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Rana margaratae antimicrobial peptide odorranain-H-OM1 as well as gene of coded sequence and application thereof

An odorranain-h-om1, coding gene technology, applied in the field of molecular genetics, can solve the problem of no report on the research on the antibacterial peptide of the green stink frog, and achieve the effects of strong killing effect, small molecular weight and low hemolytic activity

Inactive Publication Date: 2014-11-19
GUIZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there have been reports on the morphology, development and molecular evolution of green stinky frogs, but there is no report on the antimicrobial peptides of green stinky frogs

Method used

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  • Rana margaratae antimicrobial peptide odorranain-H-OM1 as well as gene of coded sequence and application thereof
  • Rana margaratae antimicrobial peptide odorranain-H-OM1 as well as gene of coded sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 of the present invention: the cloning of the green stink frog antimicrobial peptide odorranain-H-OM1 gene.

[0022] 1) Extraction of total RNA from green stink frog skin:

[0023] ①Take 300 mg of green stink frog skin tissue, put it into a mortar and add liquid nitrogen to grind it into powder, transfer it to an EP tube, add 1 ml of total RNA extraction buffer (Trizol, product of Invitrogen, USA), mix well, and then Centrifuge at 12000 rpm for 10 min at 4°C.

[0024] ② Centrifuge to get the supernatant, add 0.2 ml chloroform solution, mix vigorously, let stand at room temperature for 10 minutes, then centrifuge at 4°C, 12000 rpm for 10 minutes, discard the precipitate.

[0025] ③ Add an equal volume of isopropanol to the supernatant, place at room temperature for 10 minutes, centrifuge at 4°C, 12,000 rpm for 10 minutes, collect the precipitate, wash it once with 75% (V / V) ethanol, and dry it. The precipitate at the bottom of the tube is green Total RNA fr...

Embodiment 2

[0039] Example 2: Chemical synthesis of the green-smelly frog antibacterial peptide odorranain-H-OM1.

[0040] Ⅰ. The chemical synthesis method of the antimicrobial peptide odorranain-H-OM1 of green stink frog: according to the mature peptide amino acid sequence deduced from the gene, its complete sequence was synthesized with an automatic peptide synthesizer (433A, Applied Biosystems), and desalted by HPLC reverse-phase column chromatography .

[0041] Ⅱ. Molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).

[0042] Ⅲ. The purity of the purified green-smelly frog antibacterial peptide odorranain-H-OM1 was identified by high performance liquid chromatography (HPLC), the molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), and the isoelectric point was determined by isoelectric focusing electrophoresis , Determining the amino acid sequ...

Embodiment 3

[0044] The complete sequence of the green-smelly frog antibacterial peptide odorranain-H-OM1 is shown in SEQ ID No:2. Example 3: Pharmacological experiment of green stinky frog antibacterial peptide odorranain-H-OM1:

[0045] 1. Detection of antibacterial activity of green stinky frog antibacterial peptide odorranain-H-OM1:

[0046] The test strains (Proteus mirabilis, Enterococcus faecium, ampicillin-resistant Escherichia coli and ampicillin-resistant Candida glabrata) preserved on the slant were picked and spread evenly on LB solid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd. ) plate, put a sterilized filter paper sheet with a diameter of 0.5 cm on the surface of the culture medium, and drop 10 μl of the 2 mg / ml sample solution of odorranain-H-OM1 dissolved in sterilized deionized water , Inverted culture at 37 ℃ for 18-20 hours, observe the formation of the inhibition zone or not. If the sample has antibacterial activity, a clear and transparent bacteriost...

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Abstract

The invention discloses rana margaratae antimicrobial peptide odorranain-H-OM1, which is protein having an amino acid sequence as shown by SEQIDNO:1 in a sequence table, or protein obtained by substituting, deleting or adding more than one amino-acid residue to an amino-acid residue sequence as shown by SEQIDNO:2, having the same activity with the amino-acid residue sequence as shown by the SEQIDNO:2 and deriving from the SEQIDNO:2. The coding gene of the rana margaratae antimicrobial peptide odorranain-H-OM1 is obtained by cloning the rana margaratae antimicrobial peptide odorranain-H-OM1, and the odorranain-H-OM1 is synthesized by using a chemical synthesis method. The antimicrobial peptide is small in molecular weight, and has a strong killing effect on balcillus proteus mirabilis, enterococcus faecium, ampicillin-resistant escherichia coli and candida glabrata. Besides, the antimicrobial peptide is low in hemolytic activity.

Description

technical field [0001] The invention relates to the field of molecular genetics, in particular to a green stinky frog antimicrobial peptide and the gene and application of its coding sequence. Background technique [0002] With the large-scale and inappropriate use of traditional antibiotics such as penicillin, microorganisms have become more and more resistant to traditional antibiotics, and a large number of microorganisms that can completely tolerate traditional antibiotics such as penicillin have appeared clinically. Antibiotics are powerless against these pathogenic microorganisms. Antimicrobial peptides are a new type of antimicrobial peptides. Most antimicrobial peptides have a molecular weight of less than 10,000 Da, are positively charged, rich in hydrophobic bases, and can form an amphiphilic structure. The bactericidal mechanism of antimicrobial peptides is mainly to attract and bind to the negatively charged bacterial cell membrane surface through electrostatic ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/46C12N15/12A61K38/17A61K8/64A61P31/04A61P31/10A61P39/06
CPCY02A50/30
Inventor 周江凌桂英高久香李莉王义鹏
Owner GUIZHOU NORMAL UNIVERSITY
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