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Lipopeptides Lin-Lf4NH2 and Lin-Lf5NH2 and application thereof

A technology of lin-lf4nh2 and lipopeptide, which is applied in the direction of peptide/protein components, specific peptides, chemical instruments and methods, etc., to achieve the effect of reducing skin tissue damage, small MIC value, and reducing the amount of skin bacteria

Active Publication Date: 2020-08-25
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still problems in the clinical treatment and application of antimicrobial peptides. In order to improve the antibacterial activity of Lfcins, develop new functions, and combat the emergence of drug-resistant bacteria, it is necessary to use many methods including amino acid substitution, chemical modification, cyclization, chimerism, and polymerization. A molecular design strategy (Hao et al., 2018; Yin et al., 2014)

Method used

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  • Lipopeptides Lin-Lf4NH2 and Lin-Lf5NH2 and application thereof
  • Lipopeptides Lin-Lf4NH2 and Lin-Lf5NH2 and application thereof
  • Lipopeptides Lin-Lf4NH2 and Lin-Lf5NH2 and application thereof

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Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1 lipopeptide Lin-Lf4NH 2 and Lin-Lf5NH 2 design and synthesis of

[0037] The amino acid sequences of the polypeptides LfcinB4 and LfcinB5 are respectively shown in SEQ ID NO: 1 and 2. In order to further improve the antibacterial activity of LfcinB4 and LfcinB5, the structures of the two polypeptides were optimized and designed respectively.

[0038] Dissolve and mix Fmoc-Phe-OH, HOBT, HBTU, and DIEA in DMF, and perform condensation reaction with MBHA resin without Fmoc protecting group to obtain Fmoc-Phe-resin; Follow the same method to condense and react subsequent amino acids, Fmoc-Lf4-resin and Fmoc-Lf5-resin were obtained respectively, and the side chain protecting group was removed with DCM solution containing 1% TFA by volume fraction to obtain Lf4NH 2-resin and Lf5NH 2 -resin; the fatty acids Lin, HOBT, HBTU and DIEA were dissolved and mixed in DMF respectively, and mixed with Lf4NH 2 -resin, Lf5NH 2 -resin undergoes condensation reaction to ob...

Embodiment 2

[0040] Embodiment 2 lipopeptide Lin-Lf4NH 2 and Lin-Lf5NH 2 antibacterial activity assay

[0041] According to the antimicrobial peptide and lipopeptide (Lf4NH obtained in Example 1 2 ,Lf5NH 2 ,Lin-Lf4NH 2 and Lin-Lf5NH 2 ), use ultrapure water to prepare antimicrobial peptide solutions with a concentration of 2560 μg / mL, dilute to a final concentration of 2 μg / mL by 2 times, add different concentrations of antimicrobial peptide solutions to sterile 96-well cell culture plates, each well 10 μL, three parallels for each sample, the same amount (10 μL) of PBS was used as a negative control, and the blank control group was sterile MH medium. Prepare MIC plates. The strain was cultured in MH culture liquid medium, and cultured with shaking at 37°C until the logarithmic phase of growth (OD 600nm =0.4~0.6), the bacterial solution was prepared into a bacterial suspension with a concentration equivalent to 0.5 McFarland turbidimetric standard, and diluted to 10 5 After CFU / mL,...

Embodiment 3

[0045] Embodiment 3 lipopeptide Lin-Lf4NH 2 and Lin-Lf5NH 2 Cytotoxicity test

[0046] Antimicrobial peptides and lipopeptides (Lf4NH 2 , Lf5NH 2 , Lin-Lf4NH 2 and Lin-Lf5NH 2 ), is toxic to Hacat cells. MTT can be reduced to insoluble blue-purple crystalline formazan by succinate dehydrogenase in the mitochondria of living cells, which is deposited in cells, while dead cells do not undergo this reaction. DMSO can dissolve the blue-purple crystalline formazan in cells, and the number of viable cells can be indirectly reflected by the absorbance value of the solution after dissolution. Within a certain range of cell numbers, the amount of crystal formation is proportional to the number of cells.

[0047] 37°C, 5% CO 2 Hacat cells were cultured in MEM complete medium under saturated humidity conditions. Pipet the cells with a pipette gun, resuspend the cells in MEM complete medium, and resuspend the cells at 2.5×l0 5 The cells / mL density was seeded in a 96-well plate,...

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Abstract

The invention provides novel lipopeptides Lin-Lf4NH2 and Lin-Lf5NH2 and application thereof. The lipopeptides Lin-Lf4NH 2 / Lin-Lf 5NH2 are obtained by coupling linoleic acid to the N end of an antibacterial peptide LfcinB4 / LfcinB5 and performing amidation modification on the C end of the antibacterial peptide LfcinB4 / LfcinB 5. Experiments show that the lipopeptides Lin-Lf4NH2 and Lin-Lf5NH2 havea good inhibiting effect on gram-positive bacteria and gram-negative bacteria, have a smaller MIC (minimal inhibitory concentration) value and good thermal stability than a parent peptide, can remarkably reduce the skin bacteria carrying amount and relieve skin tissue injury in a mouse skin abscess model test, have a better treatment effect than Lf4NH2 and Lf5NH2, are small-molecule lipopeptideswith high application value, can be used for preparing novel antibacterial and anti-infection medicines and the like, and have a wide application prospect.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to lipopeptide Lin-Lf4NH 2 and Lin-Lf5NH 2 and its application. Background technique [0002] Lactoferrin peptide (Lactoferricin, abbreviated Lfcin) is a multifunctional antimicrobial peptide released from the N-terminal of lactoferrin by pepsin in an acidic environment (Gifford et al., 2005). Lfcin is closely related to the function of lactoferrin, including Most of the functional domains of lactoferrin have potent antibacterial, anticancer, antiviral, antiparasitic, and anti-inflammatory activities (Hao et al., 2018). In many cases, Lfcin not only retained the activity of lactoferrin, but was even more active than the parent protein (Arias et al., 2014). LfcinB, consisting of amino acid residues 17-41 of bovine lactoferrin, is considered the most potent among various Lfcins including cows, mice and goats (Bruni et al., 2016; Ulvatne et al., 2001). The rapid increase of bac...

Claims

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Application Information

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IPC IPC(8): C07K14/79A61K38/40A61P31/04
CPCC07K14/79A61P31/04A61K38/00Y02A50/30
Inventor 王建华刘鹤毛若雨滕达王秀敏杨娜郝娅
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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