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An antimicrobial peptide hehampii-4(63-88) and its application

A technology of antimicrobial peptides and nucleic acids, applied in the fields of application, antibacterial drugs, antifungal agents, etc., can solve the problems of natural antimicrobial peptide toxicity, eukaryotic cell hemolysis, and narrow antimicrobial spectrum of antimicrobial peptides, and achieve broad agglutination spectrum and inhibition good effect

Active Publication Date: 2022-05-17
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although antimicrobial peptides have many advantages, there are also some disadvantages. For example, a considerable part of natural antimicrobial peptides are highly toxic and can even cause hemolysis of eukaryotic cells; Specific groups of microorganisms have antibacterial activity, such as only against Gram-negative bacteria, only against Gram-positive bacteria, only against fungi, or only against one or more microorganisms

Method used

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  • An antimicrobial peptide hehampii-4(63-88) and its application
  • An antimicrobial peptide hehampii-4(63-88) and its application
  • An antimicrobial peptide hehampii-4(63-88) and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Determination of the sequence of the striated hippocampal antimicrobial peptide hepcidin (HeHampⅡ-4)

[0021] The present invention screens and obtains the cDNA sequence of HeHampⅡ-4 based on the genome and transcriptome database of the striated hippocampus, and clones the antimicrobial peptide sequence from the striated hippocampus tissue. The specific steps are as follows:

[0022] Total RNA was extracted from adult male hippocampal liver tissue using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to its attached instructions. Using the ReverAce qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) kit, 1 microgram of total RNA was used for cDNA first-strand synthesis. Primer 5.00 (Palo Alto, CA) was used to design the gene-specific primers F, R (F: 5'-ATGAAGCCCTTCAGTTTGTC-3'; R: 5'-TTAGAATCTCCATTCGCAGC-3'), which were used to amplify the opening of HeHampⅡ-4 Reading frame (ORF). In this study, the PCR reaction follows the following cycle pa...

Embodiment 2

[0023] Example 2 Detection of the biological activity of the mature peptide of the striated hippocampus antimicrobial peptide hepcidin (HeHampⅡ-4)

[0024] Synthesis of mature peptide of HeHampⅡ-4

[0025] The amino acid sequence of the mature peptide HeHamp II-4 (63-88) is: HSGPCKFCCNCCGRMHFCGFCCEWRF, or as shown in SEQ ID NO:1. It was synthesized by GenScript Biotechnology Company (Nanjing, China) by solid-phase peptide synthesis. The molecular mass and purity of the synthetic peptide were 2.747kD and 96.1%, respectively. Synthetic peptides were stored as dry powder at -80°C until use.

[0026] Microbial agglutination test

[0027] Gram-negative bacteria used in the agglutination test: Vibrio parahaemolyticus, Vibrio harveyi, Escherichia coli, multidrug-resistant Escherichia coli, Acinetobacter baumannii, resistant strains of Acinetobacter baumannii, Klebsiella pneumoniae, Salmonella typhimurium; Gram-positive bacteria: Bacillus thuringiensis, Micrococcus luteus, Staphyl...

Embodiment 3

[0032] Example 3 Bacterial endotoxin inhibition experiment

[0033] The reagents and consumables used in this experiment are sterile and free of endotoxin.

[0034] Incubate Escherichia coli, Acinetobacter baumannii and Vibrio parahaemolyticus overnight at the optimum temperature and collect the cells, wash 3 times with 1×PBS, and resuspend to 1×10 5 wxya -1. The experimental group was: synthetic peptide mixed with bacterial solution in equal volume (when dealing with Escherichia coli and Vibrio parahaemolyticus, the final concentration of protein in the mixture of protein and bacterial solution was 6 μM; The final concentration of the protein was 6 μM in the mixed solution of the liquid); the control group was: the protein solvent (DMSO solution with a volume ratio of 1 / 1000) was mixed with the bacterial solution in equal volumes, and the mature peptide of large yellow croaker hepcidin (PC- hepc) as a reference, with three replicates in each group. At room temperature, at...

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Abstract

The invention discloses an antibacterial peptide HeHampII‑4 (63‑88) and its application, belonging to the field of biotechnology. The amino acid sequence of the antimicrobial peptide of the present invention is shown in SEQ ID NO:1. The antimicrobial peptide HeHampⅡ-4 (63-88) of the present invention has obvious agglutination effect on Gram-negative bacteria, Gram-positive bacteria and fungi, has no toxicity to hippocampal embryo cells, and can significantly inhibit Vibrio parahaemolyticus, The release of the free endotoxin of Acinetobacter baumannii and Escherichia coli can be used to prepare antibacterial preparations, so as to be applied in the fields of aquaculture and medicine.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an antibacterial peptide derived from the striated hippocampus. Background technique [0002] Seahorse is the only fish with male pregnancy, which has high economic and medicinal value. In recent years, seahorse breeding has developed rapidly and gradually entered a mature industrialization stage. With the continuous expansion of breeding scale and the increase of breeding density, seahorse breeding has been hit hard by diseases, such as enteritis and skin ulcers caused by water quality and pathogenic microorganisms, causing huge economic losses. At the same time, a large amount of nutrients in the excreted feces or fish feed may promote the rapid growth of bacteria in the water and release a large amount of endotoxin, endangering the healthy growth of farmed animals. Furthermore, the extensive use of antibiotics in mariculture, the problems of drug residues and drug resistance of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/46A61K38/17A61P31/04A61P31/10A23K20/147
CPCC07K14/461A61P31/04A61P31/10A23K20/147A61K38/00Y02A50/30
Inventor 林强肖旺红秦耿张艳红
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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