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Antibacterial peptide PD22 and application thereof

A PD22 and antimicrobial peptide technology, applied in the field of genetic engineering, can solve the problems of limiting the popularization and application of antimicrobial peptides, host cell toxicity and side effects, and low antibacterial activity, and achieve the effects of convenient artificial synthesis, small molecular weight, and low cytotoxicity

Active Publication Date: 2015-10-28
江苏傲农生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the antimicrobial peptides from natural sources have defects such as large molecular weight, low antibacterial activity, toxic side effects on host cells, or hemolysis of the host, which limits the popularization and application of antimicrobial peptides in weaned piglets to a certain extent.

Method used

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  • Antibacterial peptide PD22 and application thereof
  • Antibacterial peptide PD22 and application thereof
  • Antibacterial peptide PD22 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Preparation of antimicrobial peptide PD22

[0021] The chemical synthesis method of the antimicrobial peptide PD22: according to the amino acid sequence described in the summary of the invention, the full sequence of PD22 (synthesized by the School of Life Sciences, Hunan Normal University) was synthesized with an automatic peptide synthesizer (ABI433), and purified by desalting and desalting by HPLC reverse-phase column chromatography ;

[0022] The molecular weight of the purified antimicrobial peptide PD22 was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), the isoelectric point was determined by isoelectric focusing electrophoresis, and the amino acid sequence was analyzed by an automatic amino acid sequencer.

[0023] Antimicrobial peptide PD22, the antimicrobial peptide PD22 is an active polypeptide artificially designed and synthesized, comprising 13 amino acid residues, a molecular weight of 1...

Embodiment 2

[0024] Embodiment 2: antimicrobial peptide PD22 antibacterial experiment

[0025] The antibacterial effect was reflected by measuring the minimum inhibitory concentration (MIC) of the antimicrobial peptide PD22, which was the lowest sample concentration at which no bacterial growth could be detected. Using the double dilution method, the specific method is as follows:

[0026] The test bacteria were inoculated on LB solid medium, and then cultured upside down in a 37°C incubator. After the colonies grow out, use a disposable inoculation loop to pick a single colony of each test bacterium into LB liquid medium, and culture it in a 37°C incubator with shaking until the logarithmic growth phase. Detect the OD600 of the bacterial solution on the ultraviolet spectrophotometer, according to 1OD600=1×10 9 CFU / mL Dilute the test bacteria liquid with liquid LB medium to 2×10 5 CFU / mL. Then add 100 μL of LB liquid medium to each well of a sterile 96-well plate, and then add 100 μL o...

Embodiment 3

[0032] Embodiment 3: Cytotoxicity experiment of antimicrobial peptide PD22

[0033] Pig small intestinal epithelial cells IPEC-1 (provided by the Institute of Subtropical Agroecology, Chinese Academy of Sciences) were cultured in a medium containing 5% Tai bovine serum, 200U / mL double antibody (penicillin and streptomycin each 100U / mL), 5ug / L epidermal growth factor ( EGF) cultured in HAM'S / F12 medium to logarithmic phase, washed three times with phosphate buffer (HyClone), added 0.25% (2.5g / L) trypsin to digest to a single cell, added HAM'S / F12 culture Centrifuge at 400g for 4 minutes after the base, discard the supernatant, suspend the cells with fresh HAM'S / F12 medium, and adjust the cell density to 1×10 6 cells / mL, then add 200uL to each well of a 96-well cell culture plate, add different concentrations of antimicrobial peptide PD22 after the cells adhere to the wall, and place in a carbon dioxide incubator (37°C, 5% CO 2 ) after culturing for 24 hours, add 20uL CCK-8 (Be...

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Abstract

The invention discloses an antibacterial peptide PD22 and application thereof. The amino acid sequence of the antibacterial peptide PD22 is shown as SEQ ID NO: 1. The antibacterial peptide PD22 has the advantages that the molecular mass is small, the artificial synthesis is convenient, the cost is low, the functions of killing such bacteria as colibacillus, salmonella and clostridium welchii are strong and the cytoxicity is low; when the antibacterial peptide PD22 is applied to feed for weaned pigs, the diarrhea rate of the weaned pigs can be remarkably reduced, and the growth rate of the weaned pigs can be improved.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an antimicrobial peptide PD22 and its application. Background technique [0002] In order to improve the reproductive efficiency of sows and reduce the vertical transmission of maternal diseases, piglets in pig farms in my country generally choose to be weaned at 3-4 weeks of age, while the weaning process generally occurs at 3-4 months in nature. Early weaning of piglets caused great stress to piglets. Piglets were separated from sows, nutritional sources changed from liquid breast milk to solid feed, mixed groups and environmental changes, and the digestive system and immune system of early weaned piglets were not fully developed. It is very easy to be attacked by various pathogenic bacteria and infected with diseases, among which digestive tract disease is one of the most common and serious diseases, and it is also an important cause of growth inhibition and even death of we...

Claims

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Application Information

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IPC IPC(8): C07K7/08A61K38/10A61P31/04A23K1/17A23K1/18
Inventor 杨焕胜肖丽萍施建成周盛昌杨林姜文叶俊标
Owner 江苏傲农生物科技有限公司
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