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Active polypeptide of brown-spotted torrential frog, its gene and application in drug preparation

A technology of active peptides and turbulent frogs, which is applied in the field of biomedicine, can solve the problem that there is not much research on skin active peptide substances, and achieve the effects of inhibiting the growth of bacteria and fungi, wide antibacterial spectrum, and convenient artificial synthesis.

Inactive Publication Date: 2007-11-28
NANJING AGRICULTURAL UNIVERSITY
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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 its active ingredients and pharmacological properties mainly focuses on small organic molecules such as alkaloids, and there are not many studies on its skin active peptides

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Cloning of the active polypeptide gene of the brown-spotted turbulent frog:

[0033] I. Extraction of total RNA from brown-spotted turbulent frog skin:

[0034] A. The living brown-spotted turbulent frog was cleaned with water, put into liquid nitrogen and quick-frozen for 4 hours, got the skin tissue, weighed, got 300mg skin tissue, added 10ml total RNA extraction buffer (Trizol solution, U.S. GIBCOBRL company product), in Homogenize in a 20ml glass homogenizer for 30 minutes.

[0035] B. Add an equal volume of phenol / chloroform solution, mix vigorously, leave at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, and discard the precipitate.

[0036] C. Add an equal volume of isopropanol to the supernatant, leave it at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, wash the precipitate once with 75% ethanol, and dry it in the air.

[0037] II. Purification of brown-spotted frog skin mRNA:

[0038] Brown...

Embodiment 2

[0109] Preparation of brown-spotted frog active polypeptide:

[0110] I. The preparation method of the active polypeptide of Rana variegata: infer the amino acid sequence of the active polypeptide of Rana variegata according to the gene encoding the active polypeptide of Rana variegata, and synthesize its full sequence with an automatic polypeptide synthesizer. Reversed phase C by HPLC 18 Desalted and purified by column chromatography.

[0111] II. The molecular weight is determined by fast atom bombardment mass spectrometry (FAB-MS), with glycerol: m-nitrobenzyl alcohol: dimethyl sulfoxide (1: 1: 1, V: V: V, volume ratio) as substrate, Cs + As the bombardment particles, the current is 1μA and the emission voltage is 25Kv.

[0112] III. The purity of the purified brown-spotted frog active polypeptide was identified by high-performance liquid chromatography (HPLC), the molecular weight was determined by fast atom bombardment mass spectrometry, the isoelectric point was deter...

Embodiment 3

[0115] The effect of the active polypeptide of the brown-spotted frog on inhibiting the growth of bacteria:

[0116] The antibacterial activity was detected by the cup and saucer method, and the medium was ordinary agar medium. Inject 20ml of heated and melted medium into the plate as the bottom layer, spread it evenly in the bottom of the plate, after solidification, take another appropriate amount of medium and heat and melt, add 5ml of bacterial suspension to each plate, shake well, Spread it evenly on the bottom layer as a bacterial layer. After cooling, put 6 sterilized stainless steel cups evenly in the plate at equal distances. Add 0.1ml of the test compound solution with a concentration of 0.1-0.3mg / ml to the first steel cup, add the sample solution to the other steel cups by doubling dilution method, incubate at 37°C, and observe the size of the inhibition zone. The bacteriostatic zone above 10mm was regarded as the minimum inhibitory concentration (minimal inhibito...

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PUM

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Abstract

The invention relates to an active polypeptide, its genes and use in pharmacy, wherein the active peptide is a single-chain polypeptide encoded by Amolops loloensis gnens, a Chinese amphiphyte, the molecular weight is 2664.36 Da, the isoelectric point is 9.70, the primary structure of the polypeptide sequence is Phe Leu Pro Leu Ala Val Ser Leu Ala Ala Asn Phe Leu Pro Lys Leu Phe Cys Lys Ile Thr Lys Lys Cys -AMIDATION. The gene that encodes the Amolops loloensis active peptide comprises 448 nucleotides, among which position No:247-318 nucleotides encode the ripened Amolops loloensis active peptide. The artificially synthesized Amolops loloensis active peptide has appreciable actions for inhibiting growth of bacteria and fungus, thus can be used as the medicament for treating diseases infected by pathogenic microorganism.

Description

Technical field: [0001] The invention relates to an active polypeptide of Amolops loloensis and its gene and application in pharmacy, belonging to the field of biomedicine. Background technique: [0002] Since the invention of antibiotics, humans have made great achievements in the control and treatment of microbial infection diseases. However, with the continuous use of "traditional antibiotics", many new problems continue to arise. At present, microbial resistance has become a major problem in the treatment of clinical microbial infection diseases, so that some pathogenic bacteria have no first-line drugs for clinical treatment. Vancomycin-resistant staphylococci, enterococci and other Gram-negative infectious diseases are currently clinical problems worldwide. Three types of bacteria that mainly cause meningitis have also emerged clinically with strong drug resistance. Penicillin-resistant , chloramphenicol, meningococci, pneumococci, and pneumococci resistant to neocep...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12A61K38/17A61P31/00
Inventor 赖仞徐学清梁建国韩曜平杨海龙李建许
Owner NANJING AGRICULTURAL UNIVERSITY
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