liquid chromatography HPLC method; Mobile phase acetonitrile+water+TFA0.01%, gradient elution; Chromatographic column C 18 , the detection wavelength is 220nm), the molecular weight is determined by fast atom bombardment mass spectrometry, the isoelectric point is determined by isoelectric focusing electrophoresis, and the amino acid sequence structure ...

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Abstract

The invention provides bullfrog antimicrobial peptide temporin-La and a gene and application thereof in pharmacy, which belong to the field of biomedicine. The gene of the bullfrog antimicrobial peptide is obtained by screening from a constructed bullfrog skin cDNA library, the molecular weight is 1,623.08D, the isoelectric point is 9.70, and a primary structure of a complete sequence of the peptide is Leu Leu Arg His Val Val Lys Ile Leu Glu Lys Tyr Leu-AMIDATION. The gene for encoding the bullfrog antimicrobial peptide consists of 324 nucleotides, wherein 142th to 180th of nucleotides are used for encoding the mature peptide. The bullfrog antimicrobial peptide which is artificially synthesized has obvious effect of inhibiting the growth of bacteria and fungi, has the advantage of no hemolytic activity, and can be applied to preparing a medicine for treating pathogenic microorganism infection diseases.

Description

technical field [0001] The invention relates to a bullfrog antimicrobial peptide temporin-La and its gene and its application in pharmacy, belonging to the technical field of biomedicine. Background technique [0002] Antimicrobial peptides, as a new concept of broad-spectrum antibacterial protein antibiotics, will have a broader market prospect than traditional antibiotics, and are expected to become a new generation of antibacterial agents, and their development is currently receiving extensive attention. At present, more than 300 different antimicrobial peptides have been isolated from the skin of different anura animals, and the number is still increasing. Researchers from the Department of Biotoxins, Kunming Institute of Zoology, Chinese Academy of Sciences discovered 107 new antimicrobial peptides in Odorrana grahami (fingerless frog, a kind of amphibian), and conducted research on the antibacterial mechanism. It is three times more than the number of amphibian antimi...

Claims

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Application Information

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IPC IPC(8): C07K7/08C12N15/12A61K38/10A61P31/00A61P35/00
Inventor 韩文瑜赵瑞利雷连成韩俊友冯新孙长江
Owner JILIN UNIV
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