An antimicrobial peptide hehampii-2(63-86) and its application

An antibacterial peptide and nucleic acid technology, applied in the application, antibacterial, antifungal and other directions, can solve the problems of natural antibacterial peptide toxicity, eukaryotic cell hemolysis, narrow antibacterial peptide antibacterial spectrum, etc. good effect

Active Publication Date: 2022-05-27
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-2(63-86) and its application
  • An antimicrobial peptide hehampii-2(63-86) and its application
  • An antimicrobial peptide hehampii-2(63-86) and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1 Determination of the sequence of the striae hippocampal antimicrobial peptide hepcidin (HeHamp II-2)

[0021] Based on the striae hippocampus genome and transcriptome database, the invention obtains the cDNA sequence of HeHamp II-2 by screening, and clones the antibacterial peptide sequence from striae hippocampus tissue. The specific steps are:

[0022] Total RNA was extracted from adult male hippocampus liver tissue using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the accompanying instructions. Using the ReverAce qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) kit, 1 microgram of total RNA was taken for the synthesis of the first strand of cDNA. Primer 5.00 (Palo Alto, CA) was used to design specific primers F, R (F: 5'-ATGAAGCCCTTCAGTTTGTC-3'; R: 5'-TCAATATTCGCAGCAAAAGG-3') to amplify the opening of HeHamp II-2 Reading Frame (ORF). In this study, the PCR reaction followed the following cycle parameters: 95°C for 5 min; 36 cycle...

Embodiment 2

[0023] Example 2 Biological activity detection of the mature peptide hepcidin (HeHampⅡ-2) in striae hippocampus

[0024] Synthesis of HeHampⅡ-2 Mature Peptide

[0025] The amino acid sequence of the mature peptide HeHampII-2 (63-86) is: HNRGCRFCCNCCGRTHFCAFCCEY, or as shown in SEQ ID NO:1. Using solid-phase peptide synthesis, it was synthesized by GenScript Biotechnology Company (Nanjing, China). The molecular mass and purity of the synthetic peptide were 2.834kD and 95.6%, respectively. Synthetic peptides were stored as dry powders at -80°C until use.

[0026] Microbial agglutination test

[0027] Gram-negative bacteria used in agglutination experiments: Vibrio parahaemolyticus, Vibrio harveta, Escherichia coli, multidrug-resistant Escherichia coli, Acinetobacter baumannii, drug-resistant strains of Acinetobacter baumannii, Klebsiella pneumoniae, Salmonella typhimurium; Gram-positive bacteria: Bacillus thuringiensis, Micrococcus luteus, Staphylococcus aureus, methicillin-...

Embodiment 3

[0032] Example 3 Bacterial endotoxin inhibition experiment

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

[0034] Incubate Escherichia coli, Acinetobacter baumannii and Vibrio parahaemolyticus at the optimum temperature overnight, collect the cells, wash 3 times with 1×PBS, and resuspend to 1×10 with 1×PBS 5 cfu mL -1. The experimental group was as follows: the synthetic peptide was mixed with the bacterial solution in equal volume (when Escherichia coli and Vibrio parahaemolyticus were treated, the final concentration of protein in the mixture of protein and bacterial solution was 6 μM; when Acinetobacter baumannii was treated, the final concentration of protein and bacterial solution was 6 μM; The final concentration of protein was 6 μM in the liquid mixture; the control group was: protein solvent (DMSO solution with a volume ratio of 1 / 1000) was mixed with equal volume of bacterial solution, and the mature peptide of hepcidi...

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Abstract

The invention discloses an antibacterial peptide HeHampII‑2 (63‑86) 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 antibacterial peptide HeHampⅡ-2 (63-86) 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 striae hippocampus. Background technique [0002] The hippocampus is the only fish with male pregnancy, which has extremely high economic and medicinal value. In recent years, hippocampal farming has developed rapidly and gradually entered a mature industrialization stage. With the continuous expansion of the breeding scale and the increase of breeding density, seahorse breeding has been severely hit by diseases, such as enteritis and skin ulcers caused by water quality and pathogenic microorganisms, resulting in huge economic losses. At the same time, a large amount of nutrients in excreted feces or fish feed may endanger the healthy growth of farmed animals by promoting the rapid growth of bacteria in the water and releasing a large amount of endotoxin. Furthermore, the large-scale use of antibiotics in mariculture, the problems of drug resid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/46C12N15/12A61K38/17A61P31/04A61P31/10A23K20/147A01N47/44A01P1/00A01P3/00
CPCC07K14/461A61P31/04A61P31/10A23K20/147A01N47/44A61K38/00Y02A50/30
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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