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Bullfrog skin active peptide, gene and application thereof in pharmacy

A technology of active peptides and bullfrogs, applied in the field of biomedicine, can solve the problems of not easy to form drug resistance, achieve the effects of inhibiting bacterial growth, convenient artificial synthesis, and simple structure

Inactive Publication Date: 2010-06-09
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Since the discovery of penicillin, antibiotics have always been a powerful weapon for humans to treat diseases caused by pathogenic microorganisms. However, due to the continuous use and abuse of "antibiotics" by humans, many bacteria have developed resistance to existing antibiotics, so looking for new ones The mechanism of killing pathogenic microorganisms and the new type of antibiotics that are not easy to form drug resistance have become one of the current research hotspots

Method used

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  • Bullfrog skin active peptide, gene and application thereof in pharmacy
  • Bullfrog skin active peptide, gene and application thereof in pharmacy
  • Bullfrog skin active peptide, gene and application thereof in pharmacy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Cloning of bullfrog skin active peptide gene:

[0054] I. Extraction of Total RNA from Bullfrog Skin

[0055] A. The live bullfrog was cleaned with double-distilled water, and the medullary cavity of the bullfrog was destroyed with a sterilized needle. 50-100 mg of bullfrog skin tissue was put into a dry-baked mortar, and 1 mL of TRIZOL Reagent (product of Invitrogen Company) was added to quickly Grind in an ice water bath.

[0056] 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.

[0057] C. Add 500 μL of isopropanol to the supernatant, place at 15-30°C for 5 minutes, vortex for 15 seconds to mix, and centrifuge at 12000×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 in an ultra-clean workbench for 90 seconds, di...

Embodiment 2

[0103] Preparation of bullfrog skin active peptide

[0104] I. Sample preparation method: deduce the amino acid sequence of the bullfrog active peptide according to the gene encoding the bullfrog skin active peptide, and synthesize its full sequence with an automatic peptide synthesizer APEX396. Desalted and purified by HPLC reverse phase C18 column chromatography.

[0105] II. Molecular weight was determined by electrospray ionization (ESI) with an emission voltage of 4.5Kv.

[0106] III. The purity of the purified bullfrog lectin was identified by high performance liquid chromatography HPLC (flow rate 1.0mL / min; mobile phase acetonitrile+water+TFA0.01%, gradient elution; chromatographic column C18, detection wavelength 220nm), molecular weight determination The isoelectric point was determined by fast atom bombardment mass spectrometry and isoelectric focusing electrophoresis, and the amino acid sequence structure was determined by an automatic amino acid sequencer.

[010...

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Abstract

The invention relates to a bullfrog skin active peptide, a gene and application thereof in pharmacy. The bullfrog skin active peptide is obtained by screening a constructed bullfrog skin cDNA library, and is a single-chain polypeptide coded by a bullfrog skin active peptide gene. The molecular weight is 3,144.8 Dalton; the isoelectric point is 10.16; and the complete-sequence primary structure of polypeptide is Met Met Arg Val Met Arg Arg Lys Thr Lys Val IIe Trp Glu Arg Arg Asp Phe IIe Gly Leu Tyr Ser IIe Asp (MMRVMRRKTKVIWEKKDFIGLYSID). The gene of the coded bullfrog skin active peptide consists of 369 nucleotides, wherein the coded bullfrog skin active peptide is nucleotides at positions 97 to 171. The synthetic bullfrog skin active peptide has obvious effect of inhibiting bacterial growth, can be applied to the preparation of medicaments for treating pathogenic microbial infection diseases, and has the function of clearing free radicals. The bullfrog skin active peptide has the characteristics of low molecular weight, simple synthesis, strong antibacterial activity, oxidation resistance and the like.

Description

technical field [0001] The invention relates to a bullfrog skin active peptide and its gene and its application in pharmacy, belonging to the field of biomedicine. Background technique [0002] Since the discovery of penicillin, antibiotics have always been a powerful weapon for humans to treat diseases caused by pathogenic microorganisms. However, due to the continuous use and abuse of "antibiotics" by humans, many bacteria have developed resistance to existing antibiotics, so looking for new ones New antibiotics that kill pathogenic microorganisms by mechanism and are not easy to form drug resistance have become one of the current research hotspots. Amphibian animal skin peptides not only have the characteristics of high efficiency, broad spectrum, and are not easy to produce drug resistance, but they have become the main biological resources of new biological agents for people to deal with the increasingly serious drug-resistant bacteria today when antibiotic-resistant pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/46A61K38/17A61P31/04A61P17/18
Inventor 韩文瑜韩俊友赵瑞利雷连成冯新孙长江
Owner JILIN UNIV
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