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Novel broad-spectrum antimicrobial peptide SAMP1-A4 and preparing method thereof

An antibacterial peptide, broad-spectrum technology, applied in the field of novel antibacterial peptides and their preparation, can solve the problems of unstable expression product, low product yield, easy to be degraded, etc.

Active Publication Date: 2019-04-16
HENAN UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Using genetic engineering technology to express polypeptides, the expression products are unstable, easily degraded, and difficult to purify
Chemically synthesize polypeptides with more than 30 amino acids, the product yield is low and the purity is low

Method used

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  • Novel broad-spectrum antimicrobial peptide SAMP1-A4 and preparing method thereof
  • Novel broad-spectrum antimicrobial peptide SAMP1-A4 and preparing method thereof
  • Novel broad-spectrum antimicrobial peptide SAMP1-A4 and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Preparation of Antimicrobial Peptide SAMP1-A4

[0019] The target peptide was synthesized by the solid-phase Fmoc method from the C-terminus to the N-terminus. The carboxyl group of the first Fmoc-Ile-OH at the C-terminus of the polypeptide is activated and combined with the resin, the Fmoc-protecting group is removed, and the Fmoc-Arg(Pbf)-OH activated by the second carboxyl group is combined, and the Fmoc-Ile-OH is removed. Protecting group, the cycle is repeated until all amino acids are added. Cut the peptide on the resin with cutting solution, collect the cutting solution, dry it in a centrifuge tube, wash the precipitate with ice ether 2~3 times, centrifuge to remove the supernatant, and dry the precipitate. The obtained crude product was purified by HPLC.

Embodiment 2

[0020] Example 2 Antimicrobial Spectrum Determination of Antimicrobial Peptide SAMP1-A4

[0021] 1) Weigh and dissolve protein. Prepare the SAMP1-A4 phosphoric acid solution, set the initial concentration in the first column to 500 μg / mL, and set the final concentration to 0.975 μg / mL. It is necessary to prepare 2mg / mL SAMP1-A4 stock solution. Dissolve SAMP1-A4 with 20 mmol / L sodium phosphate buffer solution pH6.0, and filter the solution through a 0.22 μm sterile filter membrane to sterilize.

[0022] 2) Using a pipette gun, add 100 μL of 20 mmol / L pH6.0 sodium phosphate buffer to each well of the 96-well plate to dilute SAMP1-A4.

[0023] 3) Use a pipette gun to pipette 100 μL of 2 mg / mL SAMP1-A4 stock solution into each well of the first column of the 96-well plate.

[0024] 4) Repeatedly blow and aspirate the solution in the first column of the plate for 6-8 times, mix well without splashing.

[0025] 5) Aspirate 100 μL from the first column and add it to the second co...

Embodiment 3

[0037] Example 3 Determination of antibacterial peptide SAMP1-A4 bactericidal and antibacterial activity

[0038] The bactericidal and antibacterial activity of SAMP1-A4 was detected by measuring the number of viable bacteria / fungi after the action of antimicrobial peptides. Candida tropicalis is the representative fungus, and Listeria monocytogenes is the representative bacterial strain.

[0039] Candida tropicalis: culture Candida tropicalis at 28°C with SD liquid medium to OD 600 ≈0.6-0.8. Add the bacterial suspension into the solution containing 1×MIC 100 In the SD liquid medium of SAMP1-A4, the bacterial concentration was about 1×10 5 CFU / mL. At the same time, a blank SD medium without SAMP1-A4 was set as a control. Cultivate at a constant temperature in a shaker at 28°C. After culturing for 4 hours, absorb 100 μL of the bacterial solution, spread it on an SD agar plate after dilution, and culture overnight at 28°C for colony counting.

[0040] Listeria monocytoge...

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Abstract

The invention discloses a novel broad-spectrum antimicrobial peptide SAMP1-A4 and a preparing method thereof. The antimicrobial peptide has the amino acid sequence of NH2-VRLLRRRI-COOH. The preparingmethod is a solid-phase polypeptide synthesis method protected by amino acid nitrogen Fmoc. The defect that a frequently-seen antibacterial peptide is poor in biological safety, prone to hemolysis ortoxic to mammal cells is overcome, and the antimicrobial peptide is a novel antimicrobial peptide which has the broad-spectrum restraining effect on non-filamentous fungi and bacteria, has no hemolysis and is free of toxin to normal mammal cells. The peptide provides a novel primer for clinical antifungal medicine research.

Description

technical field [0001] The invention belongs to biological preparations, and in particular relates to a novel antibacterial peptide and a preparation method thereof. Background technique [0002] In recent years, due to the abuse of antibiotics, the number of isolated drug-resistant strains, especially drug-resistant fungi, in infectious diseases is increasing. With the emergence of super drug-resistant bacteria, there is an increasingly urgent need for new clinical antibiotics that are not easily resistant to bacteria and fungi. Therefore, it is of great significance to research and develop new antibacterial drugs with high efficiency, low toxicity, and pathogenic bacteria are not easy to produce drug resistance to them as soon as possible. [0003] Antimicrobial peptides are an important part of the natural immune system of animals. Unlike traditional antibiotics, which mainly function by inhibiting a certain biosynthetic pathway (such as cell wall, protein), most antimi...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K1/06C07K1/04
CPCC07K7/06
Inventor 李瑞芳石薇妮常俊朋
Owner HENAN UNIVERSITY OF TECHNOLOGY
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