Rana grahami peptide, gene thereof and application in pharmacy

A stink frog and gene technology, applied in the field of biomedicine, can solve the problems of limited research on skin active peptides, achieve the effect of inhibiting the growth of bacteria and fungi, simple structure, and wide antibacterial spectrum

Inactive Publication Date: 2007-08-08
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented method describes how certain specific sequences found between DNA segments are involved with making up nonsense mutations that can stop or slow down germ formation by preventing harmful microorganisms like yeast cells (frogs) from growing too much faster than usual. These sequence-specific modifications also make it difficult for other organism types to develop resistance against them effectively.

Problems solved by technology

This patented describes how tiny catheter structures called magains (magnetic particles) were discovered during early stages of life that could help fight against harmful germs like yeast spores. These magnetosphericles made up of iron oxysulfonucleoprotein nanoparticles attached to membrane lipids protecting these metabolites from external environments. However, little work was done exploring this type of substance due largely to lack of knowledge about it.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Cloning of Frog Peptide Gene:

[0031] I. Extraction of total RNA from the skin of the fingerless frog:

[0032] A. The live fingerless frog is cleaned with water, put into liquid nitrogen and quick-frozen for 4 hours, get the skin tissue, weigh, get 300mg skin tissue, add 10ml total RNA extraction buffer (Trizol solution, U.S. GIBCOBRL company product), Homogenize for 30 minutes in a 20ml glass homogenizer.

[0033] 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.

[0034] 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. The sediment at the bottom of the tube is the total RNA of the frog skin .

[0035] II. Purification of the skin mRNA of the fingerless frog:

...

Embodiment 2

[0103] Preparation of the fingerless stinky frog stinky frog peptide:

[0104] I, the preparation method of the fingerless frog stinky frog peptide: infer the amino acid sequence of the fingerless stinky frog stinky frog peptide according to the gene encoding the fingerless stinky frog stinky frog peptide, and synthesize its full sequence with an automatic polypeptide synthesizer. Reversed phase C by HPLC 18 Column desalting and purification.

[0105] 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 bombardment particles, the current is 1μA and the emission voltage is 25Ky.

[0106] III. Use high-performance liquid chromatography (HPLC) and mass spectrometry to identify the purity of the purified stinky frog peptide. The molecular weight is determined by fast atom bombardment mass spectrometry. The isoelectric point is de...

Embodiment 3

[0109] Pharmacological experiment of stinky frog peptide:

[0110] 1. Inhibition of Bacteria Growth

[0111] 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, and spread it evenly on the bottom of the plate. After solidification, take another appropriate amount of medium and heat it to dissolve. Add 5ml of bacterial suspension to each plate, shake well to make it on the bottom layer Spread evenly on the top as a bacterial layer. After cooling, put 6 sterilized stainless steel cups equidistantly in the flat blood. Add 0.1ml of the compound solution to be tested at a concentration of 0.3mg / ml to the first steel cup, add the sample solution to the remaining steel cups by doubling the dilution method, incubate at 37°C, and measure the size of the inhibition zone after 24 hours. A bacteriostatic zone above 10mm was regarded as the minimum inhib...

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Abstract

The invention discloses a non-finger frog peptide and gene and application in the drug in the biological medical domain, which codes a single-chained peptide with molecular weight at 1814.11 and isoelectric point at 8.08, wherein the primary structure of peptide is Gly Cys Ser Arg Trp Ile Ile Gly Ile His Gly Gln Ile Cys Arg Asp (GCSRWIIGIHGQICRD); the gene of secretory peptide is composed of 312 nucleic acids, which codes 141-189th nucleic acid; the peptide possesses obvious bacteria and fungi inhibiting action, which can be applied to make microbe infection disease drug.

Description

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Claims

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Application Information

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Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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