A kind of natural antibacterial peptide and its application

A natural antibacterial and antibacterial peptide technology, applied in the field of biomedicine, can solve the problems of human health threats, antibiotic pathogenic microorganism resistance, etc., and achieve the effects of small molecular weight, good stability and simple chemical synthesis method.

Active Publication Date: 2017-11-03
王义鹏
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] In recent years, the large-scale abuse of traditional antibiotics has led to more and more serious drug resistance of pathogenic microorganisms, posing a huge threat to human health.

Method used

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  • A kind of natural antibacterial peptide and its application
  • A kind of natural antibacterial peptide and its application
  • A kind of natural antibacterial peptide and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Cloning of the gene encoding natural antimicrobial peptide QHA:

[0016] 1) Extraction of total RNA from the venom glands of the sea snake:

[0017] ①Take 250 mg of the venom gland tissue of the sea snake venom in a mortar, add liquid nitrogen and grind it into powder, transfer it to an EP tube, add 1 ml of total RNA extraction reagent (Trizol, product of Invitrogen, USA), mix thoroughly, and then heat it at 4°C , Centrifuge at 12000rpm for 10min.

[0018] ② Take the supernatant by centrifugation, add 0.2ml chloroform solution, mix vigorously, leave it at room temperature for 10 minutes, and then centrifuge at 4°C, 12000rpm for 10 minutes to discard the precipitate.

[0019] ③Add an equal volume of isopropanol to the supernatant, place at room temperature for 10 minutes, centrifuge at 4°C, 12000 rpm for 10 minutes, collect the precipitate, wash once with 75% (V / V) ethanol, and dry. The precipitate at the bottom of the tube is a green ring Sea snake venom gland total RNA.

[002...

Embodiment 2

[0035] Chemical synthesis of antimicrobial peptide QHA:

[0036] Ⅰ. Chemical synthesis method of antimicrobial peptide QHA: According to the amino acid sequence of the mature peptide derived from the gene, the complete sequence was synthesized with an automatic peptide synthesizer (433A, Applied Biosystems), and desalted by HPLC reversed-phase column chromatography.

[0037] Ⅱ. The molecular weight is determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF).

[0038] Ⅲ. The purity of the purified antimicrobial peptide QHA was identified by high performance liquid chromatography HPLC. The molecular weight was determined by matrix-assisted laser analysis and ionization time-of-flight mass spectrometry (MALDI-TOF), isoelectric focusing electrophoresis was used to determine the isoelectric point, and the automatic amino acid sequencer was used. Amino acid sequence structure.

[0039] Antimicrobial peptide QHA is a linear polypeptide encoded by...

Embodiment 3

[0042] Antibacterial activity test of antibacterial peptide QHA:

[0043] (1) Pick the test strains stored on the inclined plane and evenly spread them on the flat plate of MH solid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.), and place the sterilized 0.5cm diameter filter paper on the medium On the surface, drop 10μl of the 2mg / ml antimicrobial peptide QHA sample solution dissolved in sterilized deionized water, incubate it upside down at 37°C for 18-20 hours, and observe whether the inhibition zone is formed or not. If the sample has antibacterial activity, a clear and transparent zone of inhibition will be formed around the filter paper. The larger the zone of inhibition, the stronger the antibacterial activity of the sample.

[0044] (2) Determination of Antimicrobial Peptide QHA Minimum Inhibitory Concentration (2-fold dilution method):

[0045] Select the strain with inhibition zone in the previous experiment for MIC determination experiment. The test strain...

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Abstract

Belonging to the technical field of biomedicine, the invention in particular relates to a natural antibacterial peptide QHA and application thereof. The natural antibacterial peptide QHA is composed of 30 amino acid residues, is straight chain polypeptide, and has a molecular weight of 3628.5Da and isoelectric point of 12.61. The antibacterial peptide QHA provided by the invention has the beneficial characteristics of small molecular weight, simple chemical synthesis method, efficient, broad-spectrum and efficient antibacterial action, and good stability, etc., thus having broad application prospects. In addition, the invention also relates to a coding gene of antibacterial peptide QHA and application thereof.

Description

Technical field [0001] The invention belongs to the field of biomedical technology, and specifically is a natural antibacterial peptide QHA and its application. Background technique [0002] In recent years, the large-scale abuse of traditional antibiotics has led to more and more serious pathogenic microorganism resistance problems, posing a huge threat to human health. The clinical response to drug-resistant microbial infections is to use new or alternative antibiotics that have not been used against drug-resistant microbes. Therefore, it is necessary to continue to develop new antimicrobial drugs. [0003] Antibacterial peptide is a kind of natural small molecule polypeptide encoded by organism's gene. It is an important molecule of organism's immune system and has direct killing effect on bacteria, fungi, viruses and even protozoa. Antimicrobial peptides have the advantages of small molecular weight, simple structure, strong antibacterial activity, unique bactericidal mechanis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/46C12N15/12A61K38/17A61K8/64A61P31/10A61P31/04A23K20/147A01N47/44A01P1/00A01P3/00A23K20/195
CPCA61K8/64A61K38/00C07K14/46
Inventor 王义鹏
Owner 王义鹏
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