Antibacterial peptide and application thereof

A technology of antimicrobial peptides and drugs, applied in the direction of cationic antimicrobial peptides, applications, antibacterial drugs, etc., can solve the problems of low antibacterial activity and hemolysis

Inactive Publication Date: 2018-07-24
江西华牧生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although there are many studies on antibiotic peptides, there are still some problems, for example, l

Method used

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  • Antibacterial peptide and application thereof
  • Antibacterial peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Preparation of porcine-derived antimicrobial peptide PMAP-36 mutant

[0019] First, the porcine antimicrobial peptide PMAP-36 (SwissProt ID: P49931) is an antimicrobial peptide composed of 37 amino acids. However, structural analysis revealed that the antimicrobial peptide has multiple folded structures (see figure 1 B), it is not conducive to the expression of antimicrobial peptides and membrane penetration during the antibacterial process. In order to further improve its antibacterial activity, the spatial structure analysis of the amino acid sequence of the porcine antimicrobial peptide PMAP-36 was finally completed. Five amino acid mutations in 36 (L7V, T11S, I18E, I25A and I32A) were used to obtain the porcine antimicrobial peptide PMAP-36 mutant, whose amino acid sequence is SEQ ID NO:1.

[0020] Second, entrust a related peptide synthesis company to synthesize and prepare the porcine antimicrobial peptide PMAP-36 mutant with the amino acid sequence of...

Embodiment 2

[0021] Embodiment 2: antibacterial test

[0022] Taking the test strain Escherichia coli after activation in the laboratory as the test object, using the antibacterial operation method, setting a positive control (ampicillin solution), a negative control (distilled water), and studying the antimicrobial peptide PMAP-36 before transformation and Example 1 The antibacterial effect of the porcine antimicrobial peptide PMAP-36 mutant whose modified amino acid sequence is SEQ ID No.1 is prepared. Among them, Escherichia coli is used as the test bacterium, and the bacteriostatic test of antimicrobial peptides on Escherichia coli is carried out, and the culture condition is cultured in a constant temperature incubator at 37°C. Among them, 50 μL of the sample to be tested was added dropwise to each well, the amp concentration of the positive control group was 1 M, and the modified porcine antimicrobial peptide PMAP-36 mutant and PMAP before modification were added dropwise at a concen...

Embodiment 3

[0024] Example 3: Minimal Hemolytic Concentration Determination (MHC)

[0025] Referring to Example 5 of the invention patent application CN201410669038.8, the minimum hemolytic concentration of the antimicrobial peptide was determined. The specific method is as follows:

[0026](1) Add 100 μL of 8% porcine red blood cells resuspended in PBS to a 96-well plate, and then add 100 μL of antibacterial peptides serially diluted in PBS, so that the concentrations of antimicrobial peptides in each well are 100 μg / mL, 50 μg / mL, and 25 μg / mL, 12.5μg / mL, 6.25μg / mL, 3.12μg / mL, 1.56μg / mL and 0.78μg / mL. Add 100 μl 0.2% Triton X-100 to positive control wells, add 100 μL PBS to negative control wells, incubate at 37°C for 1 hour, centrifuge at 3000 rpm for 5 minutes, pipette 100 μL supernatant from each well into another 96-well plate, measure OD value at 550 nm wavelength Calculate hemolysis percentage=[(OD value of experimental well-OD value of negative well) / (OD value of positive well-...

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Abstract

The invention relates to an antibacterial peptide. The antibacterial peptide is characterized in that the amino acid sequence of the antibacterial peptide is shown in SEQ ID No. 1. The antibacterial peptide enables five amino acids in an amino acid sequence of a swine-origin antibacterial peptide PMAP-36 to mutate based on the spatial structure analysis of the amino acid sequence of the swine-origin antibacterial peptide PMAP-36, and the five amino acids are respectively L7V, T11S, I18E, I25A and I32A. The antibacterial peptide product with higher antibacterial activity and better safety is obtained.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to an antibacterial peptide and its application. Background technique: [0002] my country is the world's largest producer and user of antibiotics, and it is also the hardest hit area for antibiotic abuse and bacterial resistance. Abuse of antibiotics, especially in the field of animal husbandry is particularly serious. The reason why livestock farmers like to use antibiotics is that antibiotics can not only prevent and cure diseases, but also promote animal growth and increase production. Many farmers generally add antibiotics to feed as health products to promote growth. The risk of "super bacteria" caused by this and the aggravation of environmental pollution are challenging the bottom line of Chinese people's health. Bacterial resistance caused by veterinary antibiotics and the resulting "super bacteria" will not be limited to poultry and livestock, but will also affect to humans...

Claims

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Application Information

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IPC IPC(8): C07K14/47A61K38/17A61P31/04A61P1/12A23K20/147A23K50/00A23K50/70
CPCA23K20/147A23K50/00A23K50/70A61K38/00C07K14/4723
Inventor 张磊曾和华陈瑞张志刚
Owner 江西华牧生物科技有限公司
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